SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

SUNDAY, DECEMBER 4- Late Abstracts 1 

Membrane Trafficking 

SUNDAY 

1970 

A novel SNX3-dependent Retromer pathway is required for Wnt secretion. 

I. McGough 1 , M. Harterink 2 , F. Port 3 , M. Lorenowicz 2 , R. Korswagen 2 , P. Cullen 1 ; 1 Biochemistry, 

University of Bristol, Bristol, England, 2 Hubrecht Institute, Utrecht, Holland, 3 Institute of 

Molecular Life Sciences, University of Zurich 

Wnt proteins play a central role in development. Wnt secretion is mediated by the Wnt-binding 

protein Wntless (Wls), which transports Wnt from the Golgi to the cell surface for release. Wnt 

secretion requires recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking 

pathway, but the mechanism remains poorly understood. Using C. elegans and Drosophila 

genetics, we established that Wls recycling requires an evolutionarily conserved, ‘non-classical’ 

retromer pathway, that functions independently of the classical retromer sorting nexins. We 

found sorting nexin-3, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt 

secretion. SNX3 interacts directly with the cargo-selective sub-complex of retromer. Live cell 

imaging reveals WLS exits SNX3 labelled endosomes through vesicular carriers rather than 

classic retromer decorated tubular profiles. These results establish SNX3 as part of an 

alternative retromer pathway that functionally separates retrograde transport of Wls from other 

retromer cargo. 

1971 

The lectin ArtinM binds to and is internalized by hematopoietic cell lines. 

P. A. Buranello 1 , V. M. Mazucato 1 , M. R. Pinto 1 , M. C. Barreira 1 , M. C. Jamur 1 , C. Oliver 1 ; 

1 Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão 

Preto, Ribeirão Preto, Brazil 

The D-mannose binding lectin ArtinM, extracted from Artocarpus integrifolia, has an important 

role in the defense against pathogens. Artin M is imunnomodulatory and activates immune cells, 

such as mast cells, neutrophils and macrophages. It is known that ArtinM can induce cell death 

in various hematopoietic cell lines. The aim of the present study is to further characterize the 

effects of Artin M on hematopoetic cell lines. The human hematopoietic cell lines NB4, K562 

and U937 were used. Flow cytometric analysis showed that NB4 cells bind 2.4 times less ArtinM 

than K562 and U937 cells. By scanning electron microscopy, it was possible to observe that 

ArtinM activated the various cell lines. After 5 minutes of incubation, the K562 cells already 

presented ruffles on their surface. Only after 15 minutes of incubation was this activation 

observed in NB4 and U937 cells. However, the ruffling was less intense. After one hour of 

incubation in presence of ArtinM, all of the cell lines exhibited extensive ruffling. This ruffling 

was most evident in the K562 cells. Artin M internalization was investigated by fluorescence 

microscopy using biotinylated Artin M followed by streptavidin-FITC. After 15 minutes of 

incubation the K562 and U937 cells had already internalized the lectin, but internalized lectin 

was seen only after 30 minutes in the NB4 cells. One hour after incubation, ArtinM had 

accumulated in cytoplasmic vesicles in all of the cell lines. This internalization was independent 

of clathrin and caveolin. In addition, the cytoplasmic vesicles containing ArtinM did not colocalize 

with CD63, a lysosomal marker. Our results show that ArtinM activates and is 

internalized by hematopoietic cells and that the ArtinM then accumulates in cytoplasmic 

vesicles. The binding, activation and internalization of ArtinM may all contribute to the ArtinM 

induced cell death seen in hematopoietic cells.


1972 

Proteomic Characterization of the Binding Partners of Dopamine Transporter (DAT) and 

Functional DAT Mutants. 

S. M. Moore 1,2 , A. Rao 2 , A. Sorkin 1 , C. C. Wu 1 ; 1 Cell Biology and Physiology, University of 

Pittsburgh, Pittsburgh, PA, 2 Pharmacology, University of Colorado Anschutz Medical Campus, 

Aurora, CO 

Dopamine transporter (DAT) is a 12-transmembrane domain, integral membrane protein that 

acts to terminate the synaptic transmission of the neurotransmitter dopamine. DAT is normally 

expressed primarily on the plasma membrane of the cell and is internalized through clathrinmediated 

endocytosis. It has been shown that with N-terminal deletion, DAT undergoes 

increased internalization through augmented levels of endocytosis. Conversely, a C-terminal 

deletion variant of DAT has been shown to reside primarily in the endoplasmic reticulum, with 

unknown endocytic effects. The main objective of this project is to utilize mass spectrometry 

profiling methods to analyze the binding partners of these different DAT constructs in order to 

increase the understanding of DAT trafficking and endocytosis. Initial studies in mice expressing 

a full-length hemagglutinin (HA)-tagged DAT construct combined immunoprecipitation (IP) using 

an HA antibody with profiling mass spectrometry. These studies resulted in the identification of a 

variety of potential DAT binding partners. Some of the identified proteins were previously known 

to be DAT binding partners, others were known to be involved in endocytosis, and still others 

were known to be involved in synaptic transmission. These mouse studies inspired further 

proteomic studies in cell lines containing the aforementioned DAT constructs, due to the 

identification of endocytic proteins in the mouse studies along with the difference in endocytic 

patterns in the DAT construct containing cell lines. Full length (FL), N-terminally deleted (ΔN), 

and C-terminally deleted (ΔC) DAT constructs, dually tagged with YFP and HA, were stably 

expressed in porcine aortic endothelial (PAE) cells. These constructs then underwent IP using 

either HA or GFP antibodies. After IP the samples were digested with trypsin and analyzed on 

either an LTQ linear ion trap mass spectrometer, for profiling experiments, or a TSQ Vantage 

triple quadrupole mass spectrometer, for quantitative experiments. In these cell studies, several 

proteins were identified that were present in all three samples while others were expressed 

differentially between the samples when compared to the parental PAE cell line. Proteins 

present in all samples included ubiquitin, calmodulin, and alpha-tubulin. Differentially expressed 

proteins included clathrin heavy chain, Rho-GEF 12, and AP2 alpha-1 subunit, among a variety 

of others. This differential expression indicates that the endocytic changes that occur with these 

deletion mutations are accompanied by changes in the proteins that associate with DAT. 

1973 

Cell adhesion and survival requires integrin rapid recycling. 

N. Waxmonsky 1 , S. Conner 1 ; 1 University of Minnesota, Minneapolis, MN 

Integrins are transmembrane heterodimers composed of an α and a β subunit that mediate 

attachment between the cell and its environment, which is a requirement for cell survival in 

many cell types. Cells maintain adhesion by trafficking integrins to the cell surface through 

endosomal sorting pathways; however, the molecular mechanisms governing this process are 

undefined. We hypothesized that integrin recycling through the endosomal compartment is 

important for maintenance of cell adhesion. In order to test this, we depleted HeLa cells of 

factors with known roles in long- and short-loop recycling. Depletion of a long-loop recycling 

factor, EHD1 (Eps15 Homology Domain), did not lead to loss of adhesion. However, depletion 

of factors that have established roles at the early endosome, AAK1 (Adaptor-Associated Kinase 

1) and EHD3, led to cell rounding and an eventual loss of cell adhesion. Defects in integrin 

delivery to the cell surface disrupts formation of focal adhesion complexes. We investigated


whether there were differences in number of focal adhesion complexes in cells with short-loop 

recycling defects. To do so, we visualized complexes using a vinculin antibody and found a 

decrease in number of focal adhesion complexes in AAK1 and EHD3 depleted cells. To observe 

focal adhesion formation in cells with recycling defects, we used a TIRF live cell imaging 

approach to visualize the dynamics of β3 integrin-GFP positive focal complex formation. The 

experiment revealed a reduction in the rate of focal adhesion complex formation in AAK1 and 

EHD3 depleted cells. Commensurate with this observation, we found that β3 integrin total levels 

were also reduced in AAK1 and EHD3, but not EHD1, depleted cells. This raised the possibility 

that the observed reduction might result from a biosynthetic defect or a mis-sorting toward the 

degradative pathway. To distinguish between these possibilities, we disrupted the degradative 

pathway by depleting Tsg101 (Tumor susceptibility gene 101), a factor with a role in the ESCRT 

complex. In doing so, we found partial restoration of β3 integrin total levels and cell attachment 

upon depletion of AAK1/Tsg101 and EHD3/Tsg101. Depletion of factors involved in short-loop 

recycling resulted in a loss of adhesion, a reduction in focal adhesion formation rate and missorting 

to the degradative pathway. This experimental evidence indicates that cell adhesion 

requires integrin short-loop recycling. 

1974 

CFTR anion channel modulates expression of human transmembrane mucin MUC3 

through the PDZ protein GOPC. 

T. Pelaseyed 1 , G. C. Hansson 1 ; 1 Department of Medical Biochemistry, Institute of Biomedicine, 

University of Gothenburg, Göteborg, Sweden 

The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and 

rigid mucin domains, rich in proline, threonine and serine residues that carry numerous Oglycans. 

Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal 

class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different 

human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein 

GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was 

mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. 

GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator 

(CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total 

levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that 

CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two 

proteins. For the first time a direct coupling between mucins and the CFTR channel is 

demonstrated, a finding that will shed further light on the still poorly understood relationship 

between cystic fibrosis and the mucus phenotype of this disease. 

1975 

For “The Perfect Antagonists” the winners are: MyoIC and MyoIE, for their role in “Actin 

shaping for vesicle exocytosis at the immune synapse.” 

J. M. DIAZ MUNOZ 1,2 , M. I. Yuseff 1 , D. Lankar 1 , P. Pierobon 1 , M. Rosemblatt 3 , A. M. Lennon- 

Dumenil 1 ; 1 INSERM U932, Immunity and cancer, Institut Curie, Paris, France, 2 Genetica 

Molecular y Microbiologia, P. Universidad Catolica de Chile, Santiago de Chile, Chile, 

3 Fundacion Ciencia para la Vida, Santiago de Chile, Chile 

Engagement of the B Cell Receptor (BCR) by surface-tethered antigens (Ag) leads to formation 

of a synapse that promotes Ag uptake and presentation onto MHCII molecules. Here, we 

highlight the membrane trafficking events and associated molecular mechanisms required for 

efficient Ag extraction and processing at the B cell synapse. We show that MHCII-containing 

lysosomes are recruited at the synapse and locally undergo exocytosis, a process that relies on


the SNARE protein Vamp-7. Lysosome secretion allows the extracellular release of proteases, 

whose activities promote the extraction of the immobilized Ag. We further show that local reorganization 

of cortical actin by type I Myosins, which link the cortex to the plasma membrane, 

is required for lysosome exocytosis and Ag extraction. Remarkably, while the short-tail Myosin 

IC is recruited at the synapse and facilitates vesicle secretion and Ag uptake, the long-tail 

Myosin IE negatively regulates both processes. These results suggest that the two class I 

Myosins play antagonistic roles in the local reorganization of cortical actin for vesicle secretion 

and Ag uptake. The B cell synapse therefore emerges as a highly specialized site where tightly 

regulated exocytic and endocytic events take place thanks to the local shaping of the 

membrane-cytoskeleton interface by class I Myosins 

1976 

Determination of the Subcellular, Surface, and Extracellular Localization of Hsp70s in 

Mammalian Cells. 

R. Medina* 1 , M. Rashedan* 1 , N. Nikolaidis 1 ; 1 Biological Science, California State University, 

Fullerton, Fullerton, CA 

70-kD Heat shock proteins (Hsp70s) are a family of molecular chaperones that play essential 

roles in stress response by promoting protein homeostasis. Members of this family are primarily 

localized in different subcellular compartments, including the cytosol, the endoplasmic reticulum, 

and the mitochondria. Apart from their primary location in specific parts of the cell, different 

Hsp70s have also been found in other places within the cell, at the cellular membrane, and at 

the extracellular milieu. Although a few stresses and pathophysiological conditions, like cancer, 

have been related with the re-localization of Hsp70s within the cell, their translocation to the 

membrane, and their secretion from viable cells, the majority of these conditions remain 

unknown. Additionally, the signaling mechanisms involved in the trafficking of intracellular 

Hsp70s remain elusive. To this end we studied the intra-, membrane- and extra-cellular 

localization of four members of the Hsp70 family. Specifically, we determined the re-localization 

of HSPA1A and HSPA8, primarily localized in the cytosol, HSPA5, primarily localized in the 

endoplasmic reticulum, and HSPA9, primarily localized in the mitochondria. Human embryonic 

cells treated with different stressors, including heat and ethanol, or untreated were subjected to 

sub-cellular fractionation and the presence of Hsp70s was determined by Western. The amount 

of Hsp70s in the different fractions was quantified and normalized for equal loads using fractionspecific 

antibodies, e.g., beta-actin for the cytosolic fraction. These experiments revealed that 

under normal growth conditions all four proteins were present in the nuclear, cytosolic, 

mitochondrial, and membrane fractions, as well as in the extracellular medium. In heat-shocked 

cells, although all proteins were still present in all fractions their amounts in each fraction were 

significantly different from the untreated cells. These results strongly suggest that after stress 

the different Hsp70s are being re-localized within the cell, are anchored at the cellular 

membrane, and are secreted from the cell. Additional experiments using pharmacological 

manipulation of intracellular trafficking pathways are currently being performed to identify the 

molecular mechanism used by Hsp70s to achieve their translocation and subsequent secretion. 

This study is the first step towards elucidating the biological importance of the relocalization and 

membrane occurrence of these chaperones. 

*equal contribution


1977 

Fab1/PIKfyve Is Required in Multiple Organs, and is the Major Pathway for Production of 

PI(3,5)P2 and PI5P in Mammals. 

S. N. Zolov 1 , D. Bridges 1 , Y. Zhang 2 , R. Verma 1 , W-W. Lee 1 , G. M. Lenk 3 , K. Converso-Baran 4 , 

R. Albin 5 , A. Saltiel 1,6 , M. Meisler 3 , M. Russell 4 , L. S. Weisman 2 ; 1 Life Sciences Institute, 

University of Michigan, Ann Arbor, MI, 2 Department of Cell and Developmental Biology and Life 

Sciences Institute, University of Michigan, Ann Arbor, MI, 3 Department of Human Genetics, 

University of Michigan, Ann Arbor, MI, 4 Department of Pediatrics and Communicable Diseases, 

University of Michigan, Ann Arbor, MI, 5 Department of Neurology, University of Michigan, Ann 

Arbor, MI, 6 Departments of Internal Medicine, University of Michigan, Ann Arbor, MI 

Mutations that cause defects in the levels of the signaling lipids PI(3,5)P2 and PI5P lead to 

profound neurodegeneration in mice. Moreover mutations in human FIG4 predicted to lower 

PI(3,5)P2 and PI5P levels underlie some cases of Charcot-Marie-Tooth syndrome and 

Amyotrophic Lateral Sclerosis. In mammals, PI(3,5)P2 is generated by a protein complex that 

includes the lipid kinase Fab1/PIKfyve, the scaffolding protein Vac14, and the lipid phosphatase 

Fig4. Fibroblasts cultured from Vac14 -/- mutant mice have a 50% reduction in the levels of 

PI(3,5)P2 and PI5P, which leaves open the question of whether there is a second pathway to 

generate these lipids. To address this question we characterize a Fab1 gene-trap mouse 

(Fab1 β-geo/β-geo ). The homozygous Fab1 �β-geo/β-geo mouse is a hypomorph that expresses 10% of 

the normal levels of Fab1 and produces 50% of the normal levels of PI(3,5)P2. When we used 

shRNA silencing in Fab1 β-geo/β-geo fibroblasts to knock-down the remaining Fab1, we observed a 

loss of detectable PI(3,5)P2 and an 85% reduction in PI5P. These fibroblasts also have a 

substantial increase in the amount of PI3P, the substrate for Fab1. Thus, similar to yeast, 

conversion of PI3P to PI(3,5)P2 in mammals requires the Fab1 pathway. Surprisingly, much of 

the PI5P pool also requires the Fab1 pathway. Similar to Vac14 -/- and Fig4 -/- mice, the Fab1 

hypomorphic mouse exhibits neurodegeneration in the brain and in peripheral sensory neurons. 

However, these mice also have profound defects in the heart, lung, kidney, thymus, spleen and 

intestine. Importantly, we examined the heart in Vac14 -/- and Fig4 -/- mouse mutants and found 

similar defects to those observed in the Fab1 β-geo/β-geo mouse. Thus PI(3,5)P2 and possibly PI5P 

have major roles in multiple organs. 

1978 

Characterization of Trak1, a novel protein implicated in hypertonia and epilepsy. 

C. A. Lee 1 , L. Li 1 , L-S. Chin 1 ; 1 Pharmacology, Emory University, Atlanta, GA 

Hypertonia—a pathophysiological condition characterized by postural abnormalities, jerky 

movements, and tremor—is associated with a number of neurological disorders, including 

cerebral palsy, dystonia, Parkinson's disease, stroke, and epilepsy. The pathogenic 

mechanisms that trigger hypertonia remain elusive. Interestingly, a homozygous truncation 

mutation in the trafficking protein, kinesin-binding 1 (Trak1) protein causes a recessively 

transmitted form of hypertonia in mice. Moreover, recent genome wide high-density SNP-based 

linkage analysis has linked variants in Trak1 to childhood absence epilepsy in humans. Despite 

the critical importance of Trak1 in maintaining neuronal function, the mechanism and sites of 

Trak1 action remain unclear and the pathogenic mechanism by which Trak1 mutation causes 

hypertonia is unknown. We have generated a highly specific anti-Trak1 antibody and shown that 

endogenous Trak1 protein is expressed in multiple tissues, including the brain. 

Immunofluorescence confocal microscopic studies show that endogenous Trak1 protein is 

localized in axons, dendrites, and cell bodies of mouse cortical neurons, supporting a functional 

role of Trak1 in neuronal physiology. We found that endogenous Trak1 protein is associated 

with both early endosomes and mitochondria in cortical neurons. Our data suggest that Trak1


protein may play a critical role in maintaining neuronal homeostasis by regulating endosomal 

trafficking and mitochondrial transport. Further characterization of the mechanisms of Trak1 

action and its regulation will provide a better understanding of the role of Trak1 in hypertonia 

and epilepsy. 

1979 

Post-Fusion Actin Coating of Secretory Vesicles is required for Active Content Extrusion 

in Alveolar Type II Cells. 

P. Miklavc 1 , E. Hecht 1 , N. Hobi 2 , O. H. Wittekindt 1 , T. Haller 2 , P. Dietl 1 , E. Felder 1 , C. Kranz 1 , M. 

Frick 1 ; 1 University of Ulm, Ulm, Germany, 2 Innsbruck Medical University, Innsbruck, Austria 

Exocytosis of secretory vesicles is a fundamental cellular process allowing regulated secretion 

of vesicle contents in the extracellular space. Growing evidence suggests that regulation of 

fusion pore dilation during the post-fusion stage of exocytosis plays an important role in release 

of secretory vesicle contents. However, dependent on the nature of the cargo additional 

mechanisms might be essential to facilitate effective release. We have recently described in 

alveolar type II cells that surfactant-storing secretory vesicles (lamellar bodies) are coated with 

actin following fusion with the plasma membrane. Surfactant, a lipoprotein-like substance, does 

not readily diffuse out of fused lamellar bodies following opening and dilation of the fusion pore. 

Using fluorescence microscopy, atomic force microscopy and biochemical assays we present 

evidence that actin coating and subsequent compression of the actin coat is essential to 

facilitate surfactant secretion. Simultaneous imaging of the vesicle membrane and the actin coat 

revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to 

active extrusion of vesicle contents. Preventing actin coating of fused LBs with latrunculin A 

inhibits surfactant secretion almost completely. Subsequent compression of the actin coat is 

modulated by myosin II. In summary our data suggest that fusion pore opening and dilation is 

not sufficient for “passive” release of bulky vesicle cargo in alveolar type II cells and that active 

extrusion mechanisms are required. 

1980 

Synaptotagmin-7 - the link between fusion-activated Ca 2+ -entry and regulation of fusion 

pore expansion? 

N. Sharma 1 , P. Miklavc 1 , K. Thompson 1 , O. H. Wittekindt 1 , E. Felder 1 , P. Dietl 1 , M. Frick 1 ; 

1 Institute of General Physiology, University of Ulm, Ulm, Germany 

Ca 2+ is the key element in regulated exocytosis controlling multiple steps during the exocytic 

pre- and post-fusion stages. We have recently described a “fusion-activated” Ca 2+ -entry (FACE) 

via vesicular P2X4 receptors during the post-fusion stage of lamellar body exocytosis in alveolar 

type II (ATII) cells. FACE regulates fusion pore expansion and vesicle content release during 

the post-fusion phase of exocytosis (PNAS, 2011 Aug 30;108(35):14503-8). Yet, the question 

remained how this locally restricted Ca 2+ signal is translated into mechanical force promoting 

fusion pore expansion. In this study we tested whether synaptotagmins could form a molecular 

link between FACE and fusion pore expansion. Members of the synaptotagmin family that 

localize to secretory vesicles have been found to promote fusion pore expansion via Ca 2+ 

binding to C2 domains. Using RT-PCR and western-blotting we identified synaptotagmin-7 as 

the Ca 2+ -binding isoform predominantly expressed in primary ATII cells. Immunofluorescence 

confirmed for the first time that synaptotagmin-7 is localised on lamellar bodies. We next 

performed functional studies over-expressing wt synaptotagmin-7 or mutants abolishing Ca 2+ - 

binding to either the C2A, C2B or C2A and C2B domains. We measured diffusion rates of 

fluorescent dyes through the fusion pore to directly assess dynamic changes in fusion pore 

diameters. Our results show that overexpression of neither wt nor mutant synaptotagmin-7


affects fusion pore expansion in the absence of FACE. However, when fusions were followed by 

FACE fusion pore expansion was modulated by over-expression of wt or mutant synaptotagmin- 

7. 

In summary these data confirm that FACE constitutes an “autoregulatory” mechanism, 

modulating the post-fusion fate of individual vesicles. In this model synaptotagmin-7 constitutes 

the Ca 2+ sensor for FACE providing the link between FACE and fusion pore expansion in 

lamellar body exocytosis. 

1981 

Analysis of GGA null mice demonstrates a non-redundant role for mammalian GGA2 

during development. 

B. Doray 1 , J. Govero 1 , S. Kornfeld 1 ; 1 Washington University Sch Med, St Louis, MO 

The mammalian GGAs (Golgi-localized, gamma-ear containing, ADP-ribosylation factor- binding 

proteins) comprise three homologous proteins that function as monomeric clathrin adaptors. 

Numerous studies using cultured mammalian cells have shown that the three GGAs function in 

the transport of cargo proteins between the trans-Golgi network and endosomes. However, the 

in vivo role(s) of these adaptor proteins and their possible functional redundancy has not been 

analyzed. To address this issue, gene ablation studies were performed in mice using insertional 

mutagenesis. Our data demonstrate that loss of GGA1 or GGA3 alone is well tolerated whereas 

the absence of GGA2 results in embryonic lethality (C57BL/6J and 129/Ola mixed parentage), 

or neonatal lethality (C57BL/6NJ background). Out of 169 pups from Gga2 heterozygous 

crosses in the C57BL/6NJ genetic background, a single Gga2 -/- mouse escaped lethality, 

remains alive and is fertile when crossed with a Gga2 +/- male. This result indicates that GGA2 

mediates some vital function during embryogenesis and the neonatal period that cannot be 

compensated for by GGA1 and/or GGA3 but beyond this period, GGA2 may be dispensable. 

This result is also consistent with our expression studies showing that the level of GGA2 is 

highest in the brain during embryonic development and early life but decreases substantially two 

weeks after birth. In contrast, brain expression of GGA1 and GGA3 remains consistently high 

from late embryogenesis through the adult stage. The fact that all three GGAs are expressed at 

comparable levels in the brain during embryonic development excludes the possibility that 

GGA2 is the sole GGA present during this phase, at least in the brain. The combined loss of 

GGA1 and GGA3 also results in a high incidence of neonatal mortality (58% mortality within the 

first 3 weeks) but in this case the expression level of GGA2 may be inadequate to compensate 

for the loss of the other two GGAs. We conclude that the three mammalian GGAs are essential 

proteins that are not fully redundant. 

1982 

Severing of actin filaments by cofilin is required for both disassembly and assembly of 

the actin patch in fission yeast. 

Q. Chen 1 , T. Pollard 1,2 ; 1 Department of Molecular, Cell and Developmental Biology, Yale 

University, New Haven, CT, 2 Department of Molecular Biophysics and Biochemistry, Yale 

University, New Haven, CT 

We use mutant cofilins to test the proposal that cofilin severs actin filaments during endocytosis 

in fission yeast. We replaced the endogenous protein with a mutant cofilin that is defective in 

severing actin filaments. We used quantitative fluorescence microscopy to study the endocytic 

actin patches in these mutant cells. We tracked GFP tagged patch markers in the patches, 

including early endocytic adaptor proteins, activators of Arp2/3 complex and actin filaments. 

Consistent with the hypothesis, the actin patches disassembled far slower during their inward 

movement than in wild type cells. Abundant actin filaments accumulated at endocytic sites, even


after the membrane adaptor proteins dissociated from the patches. We were surprised to find 

that actin patches also assembled slowly in these cofilin mutant cells. Adaptor proteins such as 

End4p and Pan1p accumulated and persisted at the endocytic sites more than 10 times longer 

than in wild type cells, followed by delayed put persistent recruitment of activators of Arp2/3 

complex, including WASP and myosin I. We propose that severing by cofilin normally produces 

short actin filaments that diffuse out of actin patches and stimulate Arp2/3 complex in adjacent 

patches by serving as the mother filaments that initiate the autocatalytic branching reaction. 

Additional feedback mechanisms seem to prolong the early steps in the pathway until the later 

steps are executed. 

1983 

Structural and functional characterization of cargo-binding sites of the µ4-subunit of 

adaptor protein complex 4. 

B. H. Ross 1 , E. A. Corales 1 , Y. Lin 1 , J. H. Hurley 2 , J. S. Bonifacino 3 , P. V. Burgos 1 , G. A. 

Mardones 1 ; 1 Universidad Austral de Chile Sch Med, Valdivia, Chile, 2 LMB, National Institute of 

Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, 3 CBMP, National Institute of 

Child Health and Human Development, NIH, Bethesda, MD 

Adaptor protein (AP) complexes assist protein trafficking by playing key roles in the selection of 

cargo molecules to be sorted in post-Golgi compartments. The medium-sized subunit (µ1-µ4) of 

the four heterotetrameric AP complexes recognizes YXXØ-sequences (Ø is a bulky hydrophobic 

residue), which are sorting signals in transmembrane proteins. A conserved region, the µ2binding 

site, mediates recognition of YXXØ-signals. Recently we found that a non-canonical 

YXXØ-signal binds to a distinct µ4-binding site of the AP-4 complex. In this study we aimed to 

determine the functionality of both binding sites on the recognition of this non-canonical YXXØsignal. 

We used site-directed mutagenesis, yeast-two hybrid (Y2H) analyses, isothermal titration 

calorimetry (ITC), and X-ray crystallography. Substitutions in either of both binding sites on µ4 

abrogated binding to the APP-tail in Y2H experiments. Further characterization by ITC showed 

no binding only with the R283D substitution at the µ4-binding site, in contrast with a decrease in 

binding affinity with the substitution D190A at the µ2-binding site. We solved the crystal 

structure of the C-terminal domain of the D190A mutant of the µ4 subunit bound to the noncanonical 

YXXØ-signal. This structure showed no significant difference compared to that of 

wild-type µ4 subunit. Our mutational, biochemical and structural analyses established the role of 

the µ4-binding site for the non-canonical YXXØ-signal. 

FONDECYT 1100896 

1984 

Regulation of the RalGAP Complex by Akt-Catalyzed Phosphorylation. 

D. Leto 1,2 , X-W. Chen 1,2 , A. Burk 1,2 , T. Xiong 1,3 , G. Yu 1,2 , A. Saltiel 1,2 ; 1 University of Michigan, 

Ann Arbor, MI, 2 Life Sciences Institute, University of Michigan, Ann Arbor, MI, 3 Department of 

Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 

In response to feeding, the anabolic hormone insulin increases glucose uptake into adipocytes 

by stimulating exocytosis of vesicles containing the facilitative glucose transporter, Glut4. The 

small vesicle-localized G protein, RalA, is rapidly and transiently activated by insulin in an Aktdependent 

manner. When bound to GTP, RalA facilitates recognition of Glut4 vesicles at the 

plasma membrane by interacting with the exocyst, an 8-subunit tethering complex. We have 

previously shown that in the absence of insulin, RalA is retained in a largely inactive state by a 

RalGAP Complex comprised of a regulatory subunit, RGC1, and a catalytic subunit, RGC2, that 

contains a GAP domain with specific activity toward RalA. Activation of this GTPase by insulin 

requires inhibition of the RalGAP complex. Here, we show that insulin stimulates Akt-catalyzed


phosphorylation of RGC1/2 on at least three residues, and that phosphorylation inhibits the 

complex without directly inactivating its catalytic activity. We have identified a novel interaction 

between 14-3-3 and RGC1/2. This interaction is dependent on phosphorylation of RGC2 on a 

residue that was previously identified as an Akt target site. 14-3-3 binding does not interrupt the 

interaction between RGC1 and RGC2 or the catalytic activity of the complex. Thus, 14-3-3 may 

regulate RGC1/2 by altering its interaction with RalA, as has been reported for other GAP 

complexes. 

1985 

The Rab GAP Msb3/Gyp3 regulates dose-dependent Rab5/Vps21 signaling. 

D. Nickerson 1 , A. Merz 1 ; 1 Biochemistry, University of Washington, Seattle, WA 

Membrane transport into and out of endosomes in yeast requires the function of a trio of Rab5 

orthologs: Vps21, Ypt52 and Ypt53. While loss of Vps21 causes obvious trafficking phenotypes, 

the roles of its paralogs Ypt52 and Ypt53 are mostly unexplored and the regulatory logic of the 

trio is unclear. We report that Ypt52 and Ypt53 cooperate with Vps21 to mediate biosynthetic 

transport of CPY and multivesicular body cargoes to the vacuole. Cells deficient in multiple 

Rab5 paralogs display synthetic defects in both these processes and in calcium resistance. 

Ypt53 functions as a calcineurin-dependent calcium stress response factor. We also find that 

conditions of Rab5 signaling deficiency result in significant upregulation of YPT53, suggesting 

that Ypt53 functions as a positive modulator of Rab5 signal in response to both environmental 

stresses and trafficking malfunctions. We further report that Msb3/Gyp3 functions as the 

principle GAP for Vps21 in vivo. Loss of Gyp3 activity results in both greater accumulation of 

GTP-bound Vps21 on endosomes and mislocalization of Vps21 to vacuoles, suggesting that 

Gyp3 serves the dual purposes of negatively modulating the degree of Rab5 signaling and 

spatially restricting Vps21 to endosomes. 

1986 

Glycosaminoglycan attachment affects the intracellular transport of the amyloid 

precursor protein. 

D. Mihov 1 , M. Spiess 1 ; 1 Biozentrum, University of Basel, Basel, Switzerland 

Amyloid precursor protein (APP) is a type I transmembrane protein implicated in the 

pathophysiology of Alzheimer disease by releasing the amyloidogenic Aβ peptide. The 

intracellular distribution of the different secretases and the sorting and transport of APP define 

the extents of amyloidogenic and non-amyloidogenic processing of the protein. APP splice 

variants lacking exon 15 contain a glycosaminoglycan (GAG) attachment site and are modified 

with a single chondroitin sulfate (ChS) chain. We have previously shown that GAG attachment 

affects exocytic and endocytic traffic of model proteins. Therefore, we investigated the effect of 

GAG attachment on intracellular traffic and processing of APP. HeLa cells were transiently 

transfected with the neuronal-specific splice variant APP-695 or with its corresponding 

proteoglycan isoform APP-677, lacking exon 15. Since APP is naturally tyrosine sulfated, we 

used radioactive sulfate to metabolically label transfected cells and to simultaneously detect the 

GAG-containing and GAG-free forms of APP. About 50% of APP-677 was GAG-modified, 

containing ChS chains. Pulse-chase experiments showed that GAGs accelerated the 

biosynthetic exocytosis of the APP-677 proteoglycan in comparison with APP-695 and APP-677 

lacking ChS. APP contains well characterized signals for endocytosis from the plasma 

membrane. While GAG-free APP was internalized at a rate of about 6% per min, GAG 

attachment resulted in a significant reduction of endocytosis of at least threefold. The rates of 

recycling, however, were not affected. Our results indicate that exon-15 splicing and subsequent


GAG attachment alters APP intracellular transport, potentially influencing its processing and Aβ 

generation. 

1987 

Methyl esterification of retinal proteins is essential for rod-mediated vision. 

J. R. Christiansen 1 , S. Kolandaivelu 1 , M. O. Bergo 2 , S. G. Young 3 , V. Ramamurthy 1,4 ; 

1 Ophthalmology, West Virginia University, Morgantown, WV, 2 Cancer Center Sahlgrenska, 

University of Gothenburg, 3 eDepartments of Medicine and Human Genetics, David Geffen 

School of Medicine University of California, Los Angeles, 4 Biochemistry, West Virginia University 

Background: Proteins ending in a “CAAX” box are first prenylated at their c-terminal cysteine 

and then RAS-converting enzyme 1 (RCE1) cleaves the final three amino acids before 

isoprenylcysteine methyltransferase (ICMT) catalyzes the methyl esterification of the newly 

prenylated cysteine residue. Prenylation is crucial for a protein’s ability to interact with 

membrane domains. The contribution of the final two steps to a protein’s localization and 

function are variable and depends on the protein being studied. We recently demonstrated the 

importance of RCE1-mediated proteolysis in phototransduction. In the absence of Rce1, 

photoreceptors do not function and rapidly degenerate. To determine if the effects seen in the 

retina conditional knockout of Rce1 were related to the lack of proteolysis, lack of carboxyl 

methylation, or both we analyzed the retina of mice with reduced levels of Icmt. 

Methods: We utilized the hypomorphic Icmt fl allele as a tool to generate animals expressing 

various levels of Icmt in the retina. Electroretinogram (ERG) recordings were used to analyze 

visual function of littermate animals with a range of Icmt levels. Morphology and 

phototransduction protein expression profiles were investigated by immunofluorescence and 

immunoblotting respectively. 

Results: ERG recordings displayed delayed photoreceptor cell responses and reduced 

downstream electrical responses to light stimuli. The delayed photoreceptor cell response 

correlates to changes in Icmt message levels in the retina. In agreement with delayed 

photoreceptor cell responses, critical phototransduction protein levels were also altered. To 

investigate the cause of the reduced downstream neuronal responses, we are currently verifying 

the development and proper synaptic stratification of inner retinal neurons. 

Conclusion: Efficient coupling of signal transduction in photoreceptor neurons requires methyl 

esterification of a phototransduction protein. In comparison with Rce1 CKO, we did not observe 

rapid retinal degeneration or loss of visual function. At low light levels, the visual response was 

reduced corresponding to reductions in Icmt. In agreement with the loss of visual response 

phototransduction protein turnover was also increased. 

1988 

Turnover of amyloid precursor protein carboxy terminal fragment beta (C99) in lysosomal 

compartments. 

A. Rivera-Dictter 1 , H. Bustamante 1 , V. Muñoz 1 , V. Cavieres 1 , G. A. Mardones 1 , J. S. Bonifacino 2 , 

P. V. Burgos 1 ; 1 Universidad Austral de Chile Sch Med, Valdivia, Chile, 2 CBMP, National Institute 

of Child Health and Human Development, NIH, Bethesda, MD 

Proteolytic processing of the amyloid precursor protein by β-secretase generates C99, which 

subsequently is cleaved by γ-secretase, yielding the amyloid β peptide (Aβ). C99 contains 

within its cytosolic tail key signal motifs for its delivery to lysosomal compartments. Our aim was 

to investigate lysosomal turnover of C99 in a mechanism independent of γ-secretase cleavage. 

H4 neuroglioma cells stably expressing C99-EGFP-WT were analyzed in pulse-chase 

experiments with cycloheximide, in the absence or presence of γ-secretase inhibitors testing 

several conditions in order to: 1) block delivery to lysosomes and 2) disrupt lysosomal function.


Blocking delivery of C99 to lysosomes or disrupting lysosomal function caused an enhancement 

in its proteolytic processing by γ-secretase. Similar conditions but in the presence γ-secretase 

inhibitors showed a significant delay in C99 turnover with a strong accumulations in lysosomal 

compartments. Our results show that lysosomes play an important role in the turnover of C99 

suggesting that dysfunction of these organelles might have key implications in Aβ production 

and Alzheimer. 

FONDECYT 1100027 and DID-UACH 

1989 

Ciliary targeting of sensory receptors is coordinated by Arf and Rab GTPases and their 

effectors. 

D. Deretic 1 , J. Wang 1 ; 1 University of New Mexico, Albuqueruqe, NM 

Ciliopathies encompass a wide range of human diseases caused by dysfunctional primary cilia, 

however trafficking of signaling receptors to the ciliary membrane remains poorly understood. A 

recently described ciliary targeting complex organized by Arf4 binds at the trans-Golgi network 

(TGN) to the VxPx motif present in membrane proteins targeted to primary cilia, including 

rhodopsin. We now show that the ciliary targeting is initiated at the TGN, by the formation of a 

tripartite complex between rhodopsin, Arf4GTP and the Arf-GAP ASAP1, which each recognize 

one of the two ciliary targeting motifs of rhodopsin, VxPx and FR, respectively. Subsequently, 

ASAP1 recruits Rab11a and the Arf/Rab11 effector FIP3 displaces rhodopsin from ASAP1. On 

ciliary transport carriers, ASAP1 provides an activation platform for the Rab11a-Rabin8-Rab8 

ciliary destination module. Ciliary localization of rhodopsin-GFP-VxPx expressed in epithelial 

cells is lost upon ablation of ASAP1 that causes formation of actin-rich periciliary membrane 

projections, to which ciliary cargo is redirected. Furthermore, [FR-AA]rhodopsin-GFP-VxPx that 

is defective in ASAP1 binding fails to engage Rab8 and translocate across the periciliary 

diffusion barrier. As the VxPx and FR targeting motifs are present in other sensory receptors, 

comparable coupling of cargo recognition with cargo destination likely confers directionality to 

ciliary membrane transport. 

Organelles and Membrane Biology 

1990 

Determinants of ciliary identity in Giardia intestinalis. 

A. Wiedmann 1 , K. Hagen 1 , S. C. Dawson 1 ; 1 Department of Microbiology, University of California, 

Davis, Davis, CA 

Flagella (or cilia) are essential for the function of many cell types yet little is known about how 

different cell types, given the same genetic information, can build flagella of different size, 

protein composition and function. The unicellular intestinal parasite Giardia intestinalis forms 

four pairs of flagella distinct in length, behavior and protein composition (1). Identifying factors 

that allow for the formation of these flagella could inform research on human diseases caused 

by defects in flagellar growth, such as polycystic kidney disease, as well as well as lead to 

better treatment of the disease caused by this parasite. To determine how flagellar identities are 

established in Giardia we are (1) developing a novel whole-genome overexpresision anti-sense 

screen, and (2) in a candidate approach, are investigating the role of three putative NEK 

kinases in ciliogenesis. 

Giardia is a binucleate organism and thus random mutagenesis is not a feasible approach for 

identifying ciliogenesis factors. We are using T7 polymerase to direct overexpression of a library 

of small genomic DNA fragments to allow for a whole genome screen for factors important for


the establishment of ciliary identity in Giardia intestinalis. Our candidate approach focuses on 

three NEK kinases. NEK kinases were first identified for their role in mitosis but have been 

shown to play a role in ciliogenesis and flagellar length determination in model systems such as 

Tetrahymena (2,3). Similar to the Tetrahymena genome, the giardial genome encodes an 

unusually large number of these kinases, suggesting that some may have non-mitotic, 

regulatory roles. Consistent with this hypothesis, we have identified several putative NEK 

kinases associated with the giardial ventral disk and flagella. Most of these are predicted to be 

catalytically inactive and may play structural or kinase activity independent regulatory roles. 

Three of the kinases, GL50803_11311, _8445, and _95593, however, contain a canonical 

kinase active site. We are using morpholino knockdown as well as overexpression of kinaseinactive 

and constitutively active variants to assess whether these putative kinases contribute to 

Giardia motility and/or ciliogenesis. 

References: 

1. Dawson SC and House SA (2010) Methods Cell Biol. 

2. Quarmby LM and Mahjoub MR (2005) J Cell Sci. 

3. Wloga D, et al (2006) Mol Biol Cell. 

1991 

Trophic accumulation of the antidepressant sertraline/Zoloft in fungal secretory 

membranes. 

E. Perlstein 1 , J. Chen 1 , D. Korostyshevsky 1 ; 1 Lewis-Sigler Institute/Princeton University, 

Princeton, NJ 

Many antidepressants are cationic amphipaths, which spontaneously accumulate in cellular 

membranes in the absence of their protein targets. However, the clinical relevance of 

progressive cellular membrane infiltration by antidepressants in humans is poorly understood. 

Here we take an “evolutionary pharmacology” approach to studying the effects of the selectiveserotonin 

reuptake inhibitor sertraline/Zoloft on cellular membrane homeostasis in the simple 

eukaryote Saccharomyces cerevisiae, which lacks a serotonin transporter entirely. We 

biochemically characterized the association of tritiated sertraline with the membranes of the 

fungal secretory pathway, and in parallel performed a quantitative ultrastructural analysis of 

membrane quality control in untreated vs. sertraline-treated cells. These experiments have 

revealed that sertraline enters yeast cells and then reshapes the secretory pathway by a 

complex process. Internalization of the neutral species proceeds by simple diffusion amplified 

by lysosomotropism, but is counteracted by ATP-dependent xenobiotic efflux. At equilibrium, a 

small fraction (10-15%) of spontaneously reprotonated sertraline is soluble while the remainder 

partitions into secretory pathway membranes by both adsorption and intercalation. Asymmetric 

accumulation of sertraline results in concentration- and time-dependent local membrane 

curvature stresses, which in turn trigger an adaptive autophagy-dependent relief response. Our 

model appears to support the notion of a serotonin transporter-independent, “amphipath 

accumulation” component of antidepressant pharmacology in humans. 

1992 

The UBX-domain-containing protein Ubx2/ Ubxd8 regulates lipid droplet homeostasis. 

C-W. Wang 1 , S-C. Lee 1 ; 1 Institute of Plant and Microbial Biology, Academia Sinica, Nankang, 

Taipei, Taiwan 

Lipid droplets (LDs) are central organelles for maintaining lipid homeostasis. However, how cells 

control the size and number of LDs remains largely unknown. Herein, we report that Ubx2, a 

protein involved in endoplasmic reticulum (ER)-associated degradation (ERAD), is crucial for LD


maintenance. Ubx2 redistributes from ER to LDs when LDs start to form and enlarge during 

diauxic shift and in the stationary phase. ubx2Δ cells contain abnormal number and reduced 

size of LDs and their triacylglycerol (TAG) is reduced to 50% of the normal level. Deletion of 

either UBX or UBA domain in Ubx2 has no effect, but deletion of both causes LD phenotypes 

similar to that in ubx2Δ. The reduced TAG in ubx2Δ is likely due to mislocalization of Lro1, one 

of the acyltransferases required for TAG synthesis, that normally resides at the putative LD 

assembly sites abutted LDs. The mammalian Ubxd8 expressed in yeast complements the 

defect of ubx2Δ, implying a functional conservation for these UBX-domain-containing proteins in 

lipid homeostasis. 

1993 

Mitochondrial Cardiolipin and Phosphatidylethanolamine have Overlapping Functions in 

Mitochondrial Fusion in Saccharomyces cerevisiae. 

A. S. Joshi 1 , M. N. Thompson 1 , M. Hüttemann 2 , M. L. Greenberg 1 ; 1 Department of Biological 

Sciences, Wayne State University, Detroit, MI, 2 Center for Molecular Medicine and Genetics, 

Wayne State University, Detroit, MI 

The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and 

phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We 

have previously shown that these phospholipids have overlapping functions and the loss of both 

is synthetically lethal. Recent in vitro studies have shown that CL stimulates the GTPase activity 

of the fusion protein Mgm1p and is required for assembly of its isoforms. Because the lack of CL 

does not lead to defects in the mitochondrial network, we hypothesized that PE may 

compensate for CL for maintenance of mitochondrial tubular morphology and fusion. To test this 

hypothesis, we constructed a conditional double mutant crd1�psd1� containing null alleles of 

CRD1 (lacking CL) and PSD1 (lacking mitochondrial PE), in which the wild type CRD1 gene is 

present on a plasmid under control of the TET OFF promoter. Our in vivo studies demonstrate 

for the first time that mitochondrial fusion and maintenance of a mitochondrial tubular network 

require the presence of CL and mitochondrial PE. The crd1�psd1� mutant in the presence of 

tetracycline exhibited highly fragmented mitochondria, a phenotype similar to that observed in 

fusion mutants. The crd1�psd1� cells further exhibited loss of mtDNA and reduced membrane 

potential, characteristic of cells devoid of mitochondrial fusion. Deletion of FIS1 or DNM1, which 

are required for mitochondrial fission, restored the tubular mitochondrial morphology in 

crd1�psd1� cells. Loss of CL and mitochondrial PE led to reduced levels of small and large 

isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, 

these data suggest that mitochondrial phospholipids CL and PE are required to maintain tubular 

mitochondrial morphology and have overlapping functions in mitochondrial fusion. Defects in 

mitochondrial fusion may underlie the mitochondrial morphological variation observed in Barth 

syndrome (BTHS) lymphoblasts and in cardiac and skeletal muscles of the mouse model of 

BTHS. 

1994 

CPSAR1, a protein involved in vesicular trafficking inside the chloroplast. 

N. Z. Khan 1 , E. Lindquist 1 , S. Karim 1 , H. Aronsson1 1 ; 1 Plant and Environmental Sciences, 

University of Gothenburg, Gothenburg, Sweden 

COPII coated vesicles are involved in the secretory pathway transporting proteins and lipids 

from the endoplasmic reticulum (ER) to the Golgi. On the basis of Arabidopsis genome 

sequences and web prediction tools some important components of COPII i.e. the Sec23-Sec24 

complex, the Sec13-Sec31 complex, and the small GTPase SAR1 homologues were predicted 

to be chloroplast localized. It has recently been verified that CPSAR1 is involved in a vesicular


transport system inside the chloroplast similar to the transport mechanism that exists between 

the ER and the Golgi. CPSAR1 has a role in thylakoid biogenesis and in vesicle budding from 

the inner envelope membrane for fusion with the thylakoids. As this mechanism is similar to the 

cytosolic vesicular transport, a putative model is proposed in which the CPSAR1 need other 

protein partners for the vesicular formation and transport. For this purpose we are using coimmunoprecipitation 

and yeast-2-hybrid for identification of protein-protein interaction involving 

CPSAR1. Ongoing bioinformatic studies of common motifs important for interactions and cargos 

of the secretory pathway gives that they also exist for chloroplast localized proteins i.e. possible 

protein interactors to the chloroplast vesicle traffic system. In addition, we are studying the 

intraplastidial role of CPSAR1 from isolated chloroplast envelopes in vitro. 

1995 

A Novel Function of AWP1/ZFAND6: Regulation of Pex5p Export by Interacting with Cysmonoubiquitinated 

Pex5p and AAA ATPase, Pex6p. 

N. Miyata 1 , K. Okumoto 1,2 , S. Mukai 1 , M. Noguchi 2 , Y. Fujiki 1,3 ; 1 Department of Biology, Faculty 

of Sciences, Kyushu University, Fukuoka, Japan, 2 Graduate School of Systems Life Sciences, 

Kyushu University, Fukuoka, Japan, 3 JST, CREST, Tokyo, Japan 

In biogenesis of peroxisome, a subcellular organelle, Pex5p is the shuttling receptor for 

peroxisomal matrix proteins harboring peroxisome-targeting signal 1 (PTS1). Several peroxins 

are involved in the Pex5p shuttling between the cytosol and peroxisomes. However, the precise 

mechanism underlying the PTS1 receptor shuttling remains elusive. We herein suggest that 

liver cytosol contains at least two distinct factors involved in the Pex5p export. We isolate one of 

the factors by biochemical fractionation and in vitro Pex5p export assay and identify it as 

AWP1/ZFAND6 (termed p40), a ubiquitin-binding NF-κB modulator. In in vitro Pex5p export 

assay, recombinant p40, stimulates Pex5p export, whilst anti-p40 antibody interferes with Pex5p 

export. p40 interacts with AAA ATPase Pex6p, but not Pex1p-Pex6p complex. p40 binds Cysubiquitinated 

form of Pex5p more preferentially than unmodified Pex5p, apparently via its A20 

zinc-finger domain. RNA interference for p40 significantly affects the PTS1 protein import into 

peroxisomes. Furthermore, in the p40 knocked-down cells Pex5p is unstable, as in fibroblasts 

from patients each defective in Pex1p, Pex6p, and Pex26p, all prerequisite to the Pex5p export. 

Taken together, p40 is a novel cofactor of Pex6p involved in the regulation of Pex5p export in 

peroxisome biogenesis. 

1996 

Docosahexaenoic acid is required for peroxisomal elongation, a prerequisite for division 

of peroxisomes. 

A. Itoyama 1 , M. Honsho 2 , Y. Abe 2 , Y. Yoshida 2 , Y. Fujiki 2,3 ; 1 Graduate School of Systems Life 

Sciences, Kyushu University, Fukuoka, Japan, 2 Department of Biology, Faculty of Sciences, 

Kyushu University, Fukuoka, Japan, 3 CREST, JST, Tokyo, Japan 

Peroxisomal division is strictly regulated by several fission factors and cellular environments. In 

the past decade, metabolic control of peroxisomal morphogenesis is postulated but remains 

largely undefined. We herein identify docosahexaenoic acid (DHA, C22:6n-3) as the regulator of 

the peroxisomal morphogenesis. Peroxisomes are much less abundant in fibroblasts from 

patients defective in peroxisomal fatty-acid β-oxidation. Supplementation of DHA to such 

fibroblasts induces proliferation of peroxisomes up to the same level in normal fibroblasts. DHAinducible 

peroxisomal proliferation is abrogated upon treatment with dynamin-like protein 1 

siRNA, suggesting the DHA-induced division of peroxisomes. DHA-induced peroxisomal 

division is initiated by elongation of peroxisomes in a Pex11p-dependent manner. Furthermore, 

DHA augments hyper-oligomerization of Pex11pβ, giving rise to Pex11pβ-enriched regions on


the elongated peroxisome membrane. Collectively, these findings suggest that DHA is 

responsible for the elongation step of peroxisomes, the prerequisite stage for the subsequent 

fission of peroxisomes. 

1997 

Molecular Basis for Targeting of C-tail-anchored Proteins to Peroxisomes. 

Y. Yagita 1 , Y. Fujiki 2,3 ; 1 Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, 

Japan, 2 Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan, 

3 CREST, JST, Tokyo, Japan 

C-tail anchored (TA) proteins are a distinct class of membrane proteins that harbor a single 

transmembrane domain at the extreme C-terminal region and expose their N-terminal functional 

domains to the cytosol. Although TA proteins are found in all of subcellular membranes facing 

the cytosol and play pivotal roles in various biological processes, the pathways by which they 

are targeted to and inserted into specific organellar membranes, including peroxisomal 

membranes, are not fully defined. We herein show that knockdown of Pex19p, a predominantly 

cytosolic protein that functions as a chaperon and/or soluble receptor for newly synthesized 

peroxisomal membrane proteins (PMPs), eliminates the import of peroxisomal TA proteins (P- 

TAs) in vivo. Pex19p forms complexes with P-TAs in the cytosol. These results indicate that 

Pex19p is involved in the import of P-TAs as well as non-TA-type PMPs. We also show that P- 

TAs, which form complexes with Pex19p, are specifically targeted to peroxisomes, onto Pex3p, 

even under ATP-depleted condition in an import assay using semi-intact mammalian cells. The 

targeting of P-TAs to peroxisomes is driven by Pex19p-Pex3p interaction. Collectively, these 

results strongly suggest that P-TAs are, like most PMPs, targeted to peroxisomes in a Pex19p- 

and Pex3p-dependent manner. Thus, P-TAs share the import pathway with non-TA-type PMPs, 

in contrast to the TA proteins directed to the ER and mitochondrial outer membrane that do not 

share the import pathways with non-TA proteins. 

1998 

Novel Function of Peroxisome Targeting Signal Type-1 Receptor Pex5p in Pex14p 

Stability: Study Using a Newly Isolated Peroxisome-Deficient CHO Cell Mutant, ZPEG101. 

R. Natsuyama 1 , K. Okumoto 1,2 , Y. Fujiki 1,3 ; 1 Grad. Sch. of Sys. Life Sci. of Kyushu University., 

Fukuoka, Japan, 2 Dept. of Biol., Grad. Sch. of Sci., of Kyushu University., 3 JST, CREST 

Pex5p is one of the peroxins required for peroxisome biogenesis and plays an important role in 

peroxisomal matrix protein import. Majority of peroxisomal matrix proteins possess peroxisome 

targeting signal type-1 (PTS1) at the C-terminus. Pex5p recognizes PTS1 in cytosol via its Cterminal 

region consisting of seven tetratricopeptide repeats and also interacts with peroxisome 

membrane peroxins Pex14p and Pex13p via its N-terminal region. Together with extensive 

studies in yeasts and mammals, Pex5p is now proposed as a shuttling receptor between the 

cytosol and peroxisomes. Here, we found a novel function of Pex5p in stabilizing Pex14p by 

making use of a newly isolated ZPEG101, a PEX5-deficient CHO cell mutant, showing typical 

mutant phenotypes with defects in import of both PTS1 and PTS2 proteins. No Pex5p was 

detected in ZPEG101, indicating ZPEG101 is the first pex5 CHO cell mutant completely lacking 

Pex5p. Interestingly, the expressed level of Pex14p was significantly decreased in ZPEG101, as 

compared to that in wild-type CHO-K1 and other previously isolated pex5 mutants. More 

detailed analyses of ZPEG101 are in progress.

1999 

P7BP1 E3 ubiquitin ligase controls the quality of PTS2 receptor, Pex7p. 

Y. Miyauchi 1 , Y. Fujiki 1 ; 1 Kyushu University, Fukuoka, Japan 


Peroxisomes are ubiquitous single membrane-bound organelles that contain about 100 different 

enzymes catalyzing various metabolic pathways such as fatty-acid ƒÀ-oxidation and 

etherglycerolipid synthesis. Peroxisomes are formed by growth and division of preexisting 

peroxisomes after posttranslational import of newly synthesized peroxisomal matrix and 

membrane proteins. Two distinct signals, peroxisomal targeting signal type 1 (PTS1) and PTS2, 

direct proteins to the peroxisomal matrix. PEX5 and PEX7 encode the cytosolic receptors for 

PTS1 and PTS2, respectively. Mono-Ubiquitination of Pex5p is required for its export from 

peroxisomal membrane to the cytosol in yeast to mammals. However, regulation of the function 

and transport of Pex7p remains undefined. 

We first investigated whether or not Pex7p is ubiquitinated and degraded by proteasomes in 

cells. Addition of a proteasome inhibitor, MG132 to cell culture delayed the turnover rate of 

Pex7p. We also attempted to isolate any potential Pex7p-binding proteins potentially involved in 

the degradation pathway of Pex7p. From a cell line stably expressing FLAG-Pex7p, we isolated 

several Pex7p-binding proteins, termed P7BPs: Pex7p binding proteins. Suppression of P7BP1 

by RNAi resulted in a delay of Pex7p turnover rate, as observed in the MG132-treated cells, 

hence implying that P7BP1 plays a role in Pex7p ubiquitination. Furthermore, we also show that 

the degradation of dysfunctional Pex7p mediated by P7BP1 is crucially required for the 

maintenance of normal PTS2 import. Our findings may define a mechanism underlying Pex7p 

degradation and its importance in regulating PTS2 import. 

2000 

A System to Quantify the Differential Import of Peroxisomal Matrix Protein by Measuring 

Fluorescence Intensity. 

M. Noguchi 1 , Y. Fujiki 1,2 ; 1 Graduate School of Systems Life Sciences, Kyushu University, 

Fukuoka, Japan, 2 CREST, JST 

Fourteen distinct peroxins are identified as essential factors for peroxisome biogenesis in 

mammals, of which ten are involved in peroxisomal import of matrix proteins. Peroxisomal 

matrix protein import is regulated by various cellular factors. However, mechanisms underlying 

such regulations are poorly understood, partly, if not all, due to the lack of quantitative detection 

method with high resolution. Here, we developed a monitoring system that utilizes the 

differences in the stability of fluorescent reporters localized between peroxisomes and the 

cytosol. An FKBP12 variant, termed destruction domain (DD), is rapidly and constitutively 

degraded by proteasomes when expressed in mammalian cells. Degradation of DD is reversibly 

protected by addition of a specific synthetic ligand. In the absence of the ligand, a reporter DD- 

EGFP-PTS1, EGFP fused with the DD and peroxisomal targeting signal 1, is largely degraded 

in the cytosol prior to entering peroxisomes in wild-type cells. In contrast, in the presence of the 

ligand the reporter becomes stable and translocates into peroxisomes. Upon withdrawal of the 

ligand, the reporter in peroxisomes remains intact, whilst that in the cytosol is rapidly degraded. 

Thus, peroxisomal protein import can be readily quantified by simply measuring the 

fluorescence intensity of whole cells.


2001 

Molecular architecture of OPA1, the dynamin-related GTPase involved in mitochondrial 

fusion. 

A. M. Fontainhas 1 , J. A. Heymann 1 , S. Fang 1 , J. E. Hinshaw 1 ; 1 LCBB, National Institute of 

Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 

Mitochondria, the powerhouse of the cell, continually undergo fission and fusion. OPA1 (Optic 

atrophy 1) - a dynamin-related GTPase is essential for the fusion of the inner mitochondrial 

membrane and is involved in cristae maintenance. Once OPA1 is transported to the 

mitochondria, OPA1 is proteolytically processed into a long and a short form. The long form of 

OPA1 is anchored to the inner membrane of the mitochondria through a N-terminal 

transmembrane domain, while the short form of OPA1 (OPA1s) is found in the inner 

mitochondrial space. Both forms are required for mitochondrial membrane fusion in vivo. Over 

200 mutations of OPA1 have been identified that lead to vision loss, mainly through retinal 

ganglion cells. Ban, et al. (2010) showed that OPA1s preferentially tubulates and clusters large, 

negatively charged liposomes. However, the short form alone was unable to mediate membrane 

fusion. It has been proposed that tubulation may be the mechanism by which fusion of 

mitochondrial membranes occur. To elucidate OPA1’s 3D structure and how it interacts with 

lipids we used cryo-electron microscopy to visualize and analyze the topological arrangement of 

OPA1 on these protein-lipid tubules. We show that OPA1s wraps around the perimeter of these 

tubes in a helical fashion. We observed that tubulation is not GTP dependent. However, 

GMPPCP stabilizes the chevron like pattern. OPA1s also forms an organized 2D protein lattice, 

indicating that specific intermolecular interactions are at play. Tubes, under all conditions, range 

in diameter from 50 to 150 nm with a majority of them falling in the 60-70 nm range. 

Understanding how OPA1’s interactions with the membrane leads to fusion of inner 

mitochondrial membrane will give us a better understanding of how mutations in the protein 

affect its function and give rise to vision loss. 

Reference: 

Ban, T., Heymann, J. A., Song, Z., Hinshaw, J. E., Chan, D. C.: OPA1 disease alleles causing 

dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane 

tubulation. Hum. Mol. Genet. 2010, 19: 2113-22. 

2002 

Probing the dynamics of single lipids in supported lipid bilayers and living cells by 

fluorescence correlation spectroscopy and confocal microscopy. 

C-F. Hsieh 1 , L-L. Yang 1 , Y-L. Lin 2 , C-F. Chou 1 ; 1 Institute of Physics, Academia Sinica, Taipei, 

Taiwan, 2 National Taiwan University, Institute of Chemistry, Taiwan 

Lipid raft served as a platform for recruiting signaling components of effective signal 

transduction involving various cellular activities, such as cell proliferation, differentiation, 

migration, and apoptosis in living cells. However, the single-lipid dynamics of lipid raft or nonlipid 

raft domain in living cells are not well elucidated. 

We apply fluorescence correlation spectroscopy (FCS) to probe the mobility of single lipids. We 

observed the behaviors of non-raft lipids and raft lipids in supported lipid bilayers and found that 

the diffusion constant of raft lipids (~2.4 × 10 -8 cm 2 /s) is much smaller than non-raft lipids (~8.1 × 

10 -8 cm 2 /s) and spans a wide range. These results suggest that the raft lipids are in a trend to 

aggregate in supported bilayers while non-raft lipids are not. 

Confocal microscopy was used to monitor the dynamics of raft lipids induced by ligand-receptor 

binding in living cells. The RGD-coated quantum dots (Qdots) were recognized and bound to


integrin to trigger the aggregation of lipid raft. The duration of lipid-raft aggregation was 

extended from 10 minutes (induced by ligand-coated micro-bead) to 2 hours (induced by ligandcoated 

Qdot), and finally the aggregate reached a diameter around 5 micrometers. In our 

studies, RGD-coated Qdot bound to a small number of integrin proteins may provide an 

effective means to observe the early events of lipid raft aggregation in a living cell. 

2003 

Molecular Mechanism of F-BAR Protein Pacsin2 in Caveolae Biogenesis. 

Y. Senju 1 , S. Suetsugu 1 ; 1 Institute of Molecular and Cellular Biosciences, The University of 

Tokyo, Tokyo, Japan 

Protein kinase C (PKC) and casein kinase substrate in neurons 2 (Pacsin2) belongs to the 

EFC/F-BAR domain protein family and deforms membrane. The N-terminal F-BAR domain of 

pacsin2 forms a crescent-shaped dimer with a positively charged surface. The C-terminal SH3 

domain associates with dynamin2, a GTPase implicated in vesicle endocytosis. Therefore, 

pacsin2 is involved in membrane invaginations including caveolae (Senju et al., 2011). Previous 

study has demonstrated that the cells overexpressing pacsin2 showed membrane invaginations. 

However, the number of membrane invagination was larger in the cells overexpressing pacsin2 

F-BAR domain than in the cells overexpressing pacsin2 full length, which was interpreted as 

autoinhibition of pacsin2 resulted from the intramolecular interaction between F-BAR domain 

and SH3 domain. Because pacsin2 has been identified as a PKC substrate, we investigated the 

autoinhibition mechanism based on the phosphorylation of pacsin2 by PKC. First, we performed 

an in vitro kinase assay, and confirmed that PKC actually phosphorylated pacsin2. Then, we 

generated two different pacsin2 mutants by substitution with either alanine or glutamic acid, and 

identified the phosphorylation site in pacsin2 by in vitro kinase assay. Next, we generated a 

phospho-specific antibody, and confirmed that endogenous pacsin2 was phosphorylated after 

the PKC activator PMA treatment using immunofluorescence microscopy and western blotting 

analysis. The number of membrane invaginations induced by phospho-mimic mutant of pacsin2 

was lesser than that induced by wild-type pacsin2. After the PMA treatment, the number of 

membrane invagination was decreased in the cells expressing wild-type pacsin2, but there was 

no significant effect in the cells expressing either phospho-mimic, phospho-deficient mutant, or 

F-BAR domain of pacsin2. Therefore, these data suggest that phosphorylated pacsin2 prefers 

an open protein conformation that permits interaction with its partners such as dynamin2. The 

interaction of pacsin2 with dynamin2 probably caused dynmin2-induced scission of membrane 

invaginations, which in turn decreased the number of pacsin2-induced membrane invaginations. 

Further experimentation will be required to test this hypothesis and reveal the physiological role 

of pacsin2 in caveolae biogenesis. 

2004 

Identification and characterization of atypical FYVE Domains for specific interaction with 

phospholipids. 

J-E. Gil 1 , I-S. Kim 2 , B. Ku 1 , W. Park 1 , B-H. Oh 1 , S. Ryu 2 , W. Heo 1 ; 1 Korea Advanced Institute of 

Science and Technology, Daejeon, Korea, 2 Pohang University of Science and Technology, 

Korea 

Interactions of lipid binding domains with intracellular lipids play a significant role in regulation of 

numerous signaling and cellular mechanisms. To characterize interactions of lipid binding 

domains with phospholipids, five classes of lipid binding domains were screened by fluorescent 

imaging and classified based on their subcellular localizations. Six of over one hundred kinds of 

lipid binding domains were localized in the plasma membrane where a number of signaling 

cascades are generally initiated. Interestingly, we found FYVE domain of ZFYVE27 which is


localized in the plasma membrane unlike typical FYVE domains which are localized in the 

endosomes. Conventionally, it has been known that lipid binding pocket of FYVE domains has 

WXXD, R+HHC+XCG and RVC regions through which FYVE domains bind to PI(3)P. However, 

from analysis of amino acid sequences and model of 3D protein structure, we found that 

ZFYVE27-FYVE domain did not contain WXXD, R+HHC+XCG and RVC regions, reflecting it 

may have unconventional property in lipid interaction of FYVE domain. It was demonstrated that 

ZFYVE27-FYVE domain specifically interacts with plasma membrane phospholipids by using 

inducible strategies in which phospholipids are depleted by translocating phosphatase to the 

plasma membrane and specific inhibitor treatment. We also observed that growth factor-induced 

ZFYVE27 translocation was not induced when this interaction was ruined. 

2005 

Plin 2 and Plin 3 exhibit characteristics of functionally diverse C-terminal domains. 

B. Chong 1,2 , L. Cicchini 1,2 , P. Reigan 3 , J. McManaman 1 ; 1 OB/GYN, University of Colorado 

Denver, Anschutz Medical Campus, Aurora, CO, 2 Graduate Program in Molecular Biology, 

University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, 3 Pharmaceutical 

Science, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 

Perilipin family members, adipophilin (ADPH/Plin2) and TIP47 (Plin3) have the highest degree 

of sequence similarity within the perilipin family, with nearly 60% similarity, and are thought to 

be redundant lipid droplet associated proteins. Our laboratory previously demonstrated that the 

N-terminal half of ADPH is required for association with the CLD monolayer and this interaction 

protects the CLD core from lipolysis. More recent studies indicate that the C-terminal half of 

ADPH is a lipid membrane association domain that is required for milk lipid secretion. TIP47 

was originally identified as a vessicle trafficking protein but evidence supporting this is 

controversial. Additionally, less is known about the unique functional domains within TIP47. In 

mammary epithelial cells, the two family members have distinct cellular localizations and 

differential expression patterns over the course of pregnancy and lactation, which suggest that 

the two proteins are not functionally redundant. We took a structural and biochemical approach 

to understanding how these two proteins are functionally distinct. ADPH and TIP47 share 43% 

amino acid similarity in their C-terminal domain and are hypothesized to be structural homologs. 

The C-terminus of TIP47 has been crystallized and is known to form a four-helix bundle motif 

with a centrally located hydrophobic cleft motif. We constructed a homology model for the Cterminus 

of ADPH, which shows that ADPH forms a putative four-helix bundle and hydrophobic 

cleft, similar to that of TIP47. Closer examination of these structures shows that these two 

proteins have noticeable differences in overall shape, with TIP47 being more compact and 

ADPH having a slightly more extended in structure. We expressed and purified recombinant 

ADPH and TIP47 C-termini from E. coli and analytical size exclusion chromatography (SEC) 

shows that the two proteins have different retention times, suggestive of differences in overall 

shape. Additionally, native PAGE comparison shows differences in migration between the two 

proteins and unique sensitivies to urea denaturation. Analytical ultracentrifugation sedimentation 

velocity and SEC suggest that ADPH forms higher order structures and TIP47 does not. We 

provide the first side-by-side comparison of ADPH and TIP47 and show that they are not 

structurally identical. Further work is needed to determine how these structures account for 

functional diversity.


2006 

Perilipin, Moesin and Microtubule Dependence of Lipid Droplet Clustering and 

Dispersion. 

D. Orlicky 1 , A. Stefanski 2 , B. Chong 2 , J. Monks 2 , C. Monks 3 , J. L. McManaman 2 ; 1 Pathology, UC 

Denver, Anschutz Medical Campus, Aurora, CO, 2 Ob/Gyn, UC Denver, Anschutz Medical 

Campus, Aurora, CO, 3 Intelligent Imaging Innovations, Inc., Denver, CO 

Cytoplasmic lipid droplets (CLD) function in storage, trafficking and secretion of lipids as well as 

a depot for fatty acid signaling precursors. Perilipin 1, the primary CLD binding protein in 

adipocytes, is thought to contribute to CLD trafficking by directing fragmentation of CLD into 

smaller droplets, making the lipids more enzymatically available. To study the role of perilipins in 

mediating intracellular CLD trafficking we used stable ectopic expression of perilipin 1, or 

truncated perilipin 1, in HEK293 cells. Expression of either full-length or C-term perilipin 1 

(amino acids 198-517) initiated formation of single, dense CLD clusters that localize near the 

microtubule organizing center (MTOC). Using live-cell imaging, we showed these clustered CLD 

became fragmented and moved to the periphery of the cell in response to isoproterenol or 

forskolin activation of adenylate cyclase- which has previously been shown to regulate PKAmediated 

phosphorylation of perlipin 1. We further show that nocodozole disruption of 

microtubules could inhibit this process, while staurosporine blockage of PKA activity disrupted 

dispersion, but not the initial fragmentation of the CLD cluster. Finally, siRNA knockdown of the 

cortical actin-binding protein, moesin, prevented dispersed CLD from reforming dense clusters 

when isoproterenol was removed. These data suggest a model in which CLD clustering and 

dispersion are mediated by both microtubules and actin, and trafficking is controlled by PKAphosphorylation 

of perilipin 1. 

2007 

Netrin-1-induced axon branching of cerebral cortical neurons requires DCC and lipid raft 

integrity. 

H. Matsumoto 1 , M. Nagashima 1 ; 1 Department of Anatomy, Faculty of Medicine, Saitama Medical 

University, Saitama, Japan 

A multifunctional axon guidance cue netrin-1 is known to induce axon branching in developing 

cerebral cortical neurons of mammals. In this study we investigated whether netrin-1-induced 

cortical axon branching involves DCC (deleted in colorectal cancer; one of known receptors for 

netrin-1) and lipid rafts (cholesterol- and sphingolipid-enriched membrane microdomains), 

employing dissociated cortical neurons prepared from neonatal Syrian golden hamsters. 

Stimulation of neurons by netrin-1 caused an increase in the number of branch points along the 

primary axons, which is in consistent with previous reports. We then observed that this increase 

was attenuated by application of a neutralizing antibody against DCC to the culture. Attenuation 

of netrin-1-induced axon branching was also caused by disruption of lipid rafts, while 

association of DCC to lipid rafts was revealed by Western blot analysis. These data indicate a 

significant contribution of DCC to, and requirement of lipid raft integrity for, the promotion of 

cortical axon branching caused by netrin-1. Furthermore, the data suggest the possibility that 

association of DCC with lipid rafts is involved in netrin-1-induced axon branching. (Supported by 

Grant-in-Aid for Scientific Research (C) No. 23590225 from Japan Society for the Promotion of 

Science.)


2008 

HGSNAT mono-multimer formation revealed by live-cell two-photon FRET-FLIM 

microscopy. 

F. Majer 1 , J. Sikora 1 , J. Lukas 1 , D. Musalkova 1 , O. Chernyavskiy 2 , M. Hrebicek 1 ; 1 Inst Inherited 

Metabolic Disorders, Charles University, Prague 2, Czech Republic, 2 Department of 

Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., 

Prague 2, Czech Republic 

Human HGSNAT (Acetyl-Coenzyme A: Alpha-Glucosaminide N-Acetyltransferase) is a 

membrane enzyme localized in endosomal/lysosomal microdomains catalyzing transmembrane 

acetylation of heparan sulfate. This synthetic modification is mandatory prerequisite for 

subsequent intralysosomal degradation of heparan sulfate. Mutations in HGSNAT gene cause 

rare but clinically serious lysosomal storage disorder - mucopolysaccharidosis IIIC (Sanfilippo C 

syndrome). 

Our aim was to find intracellular membrane protein partners of HGSNAT. 

We have prepared plasmids for transient expression of HGSNAT with two alternative starting 

ATG codons as well as plasmids coding potentially interacting protein partners of HGSNAT. To 

detect in vivo protein-protein interactions, we used live two-photon excitation FRET-FLIM time 

correlated single photon counting technique with EGFP/mCherry donor-acceptor pairs. 

Additionally, we also prepared fraction of enriched lysosomal membranes using density gradient 

ultracentrifugation and by 2D electrophoresis (Blue Native / SDS PAGE) we identified region of 

HGSNAT protein electrophoretic mobility and controlled protein integrity. 

Basic co-localizing experiments implied LAMP1, LAMP2 and Rab7 their intracellular cooccurrence 

with HGSNAT by resolution limited light microscopy. Nevertheless two-photon 

FRET-FLIM methodology revealed only HGSNAT-EGFP and HGSNAT-mCherry as potential 

interacting partners suggesting mono-multimer formation within the endosomal/lysosomal 

membrane. 

This study was funded by the research project MSM0021620806 from the Ministry of Education, 

Youth and Sports of the Czech Republic and by the Internal Grant Agency of the Ministry of 

Health of the Czech Republic, Project IGA MZ NS / 10342 - 3 / 2009. 

2009 

Mechanism underlying the expression of Na-H Exchanger isoforms 1 and 3 in SHR rat 

colon and ileum in hypertension. 

A. A. Ghefreh 1 , I. Khan 2 , A. A. Ghefreh 1 ; 1 Biochemistry, Kuwait University, Kuwait, 2 Kuwait 

University, Kuwait 

Ileum and colon play an important role in regulating water and electrolyte homeostasis 

mechanisms which are deregulated in hypertension. Role of colonic and ileal Na-H exchanger 

(NHE) isoforms-1 and -3 which regulate water and electrolyte absorption from GI-tract and 

kidneys is poorly investigated in hypertension. In this study we investigated a putative role of 

colonic and ileal NHE-1 and -3 isoforms in hypertension using a 12-week old spontaneously 

hypertensive rat (SHR) model. A group of SHR animals was treated with captopril (300 mg/L) ad 

libitum starting week 4 post birth and followed till week 12. Age matched genetic counterparts, 

WKY animals served as controls in this study. Further to elucidate the underlying mechanism, 

expression of NHERF-1 and cGKII which regulate activity of NHE was also examined. SHR 

animals showed a significant increase in kidney and heart hypertrophy and proteinuria, while 

decrease in urine output as compared to WKY controls. These changes were reversed by 

captopril treatment. There was significant elevation in myeloperoxidase activity in SHR ileum 

and colon as compared to WKY controls. These changes were reversed by captopril treatment. 

Expression of NHE-1, NHE-3 and NHERF-1 proteins was significantly decreased, while that of


cGKII was increased in SHR ileum and colon as compared to WKY controls. Captopril treatment 

of SHR caused a partial reversal of these changes, and a significant reversal of NHERF-1. 

These changes do not seem to be due to any difference in the microsomal protein yield. We 

demonstrate evidence of mild inflammation in SHR colon and ileum due to hypertension. Our 

findings showed decreased expression of NHE-1, -3 and NHERF-1, which together would 

suppress uptake of water and NaCl from the GI-tract and hence may be a counteracting 

response in the development of hypertension in this model. 

Acknowledgments: Kuwait University Research administration (grant YM 06/2010) and College 

of Graduate Studies for financial support and Shared Core Facility, Health Science Center, 

Kuwait University for allowing to use the instrumentation. 

2010 

Gustatory receptors not G protein coupled receptors are required for the response to Lcanavanine. 

Y. Lee 1 , M. Kang 2 , J. Shim 3 , C. Cheong 2 , S. Moon 2 , C. Montell 1 ; 1 Department of Biological 

Chemistry and Neuroscience, Center for Sensory Biology, The Johns Hopkins University School 

of Medicine, 2 Department of Oral Biology, Brain Korea21 project, Yonsei University College of 

Dentistry, Seoul, Korea, 3 Department of Phamacology, Yonsei University College of Medicine, 

Seoul, Korea 

Insects respond to their surroundings through sensing chemical cues and then decide their 

behaviors. Gustatory sense is one of the most important chemical sensory systems. There are 

68 gustatory receptors (GRs) encoded by 60 gustatory receptor genes. Several gustatory 

receptors are known to be required for sensing of sugars or bitter chemicals, such as caffeine. 

However, the functions of most GRs are unknown. Here we show that Gr8a and Gr66a are 

required for sensing insecticide, L-canavainine, which is produced by plants. Gr8a is expressed 

in a subset of Gr66a expressing gustatory receptor neurons (GRNs). The action potentials to Lcanavanine 

are detected only in the Gr8a expressing neuron. The consistence of Gr8a 

expression pattern and electrophysiological response to L-canavanine suggests the possibility 

that Gr8a is the specific receptor to L-canavanine, while Gr66a is the broad receptor to bitter 

chemicals. In contrast, DmXR, one of the G-protein coupled receptors (GPCRs), has been 

reported to be required for detecting L-canavanine. However, DmXR missing flies show the 

avoidance to L-canavanine and action potentials to L-canavanine as wild type flies do in our 

results. These results imply that Gr8a and Gr66a are indispensible for detection of Lcanavanine, 

but not DmXR. 

2011 

NHERF- 1 Modulates Intestinal NaPi transporter NaPi-2b expression in Apical Microvilli. 

H. Giral 1 , Y. Caldas 1 , L. Lanzano 2 , E. Gratton 2 , M. Levi 1 ; 1 University of Colorado Denver, Aurora, 

CO, 2 University of California Irvine, CA 

The regulation of phosphate (Pi) homeostasis is maintained by the coordinated function of the renal and 

intestinal phosphate transporters. Several PDZ (PSD-95/discs large/ZO-1 homologous) domain 

proteins, including NHERF-1, PDZK1, ShanK2, and PIST play an important role in the regulation of the 

renal sodium-phosphate (NaPi) co-transporters (NaPi-2a and NaPi-2c). The main mediator of intestinal 

sodium dependent transcellular Pi transport, NaPi-2b, also contains a PDZ-binding motif consensus in 

the C-terminal region. However, interactions of the transporter NaPi-2b with PDZ proteins have been 

not described and their potential role in regulation of the intestinal transporter is not known. For this 

purpose we performed studies with knock-out (KO) mice models and cell culture to determine a 

potential role for NHERF-1 and PDZK1 in the regulation of NaPi-2b.


To study the putative interaction between NaPi-2b and PDZ proteins we determined the Forster 

Resonance Energy Transference (FRET) by using Fluorescence Lifetime Imaging Microscopy (FLIM). OK 

cells, an extensively used proximal tubule model, and CaCo-2BBE cells, an enterocyte cell model, were 

used to perform this technique. First, expression of EGFP-NaPi-2b was confirmed in the microvilli of both 

cell types, and images along a single microvillus were obtained with the novel Modulation Tracking (MT) 

method. Cells co-expressing EGFP-NaPi-2b and mCherry-NHERF-1 were analyzed by FLIM-FRET 

technique revealing significant FRET between NaPi-2b and NHERF-1. Parallel studies between the pair 

NaPi-2b and PDZK1 proteins resulted in non occurrence of FRET. To evaluate the functional significance 

of these results we study NaPi-2b expression and activity in NHERF1 KO and PDZK1 KO mice models, 

where we found that adaptation to a low Pi diet of NaPi2b was markedly impaired in the NHERF-1 KO 

mice but not in the PDZK1 KO. 

Our results therefore suggest an important role of NHERF1 in modulation of NaPi-2b expression or 

stability in the microvilli of the mouse intestine. 

This research was supported by NIH R01 DK066029 to YC, HG, ML, EG and LL; NIH445 

P41R03155 to EG and LL; and the R01 DK-080769 to BD. 

2012 

Cytoskeleton modulates Ca2+ sensor Stim1. 

C-M. Li 1 , M. Joensuu 1 , E. Jokitalo 1 ; 1 Electron Microscopy Unit, Institute of Biotechnology, 

University of Helsinki, Helsinki, Finland 

Stim1 is a 685 amino acids, single-pass transmembrane protein with N-terminal EF hand 

located in the endoplasmic reticulum (ER) lumen and C-terminal coiled-coil domains facing cell 

cytosol. As a sensor Stim1 plays essential role in Ca2+ signalling, Store-operated Ca2+ entry 

(SOCE) channel activation, and influx of Ca2+ in response to depletion of ER luminal Ca2+. 

Depletion of intracellular Ca2+ stores results in dissociation of Ca2+ from EF-hand, thus 

activating store-operated channels, and trigger translocation of STIM1 proteins to the plasma 

membrane [1]. 

Rosado and his colleagues reported in their recent study that: cytoskeleton regulates the 

interaction between Stim1 and the plasma membrane SOCE channel components such as 

Orai1 and TRPCs. In Human Embryonic Kidney (HEK-293) cells, disruption of microtubules 

enhanced both the activation of SOCE and the association between STIM1 and Orai1 or 

TRPC1 induced by thapsigargin (TG). Conversely, stabilization of microtubules or actin 

filaments attenuated both TG evoked activation of SOCE and the interaction between STIM1 

and the channel components. However, disruption of the actin filament network did not affect 

TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG stimulated 

SOCE [2]. They concluded that the microtubules act as a negative regulator of SOCE. 

We study the location of Stim1 in association with disruption/stabilization of microtubule/actin at 

immunofluorescence microscopy and immuno electron microscopy level with HEK-293 cells and 

hepatocarcinoma Huh-7 cells. We show that Stim1 proteins translocate and aggregate on the 

plasma membrane upon Calcium depletion by TG treatment. 

References: 

Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, et al. STIM1 is a Ca2+ sensor 

that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. 

Nature 2005;437:902–5.


Galán C, Dionisio N, Smani T, Salido GM, Rosado JA. The cytoskeleton plays a modulatory role 

in the association between STIM1 and the Ca2+ channel subunits Orai1 and TRPC1. Biochem 

Pharmacol. 2011;82:400-10. 

Nuclear Structure and Function 

2013 

Proteostatic control of nuclear targeting pathways by the nuclear pore complex protein, 

Nup42. 

L-K. LIU 1 , C-C. LIN 1 , D. S. Goldfarb 1 ; 1 Biology, University of Rochester, Rochester, NY 

Multiple transport receptors ferry nuclear localization signal (NLS)-bearing cargos across the 

nuclear pore complex (NPC). The FG-domains of nucleoporins in the NPC are thought to be 

required in specific combinations for efficient transport receptor-mediated transports. A model of 

multiple independent NPC translocation routes exist for different transport receptors was 

presented. Based on this model, direct competition for the same translocation pathway shared 

by multiple transport receptors can be expected. In this study, we have found that Nup42 

governs a unique stress response that differentially controls import mediated by Kap95/60 and 

Kap104. In vivo kinetic analyses show that Kap95/60 and Kap104 compete for limiting docking 

sites at Nup42. Binding to Nup42 is the rate-limiting step in import, since the import rates of 

Kap95/60 and Kap104 NLS-cargos are faster in nup42 DFG cells. Nup42 collaborates with the 

Hsp70s Ssa1/Ssa2 (but not Ssa3/Ssa4) and the co-chaperone Swa2 to differentially control 

nuclear targeting by Kap95/60 and Kap104. Specifically, Kap95/60 import is favored over 

Kap104 import when Ssa1/Ssa2 levels are high, such as in response to heat shock or nutrient 

depletion. In contrast, Kap104 import is favored when the free Hsp70 pool is depleted, for 

example, by sequestration into Huntingtin polyQ protein aggregates. Kap123-mediated import 

rates are unaffected by this regulatory circuit. Here we present the first evidence that two 

specific transport receptors, Kap95/60 and Kap104, share and compete for similar translocation 

route in the NPC. We also conclude that Nup42 is a Kap-specific gatekeeper that controls 

import rates in response to proteostatic stress. 

2014 

Evidence of asymmetric assembly pathway of Nuclear Pore Complex. 

P. Colombi 1 , C. Vogel 1 , P. Lusk 1 ; 1 Department of Cell Biology, Yale School of Medicine, New 

Haven, CT 

In an effort to investigate regulatory mechanisms governing nuclear pore complex (NPC) 

assembly, we investigated alterations in the subcellular distribution of multiple nucleoporins 

(Nups) through the cell cycle in the budding yeast S. cerevisiae. Consistent with the idea that 

NPC assembly is under the control of cell cycle regulators, we observe that several specific 

Nups accumulate in the cytoplasm of cells arrested in S-phase but not in G1. Interestingly, 

these likely NPC intermediates accumulate asymmetrically between the mother and bud and 

appear to be strikingly biased towards the bud, often accumulating at the bud cortex. These 

intermediates can often be observed integrating into the daughter cell nuclear envelope during 

anaphase supporting the idea that there is a pathway in which Nups are specifically targeted to 

the bud to be incorporated in the NPC of the daughter cell. Perturbing this pathway by 

conditionally sequestering specific newly synthesized Nups results in a decrease in the size of 

daughter nuclei. Most strikingly, these daughter cells are no longer able to progress through the 

cell cycle, whereas the mother cells continue to divide. Together these results support the


existence of a pathway critical for continued daughter cell proliferation linked to the assembly of 

NPC. 

2015 

Temporal Control of Nuclear Envelope Assembly by Phosphorylation of Lamin B 

Receptor. 

L-C. Tseng 1 , R-H. Chen 1 ; 1 Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase 

after sister chromatids have well separated. The coordination of these mitotic events is 

important for genome stability, yet the temporal control of nuclear envelope reassembly is 

unknown. Although the steps of nuclear formation have been extensively studied in vitro using 

the reconstitution system from egg extracts, the temporal control can only be studied in vivo. 

Herein, we use time-lapse microscopy to investigate this process in living HeLa cells. We 

demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that 

phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 

contributes to the temporal control. We further identify a region in the nucleoplasmic domain of 

LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory 

effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced 

chromatin-binding ability of LBR together with Aurora B activity contribute to nuclear envelope 

breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear 

envelope reassembly through modification of an integral nuclear membrane protein. 

2016 

Amphiregulin, an EGF family protein localized to the nuclear envelope, activates 

directional cell migration via histone H3K9 methylation. 

M. Hieda 1 , Y. Yokoyama 1 , Y. Nishioka 1 , H. Tanaka 1 , S. Higashiyama 2 , N. Matsuura 1 , S. 

Matsuura 1 ; 1 Graduate School of Medicine, Osaka University, Suita, Japan, 2 Proteo Medicine 

Research Center, Ehime University, Toon, Japan 

EGF family members are expressed on the cell surface in pro-form, comprising an extracellular 

EGF-like domain, a transmembrane segment, and a short cytoplasmic tail. The EGF-like 

domain is sufficient to activate the EGF receptor, whereas the function of the cytoplasmic tail 

remains unknown. Previously we showed that in the case of one EGF family member, 

amphiregulin (AREG), shedding stimuli result in release of soluble EGFR ligand and migration 

of the pro-form (proAREG) to the nuclear envelope (NE) and induction of global histone 

H3K9methylation. 

Here we found that NE-localized proAREG activates cell migration. This activation was 

dependent on the cytoplasmic tail of proARG but independent of the extracellular growth factor 

domain. The activation of cell migration did not require EGFR function, but did occur 

concomitantly with a slight increase in a specific histone modification, histone H3 lysine9 

methylation (H3K9me3). Expression of the H3K9 methyltransferase SUV39H1 activated cell 

migration; inversely, knockdown of SUV39H1 or chemical inhibition by chaetocin reduced cell 

migration. Chaetocin also suppressed the cell migration activated by NE-localized proAREG. 

Moreover, impairment of H3K9me3 suppressed the proper formation of cell polarity and led to a 

reduction in directional cell movement. Thus, NE-localized proAREG regulates cell migration via 

histone modification, indicating that chromatin organization influences both cell polarity and 

motility.


2017 

Samp1 is functionally associated with the LINC complex and A-type lamina networks. 

S. Gudise 1 , R. Figueroa 2 , V. Larsson 3 , E. Hallberg 3 ; 1 Department of Biosciences and Nutrition, 

Karolinska Institute, Sweden, 2 Department of Biochemistry and Biophysics, Stockholm 

University, 3 Department of Neurochemistry, Stockholm University, Stockholm, Sweden 

The transmembrane inner nuclear membrane (INM) protein Samp1 is required for anchoring 

centrosomes near the nuclei. Using highresolution 

fluorescence microscopy we show that Samp1 is distributed in a distinct and characteristic 

pattern in the nuclear envelope (NE), where it partially colocalizes with the LINC complex 

protein Sun1. By studying the localization of Samp1 deletion mutants and fusion proteins, we 

conclude that the cysteine-rich N-terminal half of Samp1 is nucleoplasmically exposed and is 

responsible for targeting to the INM. It contains four conserved CxxC motifs with the potential to 

form zinc fingers. The distribution of cysteine-toalanine substitution mutants, designed to 

prevent zinc finger formation, showed that NE localization of Samp1 depends on intact CxxC 

motifs. Overexpression of Samp1 zinc finger mutants produced an abnormal dominant 

phenotype characterized by disrupted organization 

of a selective subset NE proteins, including emerin, Sun1, endogenous Samp1 and, in some 

cases, lamin A/C, but not lamin B, Sun2 

or nucleoporins. Silencing of Samp1 expression showed that emerin depends on Samp1 for its 

correct localization in the NE. Our results demonstrate that Samp1 is functionally associated 

with the LINC complex protein Sun1 and proteins of the A-type lamina network. 

2018 

NE localization of the LINC complex requires multiple elements of SUN and KASH 

proteins. 

Y. G. Turgay 1 , R. Ungricht 2 , A. Rothballer 2 , A. Kiss 2 , G. Csucs 2 , P. Horvath 2 , U. Kutay 2 ; 1 Institute 

of Biochemistry, ETH Zurich, Zurich, Switzerland, 2 ETH Zurich, Zurich, Switzerland 

Inner nuclear membrane (INM) proteins are co-translationally inserted into the endoplasmic 

reticulum membrane (ER) and need to cross the nuclear pore complex (NPC) to reach their final 

destination. Originally, targeting of INM proteins to the INM was solely explained by a “diffusionretention” 

model. However, recent studies on yeast INM proteins revealed the existence of 

classical nuclear localization signals (cNLS) that mediate the active transport of the proteins 

across the NPC. Whether active transport mechanisms also play a role in the targeting of INM 

proteins in mammalian cells is still unclear. We have used human SUN2 to study INM targeting 

and identified three different elements, which collectively mediate NE targeting. The N-terminal 

nucleoplasmic domain of SUN2 comprises a cNLS and a Golgi retrieval signal. The cNLS was 

shown to constitute a functional binding site for the heterodimeric transport receptor importin 

α/β. A nearby arginine cluster was found to serve as binding platform for the coatomer complex, 

which mediates retrieval of SUN2 from the Golgi to the ER and ensures efficient INM targeting. 

The conserved SUN domain at the C terminus represents the third targeting element we have 

identified in our study. Together, our study shows that multiple elements in SUN2 contribute to 

NE targeting and that these elements are not limited to cytoplasmic or transmembrane domains.


2019 

LINC complexes mediate the positioning of cone photoreceptor nuclei in mouse retina. 

D. Razafsky 1 , S. Zang 1 , D. Hodzic 1 ; 1 Ophthalmology and Visual Sciences, Washington 

University School of Medicine, St Louis, MO 

LINC complexes form through the interaction between inner nuclear membrane Sun proteins 

(Sun 1 and Sun 2) and outer nuclear membrane Nesprins (Nesprin 1, 2, 3 and 4). The Suns and 

Nesprins interact within the perinuclear space thereby connecting the interior of the nucleus to 

the cytoplasm. In C.elegans and D. melanogaster, mutation of Sun and Nesprin orthologs 

prevent nuclear anchorage and/or migration within cells or syncitia. Mouse models deficient for 

both Sun1 and Sun2 expression display abnormal migration of cortical neurons and thereby 

phenocopies human lissencephaly phenotypes associated with mutations in Lis1 and Dcx. Our 

goal is to identify the role of the LINC complexes in mammalian tissue development and 

homeostasis. To this end, retinal development provides an excellent model for two reasons: 1) 

different types of nucleokinetic events such as interkinetic nuclear migration and postmitotic 

neuronal migration can be studied and 2) several waves of neuronal migration culminate in the 

development of a stratified tissue whereby nuclei of various cell types are anchored specific 

spatial positions. Here, we characterized the expression of LINC complex components during 

mouse retinal development and present a new versatile transgenic mouse model allowing for 

the conditional expression of a dominant negative EGFP-KASH2 protein. Using this model, we 

disrupted LINC complexes within the photoreceptor layer of mouse retina and report the ectopic 

positioning of cone photoreceptor nuclei. These studies show, for the first time, the relevance of 

LINC complexes in the positioning of cell nuclei in a mammalian CNS tissue. 

2020 

Molecular Basis of the SUN-KASH interaction. 

B. A. Sosa 1 , A. Rothballer 2 , U. Kutay 2 , T. U. Schwartz 1 ; 1 Biology, MIT, Cambridge, MA, 2 ETH 

Zurich, Switzerland 

The position of the nucleus within the eukaryotic cell is defined. To achieve specific anchorage 

and movement nucleoskeleton and cytoskeleton are mechanically connected across the doublelayered 

membrane of the nuclear envelope. These bridges, also called LINC-complexes, are 

formed by KASH-peptide containing proteins spanning the outer nuclear membrane tightly 

bound to SUN-domain proteins spanning the inner nuclear membrane. We present the crystal 

structure of the SUN domain of human Sun2 in complex with the KASH peptides of Nesprin-1 

and -2, respectively. The unexpected structure reveals several key features of this novel 

interaction that help explain how it can work as the mechanical tether it is proposed to be. 

2021 

Building SUN-KASH Complexes at the Nuclear Envelope. 

A. Rothballer 1 , B. A. Sosa 2 , T. U. Schwartz 2 , U. Kutay 1 ; 1 Institute of Biochemistry, ETH Zurich, 

Zurich, Switzerland, 2 Department of Biology, Massachusetts Institute of Technology, 

Cambridge, MA 

Physical connections between the nuclear envelope and the cytoskeleton are an essential 

feature of eukaryotic cells. They are required for diverse biological processes such as nuclear 

anchorage and migration, or homologous chromosome pairing. Nucleo-cytoskeletal connections 

are built by SUN and KASH domain proteins, transmembrane proteins of the inner and the outer 

nuclear membrane, respectively. A key element lies in the perinuclear space where the 

conserved SUN and KASH domains form a complex. Thereby, SUN-KASH protein assemblies 

bridge the entire nuclear envelope to connect nuclear structures with the cytoskeleton.


We set out to identify the determinants of SUN-KASH interaction and to understand complex 

formation in molecular detail. Using pulldown experiments, we were able to reconstitute SUN- 

KASH binding in vitro. We analyzed luminal constructs of human SUN1 and SUN2 expressed 

either in the ER of Hela cells or purified from E. coli. Both the conserved SUN domains and the 

preceding coiled-coil regions were required for interaction. Replacing the endogenous coiledcoils 

of SUN1 or SUN2 by an unrelated coiled-coil domain allowed efficient KASH binding. This 

indicates that oligomerization of SUN domains by coiled-coils is a key determinant of SUN- 

KASH interaction. 

The crystal structure of the complex between the SUN domain of human SUN2 and the 

Nesprin-2 KASH domain (see poster presented by Brian Sosa and Thomas Schwartz) revealed 

essential features of SUN-KASH interaction in molecular detail. Using mutational analyses of 

both binding partners, we confirmed the key determinants in in vitro binding assays. 

Furthermore, we could show that the disruption of SUN-KASH interaction affects nuclear 

envelope targeting of both SUN and KASH proteins. 

2022 

NSrp70 is a novel nuclear speckle-related protein and modulates alternative pre-mRNA 

splicing in vivo. 

Y-D. Kim 1 , J-Y. Lee 1 , K-M. Oh 1 ; 1 Life Science, Gwangju Inst Science/Technol, Gwangju, Korea 

Nuclear speckle is known to serve as the storing place of mRNA splicing regulators. We report 

here the identification and characterization of a novel speckle protein, referred to as NSrp70, 

based on its sub-cellular location and apparent molecular weight. This protein was firstly 

identified as the NSrp70, as the product of the NIH Mammalian Gene Collection, while no 

function has been assigned yet. NSrp70 was co-localized and physically interacted with both 

SC35 and ASF/SF2 in speckles. NSrp66 has putative RNA recognition motif, RE/RD rich 

domain, and two coiled-coil domains, suggesting role in RNA processing. Accordingly, using 

CD44, Tra2b1 and Fas constructs as splicing reporter minigenes, we found that NSrp70 

modulated alternative splice site selection in vivo. The C-terminal ten amino acids (aa) (531- 

540) including 536 RD 537 were identified as the novel nuclear localization signal, and the region 

spanning 290 to 471 aa was critical for the speckle localization and the binding to SC35 and 

ASF/SF2. The N-terminal region (107-161) was essential for the pre-mRNA splicing activity. 

Finally, we found that knockout of NSrp70 gene in mice produced no progeny including fetal 

embryos. Collectively, we demonstrate that the NSrp70 is a novel splicing regulator and 

essentially required earlier during embryonic development. 

2023 

Quality control and stress response in mammalian ribosomal biogenesis. 

M. Wang 1,2 , D. Pestov 2 ; 1 UMDNJ-GSBS, Stratford, NJ, 2 Cell Biology, UMDNJ-SOM, Stratford, 

NJ 

Ribosome is the subcellular component that translates mRNA to proteins. This complex 

molecular machine is composed of two subunits, and contains four different ribosomal RNAs 

(rRNAs) and 79 ribosomal proteins (RPs). The assembly of eukaryotic ribosomes is a multi-step 

process involving numerous of proteins and RNA factors. Compromised ribosome integrity 

contributes to premature aging syndromes, cancer and other diseases. Surveillance 

mechanisms act throughout the entire ribosome synthesis pathway to eliminate defective 

intermediates and thereby promote structural and functional integrity of the final ribosomal 

particles. However, ribosome biogenesis is very sensitive to various disturbances of cellular


metabolism. Multiple studies have shown that “nucleolar stress” caused by disruption of 

ribosome biogenesis activates the p53 pathway. However, the critical component(s) in ribosome 

synthesis machinery that trigger the p53 response are unknown. We propose that 

nucleophosmin (NPM), a molecular chaperone with predominant nucleolar localization, is able 

to transmit stress signal via binding to ribosomal proteins or other factors involved in ribosome 

synthesis. Although NPM is thought to be required for ribosome biogenesis, its down-regulation 

in NIH3T3 cells, unlike other ribosome synthesis factors, does not trigger cell growth arrest. Our 

immuno-precipitation data show that certain amount of NPM could be pulled down by ribosomal 

proteins RPL5 and RPL11. This suggests that instead of participating in pre-ribosome 

processing directly, NPM may associate and stabilize free RPs in the nucleolus, thereby 

preventing binding of Mdm2 to RPs. We also found that expressing a mutant form of NPM with 

two amino acids substitutions in its oligomerization domain triggered cell growth arrest but did 

not affect pre-rRNA processing. Immunostaining also indicates that the mutant NPM becomes 

distributed over the whole nucleus. We are currently testing the ability of NPM (both wild-type 

and mutant forms) to bind RP in the in vitro system. Our data support the idea that under normal 

growth condition, free ribosomal proteins (RPL5 or RPL11) are sequestered by nucleolar NPM 

and thus do not interact with Mdm2, an inhibitor of p53. Upon stress, delocalization of NPM may 

liberate ribosomal protein and make them available for binding to Mdm2 in the nucleoplasm 

which eventually leads to stabilization p53. 

2024 

Nucleologenesis in Giardia lamblia by light, electron and atomic force microscopy 

Nucleolog. 

L. F. Jiménez-García 1 , R. Lara-Martínez 2 , I. de la Mora-de la Mora 2 , H. Reyes-Vivas 3 , G. López- 

Velázquez 4 , M. Segura-Valdez 2 ; 1 Cell Biology, UNAM, México City, Mexico, 2 Cell Biology, 

UNAM, Mexico, 3 Instituto Nacional de Pediatría, 4 Instituto Nacional de Pediatría, Mexico 

The nucleolus is the site of most ribosome biogenesis in eukaryotes. During cell division, the 

nucleolar elements are redistributed from prophase and they reorganize during telophase by a 

process called nucleologenesis. There are several studies on the nucleologenesis in animal and 

plant cells Nucleologenesis has been studied in animal and plant cells, but less work is known in 

protest cells. The smallest nucleolus was described recently in Giardia lamblia. Here we show 

by light, electron and atomic force microscopy, that nucleolar elements are present during cell 

division in G. lamblia. Samples of trophozoites growing onto glass coverslips were processed 

for standard transmission electron microscopy and silver staining for NOR both for light and 

electron microscopy. Atomic force microscopy was conducted as described using semithin 

sections intended for electron microscopy. During cell division, silver staining was observed at 

the internal periphery of each cell nuclei. Atomic force microscopy revealed some nuclear 

peripheral material. We conclude that nucleolar elements are present throughout cell division of 

G. lamblia (This work is supported by UNAM DGAPA PAPIIT IN227810). 

2025 

Mitotic Lamin disassembly is triggered by a lipid-mediated mitotic Lipin signaling 

pathway. 

M. Mall 1 , T. Walter 1 , M. Gorjánácz 1 , J. Ellenberg 1 , I. W. Mattaj 1 ; 1 European Molecular Biology 

Laboratory (EMBL), Heidelberg, Germany 

Disassembly of the lamina, an intermediate filament meshwork that underlies the nuclear 

envelope, is a key step in early mitosis. While the activity of several kinases including cyclindependent 

kinase 1 (CDK1) and protein kinase C (PKC) have been reported to trigger this 

process, the relative contribution of these kinases to lamina disassembly remains unclear.


We have established an automated image-processing pipeline that allows us to quantitatively 

monitor lamina disassembly in live HeLa cells. Using kinase inhibitors, we could show that both 

CDK1 and PKCs significantly contribute to efficient Lamin B1 disassembly in HeLa cells. 

Similarly, overexpression of Lamin B1 reporters with mutated CDK1 or PKC consensus 

phosphorylation sites also impeded Lamin B1 disassembly. Since the role of PKC-dependent 

pathways during mitosis and their effects on lamina disassembly are poorly understood, we 

focused on elucidating this process in more detail. Using RNAi we found that specific PKC 

isoenzymes that require diacylglycerol (DAG) for their activation trigger rate-limiting steps during 

mitotic Lamin B1 disassembly in our in vivo assay. 

Interestingly, we recently showed that the enzyme Lipin, which produces DAG by 

dephosphorylation of phosphatidate, is essential for Lamin disassembly in the nematode C. 

elegans (Gorjánácz and Mattaj, 2009). This suggested that Lipin might act upstream of DAGdependent 

PKCs. To investigate this possibility we used RNAi to deplete the three redundant 

human Lipin enzymes and monitored Lamin B1 disassembly. Strikingly, Lipin depletion inhibited 

Lamin B1 disassembly to a similar extent as did PKC depletion. Furthermore, the effect of Lipin 

RNAi could be rescued by the addition of a DAG analogue. 

This supports a model where Lipins function in a lipid-mediated manner to activate a PKCdependent 

pathway of mitotic Lamin disassembly. 

Gorjánácz, M., and Mattaj, I.W. (2009). Lipin is required for efficient breakdown of the nuclear 

envelope in Caenorhabditis elegans. J Cell Sci 122, 1963-1969. 

Signal Transduction and Signaling Networks I 

2026 

Negative regulation of Notch1 signaling by Serum- and glucocorticoid-inducible kinase 1. 

J-H. Yoon 1 , E-J. Ann 1 , J-S. Ahn 1 , H-S. Park 1 ; 1 Hormone Research Center, School of Biological 

Sciences and Technology, Chonnam National University, Gwangju, Korea 

Notch is a transmembrane protein that acts as a transcriptional factor in the Notch signaling 

pathway for cell survival, cell death, and cell differentiation. However, little is known about 

mechanisms and regulators that are responsible for attenuating the Notch signaling pathway 

and protein stability. Here, we report that SGK1 remarkably reduced the protein stability of 

Notch1 through Fbw7. The protein level and transcriptional activity of the Notch1 intracellular 

domain were higher in SGK1 null cells than in SGK1 wild-type cells. Notch1-IC was able to form 

a trimeric complex with Fbw7 and SGK1, thereby SGK1 enhanced the protein degradation of 

Notch1-IC via a Fbw7-dependent proteasomal pathway. Furthermore, activated SGK1 

phosphorylated Fbw7 at Ser227, an effect inducing Notch1-IC protein degradation and 

ubiquitination. Moreover, accumulated dexamethasone-induced SGK1 facilitated the 

degradation of Notch1-IC through phosphorylation of Fbw7. Altogether our results suggest that 

SGK1 inhibits the Notch1 signaling pathway via phosphorylation of Fbw7.


2027 

Secretory Protein Expression Under Microgravity Conditions. 

M. Mednieks 1 , I. Khan 2 , A. Khatri 3 , J. Perrier 1 , A. Hand 4 ; 1 Oral Health and Diagnostic Sciences, 

University of Connecticut Health Center, Farmington, CT, 2 University of Connecticut School of 

Dental Medicine, Farmington, CT, 3 Wesleyan University, Middletown, CT, 4 Departments of 

Craniofacial Sciences and Cell Biology, University of Connecticut Health Center, Farmington, 

CT 

A major goal of the space shuttle biological experiments is to identify the physiological 

subsystems that require in-flight monitoring and post-landing testing to evaluate spaceflight 

crew status and to devise countermeasures for longer duration missions. Previous studies have 

shown that exposure of rodents to microgravity results in changes in the regulation of protein 

secretion. To test the hypothesis that signaling via cyclic AMP-dependent pathways is affected 

by microgravity the expression of several salivary proteins (both regulated by cyclic AMP and 

not) was determined. Methods: Adult female C57Bl/6J mice housed in Animal Enclosure 

Modules (AEMs) were flown on the space shuttles Discovery and Atlantis. Findings from flight 

animals were compared with those from ground control animals housed in AEMs and from 

vivarium-housed controls. Tissue samples were collected within 5 hours of landing, and glands 

were fixed and processed for ultrastructure and frozen for biochemistry. Specific proteins, 

including the PKA type II regulatory subunit (RII), were identified by immunocytochemistry and 

by electrophoresis and Western Blotting (WB), digitized and quantified using NIH ImageJ. 

Results: The ultrastructure of serous and mucous secretory cells, as well as ducts, was similar 

in flight and control animals. Quantitative analysis of immunogold labeled thin sections of parotid 

acinar cells of flight animals showed a decrease (p < 0.01) in the expression of RII, amylase, 

and proline-rich proteins (PRPs), whereas parotid secretory protein (PSP) expression was 

unchanged. In the sublingual gland the expressionof PSP in serous demilune cells and mucin 

(Muc-19) in mucous acinar cells was increased (p < 0.001), while RII was slightly increased in 

demilune cells (p = 0.23). Demilune cell and parotid protein (DCPP) was increased in 

intercalated ducts of the parotid (p < 0.001), whereas a slight decrease occurred in the 

sublingual gland (p = 0.27). Electrophoretic banding patterns were not markedly altered in flight 

parotid or submandibular glands compared to controls. Analysis by WB, however, showed that 

in the submandibular gland expression of IgA (p < 0.05) was decreased while there was an 

increase in RII (p < 0.02). Recent data from the last flight of the space shuttle Discovery show 

that protein expression in flight samples begins to return to normal by day 5 after landing and 

approaches control values by day 7. Conclusions: The expression of several parotid, 

submandibular and sublingual gland secretory proteins is altered by travel in space. These data 

also indicate that the responses of the different salivary glands to microgravity are cell, tissue, 

and organ specific. As these secretory proteins are present in saliva, these findings can be used 

to establish this easily collected biofluid as a diagnostic tool to assess physiological responses 

of astronauts as well as for clinical applications on Earth. Support: NASA grant NNX09AP13G 

2028 

A-kinase Anchoring Proteins: Mediators in Neonatal Rat Schwann Cell Proliferation. 

A. L. Asirvatham 1 , R. Stahl 2 , C. M. Schworer 2 , E. Shoemaker 1 , D. J. Carey 2 ; 1 Biology, 

Misericordia University, Dallas, PA, 2 Weis Center for Research, Geisinger Clinic, Danville, PA 

A-kinase Anchoring Proteins: Mediators in Neonatal Rat Schwann Cell Proliferation 

Schwann cell proliferation in the peripheral nervous system is mediated by the 

heregulin/neuregulin family of growth factors that are secreted by neurons. Previous studies 

have revealed that culture of neonatal rat Schwann cells exhibit a synergistic response in 

growth when treated with both heregulin and forskolin, in comparison to cultures incubated with


either heregulin or forskolin. The molecular mechanisms that mediate the proliferation of 

Schwann cells in response to heregulin and forskolin are not well known. This study was 

undertaken to identify the role of the cyclic AMP protein kinase A signaling pathway in Schwann 

cells that are cultured with heregulin and forskolin. Studies conducted previously have shown 

that the A-Kinase anchoring proteins of the cAMP/PKA pathway play an important role in 

Schwann cell division. Incubation of neonatal rat Schwann cells with SiRNA oligos synthesized 

against AKAP95 and AKAP150 resulted in a reduction in protein levels of both AKAPs 

accompanied by a decrease in cell proliferation. In addition to a decrease in AKAP protein 

levels, Schwann cells cultured with the SiRNA oligos against AKAP95 and AKAP150, exhibited 

a reduction in protein levels of cyclin D3 and PKB/Akt respectively. To investigate if the loss of 

AKAPs along with cyclin D3 occurred at the level of synthesis or degradation, neonatal rat 

Schwann cells were cultured with the protein synthesis inhibitor cycloheximide and the 

proteasome inhibitor lactacystin. Immunoblot analysis of Schwann cells incubated with 

cycloheximide and SiRNA oligos or cycloheximide alone showed a reduction in both AKAP95 

and cyclin D3 levels. Schwann cells cultured with lactacystin and SiRNA oligos showed a 

marked reduction in AKAP95 and cyclin D3 levels in comparison to cultures incubated with 

lactacystin only. These preliminary observations suggests that the loss of AKAP95 along with 

the cyclin D3 proteins does not occur at the synthesis or degradation levels and instead may be 

mediated by a different mechanism. To examine if AKAP150 associates with PKB/Akt to 

stimulate Schwann cell proliferation, cells were incubated with modified N2 media or modified N2 

media with heregulin and forskolin. Using immunofluorescent staining techniques, preliminary 

results demonstrate that Schwann cells treated with heregulin and forskolin exhibit distinct 

colocalization patterns for AKAP150 and PKB/Akt. These results suggest that in the presence of 

forskolin and heregulin, AKAP150 may associate with PKB/Akt to mediate cell proliferation. 

2029 

Turn motif phosphorylation negatively regulates activation-loop phosphorylation in Akt. 

D. Hiraoka 1 , E. Okumura 1 , T. Kishimoto 1 ; 1 Tokyo Institute of Technology, Yokohama, 

Kanagawa, Japan 

Akt, also known as PKB, plays a central role in various signaling pathways that regulate cellular 

processes such as metabolism, proliferation and survival. Under stimulation, phosphorylation of 

the activation loop (A-loop) and hydrophobic motif (HM) of Akt by the kinase, PDK1, and the 

mammalian target of rapamycin complex 2 (mTORC2), respectively, results in its activation. A 

well-conserved threonine in the turn motif (TM) is also constitutively phosphorylated by 

mTORC2 and contributes to the stability of Akt. However, the relationship between TM 

phosphorylation and the phosphorylation of Akt on the HM and A-loop has not been sufficiently 

evaluated. Using starfish oocytes as a model system, our study provides the first evidence that 

TM phosphorylation plays a negative role in A-loop phosphorylation. In this system, the 

maturation-inducing hormone, 1-methyladenine, stimulates Akt to induce meiotic reinitiation 

through the activation of cyclin B-Cdc2. The phosphorylation status of Akt was monitored via 

introduction of exogenous human Akt in starfish oocytes. Injection of an anti-starfish TOR 

antibody inhibited TM and HM phosphorylation, suggesting that phosphorylation of these sites 

depends on TOR, as reported in mammalian cells. Precise analyses of single or double alanine 

substitution mutants at each of three phosphorylation residues revealed that TM 

phosphorylation renders Akt susceptible to dephosphorylation on the A-loop. When A-loop 

phosphatase was inhibited by okadaic acid, TM phosphorylation still reduced A-loop 

phosphorylation, suggesting that the effect is caused at least partially through reduction of 

sensitivity to PDK1. By contrast, HM phosphorylation enhanced A-loop phosphorylation and 

achieved full activation of Akt via a mechanism at least partially independent of TM


phosphorylation. These observations provide new insight into the mechanism controlling Akt 

phosphorylation in the cell. 

2030 

MK-STYX Reduces eIF2α Phosphorylation. 

J. E. Barr 1 , S. D. Hinton 1 ; 1 Biology, College of William and Mary, Williamsburg, VA 

The pseudophosphatase MK-STYX [MAPK (mitogen-activated protein kinase) 

phosphoserine/threonine/tyrosine-binding protein] has been implicated in the stress response 

pathway. MK-STYX interacts with Ras-GTPase activating protein SH3 domain binding protein-1 

(G3BP-1), and inhibits stress granule assembly. Stress granules, cytoplasmic storage sites for 

mRNA, form as a protective mechanism against stress caused by UV irradiation, hypoxia, and 

heat shock. In addition, the overexpression of G3BP-1, when dephosphorylated induces stress 

granule assembly. Initially, we hypothesized that MK-STYX inhibition of stress granules was 

G3BP-1 phosphorylation-dependent. However, data with G3BP-1 phosphomimetic and nonphosphorylatable 

mutants suggest differently so we turned our focus to the eukaryotic initiation 

factor 2 alpha (eIF2�). eIF2�� initiates translation by forming a ternary complex with methionine 

bound to tRNA and GTP. Phosphorylation of eIF2� arrests translation and results in stress 

granule formation, whereas dephosphorylation promotes polysomes. To determine if MK-STYX 

inhibits stress granule assembly via upstream interactions, we investigated its affects on eIF2� 

phosphorylation. HeLa cells were transfected with pMT2-FLAG-MK-STYX-FLAG or pMT2 

expression vectors, and heat shocked for an hour to induce stress. Cells were lysed and 

immunoblots for phosphorylation of eIF2� were performed. We show that the presence of MK- 

STYX reduced the phosphorylation of eIF2�. Furthermore, MK-STYX also reduced the eIF2� 

phosphorylation in heat shocked cells, which showed a drastic increase in phosphorlyation in 

the absence of MK-STYX. These data are significant because they provide more insight into 

how MK-STYX inhibits stress granule assembly. 

2031 

VEGF modulates Col I expression in human adipose derived stem cells through Akt 

dependent mechanisms. 

C-J. Li 1 , V. Madhu 1 , A. Dighe 1 , G. Balian 1,2 , Q. Cui 1,3 ; 1 Orthopaedic Research Labs, University of 

Virginia, Charlottesville, VA, 2 Biochemistry and Molecular Genetics, University of Virginia, 

Charlottesville, VA, 3 Orthopaedic Surgery, University of Virginia, Charlottesville, VA 

Objective: 

We earlier demonstrated that in vivo osteogenesis induced by mouse bone marrow derived 

osteoprogenitor cells expressing bone morphogenetic protein – 6 (BMP-6) or Lim Mineralization 

Protein - 1 (LMP-1) was enhanced significantly by co-expressing vascular endothelial growth 

factor (VEGF) gene. The facts that increase in osteogenesis did not completely correlate with 

VEGF-induced angiogenesis and that LMP-1 is downstream activator of BMP-6 signaling 

pathway clearly indicated interaction between VEGF and other signaling pathways. In order to 

understand mechanisms of the interaction and to determine if similar interactions occurred in 

human mesenchymal stem cells we determined in vitro osteogenesis of human adipose derived 

stem cells (hADSCs) in presence of purified VEGF and BMP-6 proteins. 

Results: 

The results of calcium deposition showed no significant difference between control, BMP-6, 

VEGF or BMP-6+VEGF group in hADSC at day 14th. However, significant increase in 

expression of Col I α2 gene was observed in VEGF group. At day 14, a synergistic increase in 

Col I α2 expression was observed in VEGF+BMP-6 group. Addition of Akt1/2/3 inhibitor


suppressed Akt and Smad1/5 phosphorylation, calcium deposition and Col I α2 mRNA 

expression in hADSCs. Most importantly, blockage of Akt activation significantly inhibited the 

synergistic enhancement of Col I α2 mRNA expression, which was induced by BMP-6 and 

VEGF combined treatment. 

Conclusion and Discussion: 

Our data showed a striking observation that VEGF and BMP-6 synergistically enhanced Col I α2 

gene expression in hADSCs. Furthermore, biochemical blockage of Akt activation significantly 

inhibited the enhancing effects of Col I α2 mRNA expression by BMP-6 and VEGF combined 

treatment. It is reported that PI3-kinase catalytic isoform is coupled to VEGFR2 and regulates 

the bulk of Akt activity. Moreover, dominant-negative Akt inhibited Smad5 phosphorylation. Our 

data and these reported observations together establish a novel axis of control of Col I 

expression as VEGF-VEGFR2-PI3K-Akt-Smad1/5-Col I in hADSCs. However, Akt-dependent 

BMP-6 modulation of this axis remains to be investigated in near future. 

2032 

A novel gene encoding a candidate CK2 substrate involves in cancer cell proliferation. 

H. Yang 1 , S. Liu 1 , H. Zheng 1 , H. Jiang 1 , Y. Qu 1 , Y. Li 1 ; 1 Harbin Institute of Technology, Harbin, 

China 

CK2, a ubiquitous protein serine/threonine kinase, is essential for the decision of cell survival 

and cell death. In the present study, we identified a novel gene, Homo Sapiens Chromosome 1 

ORF109 which protein product might be a candidate CK2 substrate. The C1ORF109 protein 

presented in the phosphorylated form in vivo, and could be phosphorylized by the protein kinase 

CK2 in vitro. Truncated constructs and site direct mutagenesis analysis revealed that Ser104, 

Ser 134, and Ser182 were phosphorylated sites. Both TATA box and CAAT box within the 

crucial region of c1orf109 promoter are required for maximal transcription of the c1orf109 gene. 

The C1ORF109 protein was mainly located in the nucleus and cytoplasm. Upregulated 

expression of c1orf109 was detected in multiple breast cancer cells. Exogenous expression of 

C1ORF109 in breast cancer Hs578T cells promoted colony formation and cell proliferation 

along with increased level of PCNA and cyclinD1. Meanwhile, knockdown of c1orf109 by siRNA 

in breast cancer MDA-MB-231 cells resulted in inhibition of cell proliferation. Taken together, our 

findings suggest that C1ORF109 may be the down stream target of protein kinase CK2 and 

involved in the regulation of cancer cell proliferation. 

Key words: promoter, transcription, CK2 kinase, c1orf109, proliferation 

2033 

Quantitative detection of phosphorylation of STAT3 and p38 by Western blotting. 

K. Söderquist 1 , S. Grimsby 1 , M. Winkvist 1 ; 1 GE Healthcare, Uppsala, Sweden 

Introduction 

Western blotting is a well established technique used to study proteins from a wide variety of 

sources. The technique is used throughout life sciences from basic research to medical 

diagnostic applications. Western blotting is at best considered as semi-quantitative and hence 

limited to studies involving large protein differences. However development of detection 

reagents as well as imagers has open up for quantitative applications when high sensitivity and 

broad dynamic range is needed. 

Here we demonstrate how the Western blotting technique can be used to determine 

phosphorylation levels of various proteins in whole cell lysates upon stimulation with various 

well known inducers.


Methods 

HeLa cells were stimulated with INF-β. Phosphorylation level of STAT3 was determined by 

Western blotting performed according to standard procedure and protocol supplied with the 

Amersham ECL Prime reagent using optimized blocking solution and antibodies 

concentrations. 

293T cells where stimulated with TGF-β. Phosphorylation level of p38 was determined by 

Western blotting performed according to standard procedure and protocol supplied with the 

Amersham ECL Prime reagent using optimized blocking solution and antibodies concentrations. 

Detection reagent was added to the membrane and the chemiluminescent signal was captured 

using CCD cameras. 

Results 

The result demonstrates that phosphorylation of STAT3 upon stimulation with INF-β can be 

quantitatively determined by Western blotting. In addition, the result demonstrates that time 

dependent phosphorylation of p38 upon stimulation with TGF-β can be quantitatively 

determined by Western blotting. 

Conclusions 

The Western blotting technique can be used for accurate quantitative analysis of 

phosphorylation level of various proteins, such as p38 and STAT3, by combining the use of a 

detection reagent, providing high sensitivity and broad dynamic range, with a CCD camera. 

2034 

A distinctive salt bridge arrangement in RSK P-loop enables discovery of isoformselective 

RSK1/2 inhibitors that protect from myocardial infarction injury. 

V. O. Paavilainen 1 , I. Serafimova 2 , M. S. Cohen 3 , M. Spreafico 2 , J-I. Abe 4 , M. Jacobson 2 , J. 

Taunton 2 ; 1 Cellular and Molecular Pharmacology, UC-San Francisco, San Francisco, CA, 2 UC- 

San Francisco, 3 Oregon Health and Science University, Portland, OR, 4 University of Rochester, 

Rochester, NY 

p90 ribosomal protein S6 kinases (RSK) are serine/threonine kinases under the MAPK/ERK 

signaling pathway, which have been implicated in a variety of cellular functions such as cell 

proliferation and survival. Their expression is upregulated in primary breast and prostate 

cancers, and mutations in RSK2 gene lead to Coffin-Lowry syndrome, an X-linked form of 

mental retardation. We sought to discover potent RSK1 inhibitors with high selectivity over 

RSK2. These inhibitors utilized diverse electrophilic warheads that covalently target a nonconserved 

cysteine in the RSK active site and led to the identification of an irreversible RSK 

inhibitor, fmk, with ~20-fold selectivity difference between the highly identical RSK1 and RSK2 

isoforms both in vitro and in vivo. Detailed biophysical and structural experiments showed that 

this selectivity arises from differences in the reversible binding affinity mediated by subtle 

differences in the structure and dynamics of the kinase P-loop region due to a specific salt 

bridge arrangement around the P-loop region. In a mouse model of myocardial infarction (MI), 

fmk treatment resulted in a dramatic improvement in survival and heart function, possibly by 

inhibition of NHE1 phosphorylation. Future studies will aim to dissect the roles of different RSK 

isoforms during MI injury.


2035 

Inhibition of Notch1 signaling by APP intracellular domain. 

E-J. Ann 1 , J-H. Yoon 1 , J-S. Ahn 1 , H-S. Park 1 ; 1 Chonnam National University, Gwangju, Korea 

The Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of 

cell growth and differentiation in a variety of contexts. In this study, we have demonstrated that 

the APP intracellular domain (AICD) attenuates Notch1 signaling by accelerated degradation of 

the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation 

pathways. AICD suppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC– 

RBP-Jk complex after processing by gamma-secretase. Notch1-IC is capable of forming a 

trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1- 

IC through an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP- 

Jk protein through the lysosomal pathway. AICD-mediated degradation is involved in the 

preferential degradation of non-phosphorylated RBP-Jk. Collectively, our results demonstrate 

that AICD functions as a negative regulator in Notch1 signaling through the promotion of 

Notch1-IC and RBP-Jk protein degradation. 

2036 

Isoprenylcysteine methylation is required for growth and endocytosis in Dictyostelium 

discoideum. 

K. J. McQuade 1 , J. Bollan 1 , K. Bailey 1 , J. Fritz 1 ; 1 Colorado Mesa University, Grand Junction, CO 

Many members of the Ras superfamily of small monomeric GTPases and other membraneassociated 

proteins ending in carboxy-terminal –CAAX motifs are proteolytically processed and 

reversibly methylated on isoprenylated terminal cysteine residues. Methylation is thought to 

regulate localization and activity of these proteins. Ras GTPases are often overactive in cancer 

cells and inhibitors of isoprenylcysteine methyltransferase are being tested as potential 

chemotherapies, but the cellular effects of these inhibitors are unclear. We are characterizing 

the effects of loss of isoprenylcysteine methyltransferase activity in the social amoeba, 

Dictyostelium discodeum. Amoebae lacking methyltransferase activity grow slowly on bacterial 

lawns and do not grow in shaking or adherent axenic culture. Methyltransferase-deficient cells 

also migrate slowly towards folate, have reduced rates of endocytosis and are sensitive to 

osmotic shock. The mechanisms underlying these defects are being investigated. 

2037 

Understanding the cellular function of poly(ADP-ribose) and its importance in cancer. 

K. Krukenberg 1 , T. Mitchison 2 ; 1 Systems Biology, Harvard Medical School, Boston, MA, 

2 Systems Biology, Harvard Medical School 

Poly(ADP-ribose) is a unique but poorly understood post-translational modification. It has been 

implicated in multiple cellular processes including DNA damage, cell death, inflammation, 

protein stability and cell division. In the clinic, inhibition of poly(ADP-ribose) formation is showing 

promise as a cancer therapeutic. Poly(ADP-ribose) polymerases (PARPs) catalyze the addition 

of ADP-ribose onto acceptor proteins using NAD+ as a substrate. Though the importance of 

PARPs and poly(ADP-ribose) is clear, their biological functions are not well understood. We are 

working on developing tools for characterizing the function of PARPs and poly(ADP-ribose) and 

are using these tools to further identify and define the biological roles of these intriguing and 

essential molecules. Here we describe an assay for quantifying the levels of poly(ADP-ribose) in 

cells that has increased sensitivity as compared to previous approaches. Using this assay, we 

quantified the extent to which poly(ADP-ribose) levels vary throughout the cell cycle. We have 

also found that different cancer cell types have different amounts of poly(ADP-ribose) and these


levels appear to be largely attributable to PARP1 activity. Current efforts are focused on better 

understanding the molecular basis for the differences in polymer levels. Not only will this provide 

insight into the biological function of poly(ADP-ribose) but poly(ADP-ribose) levels may be 

useful as an indicator of which patients will respond best to PARP inhibitors or other treatments. 

2038 

Monitoring Post-Translational Modifications Using Antibody-Based Real-Time PCR. 

R. Bruinsma 1 , K. G. Huwiler 1 , B. D. Marks 1 , M. Shannon 2 , D. Ruff 2 , B. Schweitzer 3 ; 1 Life 

Technologies, Madison, WI, 2 Life Technologies, Foster City, CA, 3 Life Technologies, Carlsbad, 

CA 

Protein post-translational modifications (PTM) are covalent modifications to specific amino acids 

within a protein. PTMs often occur via reversible enzymatically catalyzed reactions and 

numerous types of PTMs exist, including phosphorylation, acetylation, methylation, and 

ubiquitination. The importance of PTMs resides in their ability to influence the function, stability, 

and localization of the modified protein within cells. These changes in protein properties due to 

PTMs are critical to the regulation of cellular signaling networks and the deregulation of protein 

PTMs is associated with many diseases, including cancer. Two proteins that are recognized to 

be important in cancer biology and that undergo PTMs are p53 and AKT. p53 is central to 

maintaining the integrity of the genome and is found to be mutated in ~50% of all cancers. 

Phosphorylation of p53 at Ser15 has been shown to result in p53 protein stabilization, which can 

result in transcriptional activation. The PI3K/AKT pathway is critical for normal cell growth and 

survival, but persistent uncontrolled activation of this pathway is associated with cancer. Full 

kinase activity of AKT and downstream cellular signaling involves phosphorylation of Ser473 

and Thr308. We report the application of a technique that partners the specificity of antibody 

detection with the signal amplification of real-time PCR (RT-PCR) to monitor changes in PTMs. 

Specifically, we were able to quantitatively monitor changes in p53 Ser15 phosporylation and 

AKT Ser473 phosphorylation from very small cellular samples. To our knowledge, this is the first 

demonstration of monitoring PTMs for p53 and AKT via RT-PCR. This is a powerful method for 

monitoring PTMs due to the high specificity, high sensitivity, and small sample requirement. 

2039 

Association of Connexin 36 with synaptic scaffolding proteins in the retina. 

A. Vila 1 , C. Whitaker 1 , J. O'Brien 1 ; 1 Opthalmology and Vision, University of Texas Health 

Science Center, Houston, TX 

Our visual world contains an incredible range of light intensities. This necessitates adaption at 

many levels of the visual system to produce useful vision. In the retina, electrical synapses 

composed of gap junctions display extensive plasticity associated with light adaptation. Recent 

studies have found that NMDA-type glutamate receptors regulate coupling among AII amacrine 

cells and that NMDA receptors are localized near Cx36 gap junctions on AII amacrine cells. 

These studies indicate that a localized Ca 2+ signal derived from NMDA receptor activation drives 

Cam kinase II-dependent phosphorylation of Cx36 to produce activity-dependent enhancement 

of coupling. To maintain the integrity of this signaling mechanism, we hypothesized that 

scaffolding proteins may assemble Cx36 gap junctions and NMDA receptors into a complex 

analogous to the post-synaptic density of conventional synapses. To explore this hypothesis, we 

examined the association of Cx36 with traditional synaptic scaffold proteins Synapse- 

Associated Protein 97 (SAP97), SAP102, and Post-Synaptic Density 95 (PSD95) by 

immunofluorescence in rabbit retina and 3-D image reconstruction. We further examined the 

association of these proteins by co-immunoprecipitation and western blot techniques. 

Antibodies to SAP97 and SAP102 gave punctate labeling throughout the inner synaptic layer of


the retina, in which AII amacrine cells make their gap junctions. Antibodies to PSD95 did not 

label the inner synaptic layer, but did label the outer synaptic layer, where photoreceptor 

synapses are located. In the inner synaptic layer, approximately 50% of the Cx36 gap junctions 

were co-localized with either SAP97 or SAP102, and 15% co-localized with both. SAP102 colocalized 

with about twice as many Cx36 gap junctions as did SAP97. Furthermore, of the 

SAP102 puncta in the inner synaptic layer, fully one third were associated with Cx36 gap 

junctions. In the outer synaptic layer, some PSD95 immunoreactivity was closely associated 

with Cx36. Our data show that synaptic scaffolding proteins are associated not only with 

conventional synapses, but also with electrical synapses. These scaffolding proteins may 

facilitate assembly of non-synaptic neurotransmitter receptors, such as the NMDA receptors on 

AII amacrine cells, with Cx36 gap junctions. They may alternatively, or in addition, facilitate the 

association of conventional synapses and gap junctions to form mixed synapses. 

2040 

A structural biology approach to predict proteolytic sites of accessory gland proteins in 

D. Melanogaster. 

C. A. Del Carpio 1 , M-T. Yamamoto 1 ; 1 Drosophila Genetic Resource Center (DGRC), Kyoto 

Institute of Technology, Kyoto, Japan 

Extra and intra-cellular protein processing and degradation accounts for one of the main 

biochemical processes involved in series of signal transduction and regulation pathways. 

Moreover, genome-wide studies of higher organisms have unveiled that a large proportion of 

encoded proteins are enzymes oriented to the cleavage of other proteins. Thus predicting the 

cleavage sites of substrate proteins and the enzyme involved is highly required in functional 

genome analyses including the elucidation of intra and extra-cellular mechanisms at the origin 

of cellular malfunction and diseases. Sequence based inference of proteolytic cleavage sites in 

polypeptides frequently leads to several sites that are located in the core of the molecule or are 

sterically hindered and hardly accessible to the cleaving enzyme. Catering to the need of 

including 3D structural factors in the assessment of cleavage sites in polypeptides and reduce 

the number of candidates output at the primary structure level, we propose a bio-computational 

strategy to firstly evaluate substrate-enzyme affinity and subsequently infer putative scissible 

bonds. Affinity is evaluated by docking the 3D structures of the substrate and the enzyme, and 

molecular dynamics (MD) simulation is performed to assess the formation of the transition state 

(TS) complex triggering the proteolytic reaction. The cleavage sites are then inferred by tracing 

the main atomic interactions throughout the MD simulation leading to formation of the TS 

species. The methodology involves protein structure prediction, as well as protein docking to 

assess substrate-enzyme interaction. As a case study we applied the methodology to the 

prediction of cleavage sites for Acp36DE, a D. Melanogaster seminal fluid protein that plays an 

important role in sperm storage and competition in multiply mated females. The results show 

that among several candidate regions amenable to protease attack, the region LEU514- 

ASP517, located at a disordered region between two alpha helices shows geometrical 

complementarity and energetic affinity for an astacin like protease. The TS species derived 

through the MD simulation shows the metal (Zn) in the astacin pulling the LYS515.O, and the 

contraction of the distance between the SER516.N and GLU101.OE2 in astacin suggests the 

scissible peptide bond. Repeating the calculations with serine proteases reported to be 

expressed at copulation shows a higher affinity region for the hydrophobic region that includes 

amino acids SER21-GLU22-SER23-PHE24 the cleavage bond being GLU22-SER23. Cleavage 

of Acp36DE at LYS515-SER516 generates fragments of Acp36DE of molecular weights that 

compare well with reported experimental findings


2041 

Evolutionary analysis of the phosphorylation signaling reveals functionally conserved 

motifs. 

H. Yoshizaki 1 , S. Okuda 2 ; 1 Department of Biomedical Sciences, College of Life Sciences, 

Ritsumeikan University, Kusatsu, Shiga, Japan, 2 Department of Bioinformatics, College of Life 

Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan 

(Background) Protein kinases regulate various cellular processes by phosphorylating specific 

substrates. Phosphoproteomic studies recently have shown that tens of thousands of sites are 

phosphorylated. In addition, in silico systemes biological analyses have revealed protein 

phosphorylations dynamically regulate cellular networks. These studies provide powerful tools 

for studying intra-cellular signal transduction pathways. High throughput proteomic techniques 

provide a massive amount of protein information but we can not directly retrieve protein-protein 

interactions organizing cellular processes. Toward understanding the complicate intracellular 

signaling networks, we performed a comparative analysis about evolutionally conserved 

phosphorylation motifs. (Methods and Results) First, we retrieved the known phosphorylated 

protein sequences from the PhoshoSitePlus database (http://www.phosphosite.org/) and 

determined phosphorylation motifs by clustering them. As a result, we acquired 186 original 

phosphorylation motifs from 434 clusters obtained by the MCL clustering method. Subsequently, 

we assigned kinase information to a motif based on the known kinase-subustrate combinations 

stored in the PhosphoSitePlus database and predicted a probable kinase phosphorylating the 

motif. Furthermore, we evaluated the evolutionary conservation of the phosphorylation motifs by 

a comparative sequence analysis from yeast to human. We found that most of the motifs 

conserved along the general evolutionary scenario, but some motifs showed the evolutionary 

patterns different from them. Additional protein functional analysis showed that these 

phosphorylation sites were involved in a specific protein function. Our comparative evolutionary 

analysis of phosphorylation motifs is useful methods to highlight protein groups with specific 

functions from complicated signal transduction pathways. 

2042 

Network topologies evolve during neutrophil polarization. 

C-J. Ku* 1 , Y. Wang* 1 , O. Weiner 2 , S. J. Altschuler 1 , L. F. Wu 1 ; 1 Dept of Pharmacology, Green 

Ctr for Systems Biology, Simmons Cancer Ctr, University of Texas Southwestern Med Ctr, 

Dallas, TX, 2 Cardiovascular Research Institute and Department of Biochemistry, University of 

California, San Francisco, San Francisco, CA 

*These authors contributed equally to the work. 

Much attention has focused on how complex cellular behaviors can emerge from static 

networks. Here, we found that the rapid response of neutrophils to stimulus arises from a timevarying 

series of distinct signaling network topologies. Not only do the signaling topologies 

change over time, but the topology also depends on the phenotype. Surprisingly, the set of 

persistent links revealed an underlying simplicity of the network structure: a linear cascade 

controlling signaling activities, and a feed-forward network in the opposite direction controlling 

signaling polarity. Our work provides a clear example that simple circuits orchestrating cellular 

behaviors may lay hidden beneath the complexity of comprehensive “everything-connects-toeverything” 

networks obtained by combining links from different times and phenotypes.

2043 

The Drosophila Transcription Factor Protein Interactome. 

D. Y. Rhee 1 , B. Zhai 1 , C. Wong 1 , C. Beekman 1 , S. Gygi 1 , R. Obar 1 , S. Artavanis-Tsakonas 1 ; 

1 Department of Cell Biology, Harvard Medical School, Boston, MA 


From the earliest embryo to the adult, the spatiotemporal expression of genes is essential for 

normal development and physiology. At the basis of this is the regulation of transcription via 

transcription factors (TFs), proteins that physically bind DNA to activate or suppress gene 

expression. As the target of signaling pathways, TFs represent a crucial point of regulation 

relating to the vast majority of cellular processes and as a rule function through interactions with 

other proteins. Consequently, the characterization of protein-protein interactions involving TFs is 

essential for understanding how they function to regulate gene expression and, in turn, the 

biology of the cell. Towards this end, we have generated a co-AP/MS-based interaction map 

encompassing more than 500 from the total of 750 TFs estimated, by various criteria, to 

represent the entire gamut of TFs in Drosophila melanogaster. 

Cell Migration and Motility 

2044 

Macrophage Strength: Quantifying Phagocytic Forces and Kinetics. 

N. Sosale 1 , T. Rouhi 2 , P. Rodriguez 1 , R. Lipowsky 2 , D. Discher 1 ; 1 Biophysical Engineering Lab, 

University of Pennsylvania, Philadelphia, PA, 2 Max Planck Institute of Colloids and Interfaces, 

Potsdam, Germany 

Macrophages act as immunological gatekeepers at the interface of tissue, blood, and lymph as 

these cells take up antigens from the extracellular environment and then present them to the 

immune system stimulating lymphocytes. We have studied the force and kinetics of 

phagocytosis by human macrophages. To this aim, we studied phagocytosis of opsonized red 

blood cells. The force calculation is based on blood cell's shape deformation and elastic 

properties. Our analysis shows that the range of the resultant force imposed by Macrophages is 

up to 100 pN and is imparted within 10 minutes after initial contact. 

2045 

Integrin Linked Kinase Modulates Phagocytosis through Rac1 Activation in Epidermal 

Keratinocytes. 

S. Sayedyahossein 1,2 , L. Nini 1 , L. Dagnino 1,2 ; 1 Physiology and Pharmacology, The University of 

Western Ontario, London, ON, Canada, 2 Children’s Health Research Institute, London, ON, 

Canada 

Integrin linked kinase (ILK) is an adaptor protein that regulates numerous biological processes, 

such as cell survival and migration. ILK is also essential for keratinocyte interactions with the 

extracellular matrix and development of polarity, but the exact role of ILK in other epidermal 

functions, such as melanosome phagocytosis, is not clear. We examined alterations in 

phagocytic capacity in ILK-deficient epidermal keratinocytes, using 0.5-µm latex microspheres. 

Inactivation of the Ilk gene resulted in severe impairment of microsphere uptake. Further, 

stimulation of the keratinocyte growth factor (KGF) receptor or PAR-2 failed to increase 

phagocytosis in these cells. Activation of ERK in response to KGF stimulation is unaltered in 

ILK-deficient keratinocytes, indicating that the KGF receptor likely signals normally in the 

absence of ILK. In contrast, ILK deficiency was associated with impaired Rac1 activation in 

response to KGF. The latter was accompanied by reduced formation of membrane ruffles and


abnormal F-actin dynamics. Thus, ILK is essential for normal keratinocyte phagocytosis in 

response to KGF. 

Supported by the Canadian Institutes of Health Research (CIHR). 

2046 

Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) is an Important Regulator of 

Equine Neutrophil Migration and Adhesion. 

M. K. Sheats 1 , K. L. Carren 2 , E. M. Hefner 3 , E. J. Sung 2 , K. B. Adler 4 , S. L. Jones 2 ; 1 Molecular 

Biological Sciences, North Carolina State University, Holly Springs, NC, 2 Clinical Sciences, 

North Carolina State University, 3 Animal Science, North Carolina State University, 4 Molecular 

Biological Sciences, North Carolina State University 

Neutrophil infiltration is a prominent feature in a number of pathologic conditions including 

recurrent airway obstruction and ischemic reperfusion injury. Cell signaling components 

involved in neutrophil migration represent targets for novel anti-inflammatory therapies. This 

study was conducted to test the hypothesis that the 32 kD protein, Myristolated Alanine-Rich C- 

Kinase Substrate (MARCKS) is involved in equine neutrophil migration and adhesion. 

In other species (i.e. human) MARCKS phosphorylation and dephosphorylation causes the 

protein to cycle between the cell membrane and cytosol, respectively. To investigate MARCKS 

phosphorylation in horses, neutrophils were isolated from whole blood using Ficoll gradient 

centrifugation and stimulated with platelet activating factor (PAF), leukotriene B4 (LTB4) or 

phorbol myristate acetate (PMA). Western blot was performed using specific phospho-MARCKS 

and total MARCKS primary antibodies. These results determined that MARCKS phosphorylation 

in equine neutrophils is maximal 30 seconds following stimulation with 100 nM PAF or LTB4 and 

that dephosphorylation occurs within 3 minutes. 

MARCKS interacts with the resting neutrophil cell membrane through the combined efforts of its 

myristolated N-terminus and basic amino acid effector domain; potentially regulating interactions 

between PIP2 and actin binding proteins. To investigate the requirement for MARCKS in equine 

neutrophil chemotaxis, isolated neutrophils were pre-treated with MANS (a cell permeant 

peptide identical to the N-terminal 24 amino acids of MARCKS), RNS (a scrambled sequence 

control peptide) or vehicle control prior to migration toward known neutrophil chemoattractants 

(LTB4 or PAF). Pre-treatment of equine neutrophils with the MANS peptide significantly inhibited 

LTB4 and PAF stimulated migration while the control RNS peptide had no effect. 

Beta-integrin upregulation and adhesion is an essential process to neutrophil chemotaxis. To 

investigate the requirement for MARCKS in equine neutrophil adhesion, isolated neutrophils 

were pretreated with MANS, RNS or vehicle control and stimulated to adhere to Immulon 2 

plates coated with 5% FBS (a β-integrin dependent substrate) in PBS with 10 ng/ml PMA or 

vehicle control. Pre-treatment of equine neutrophils with the MANS peptide significantly inhibited 

PMA induced adhesion; while the control RNS peptide had no effect. 

Pre-treatment of equine neutrophils with a cell permeant peptide identical to the N-terminus of 

MARCKS significantly inhibited equine neutrophil PMA induced adhesion and LTB4 and PAF 

induced migration. These results indicate that MARCKS is an important regulator of equine 

neutrophil migration and adhesion and MARCKS regulation of neutrophil chemotaxis has been 

conserved across evolutionary lines.


2047 

The Scar/WAVE complex is necessary for proper regulation of traction stresses during 

amoeboid motility. 

E. E. Bastounis 1 , R. Meili 2 , B. Alonso Latorre 2 , J-C. del Alamo 2 , J. Lasheras 2 , R. Firtel 2 ; 

1 Bioengineering, University of California, San Diego, San Diego, CA, 2 University of California, 

San Diego 

Chemotaxis, or directed cell migration, is involved in a broad spectrum of biological phenomena, 

ranging from the metastatic spreading of cancer to the active migration of neutrophils during 

wound healing or in response to bacterial infection. Chemotaxis requires a tightly regulated, 

spatiotemporal coordination of underlying biochemical processes. SCAR/WAVE-mediated 

dendritic F-actin polymerization at the cell’s leading edge plays a key role in cell migration. Our 

study identifies a mechanical and biochemical role for the SCAR/WAVE complex in modulating 

the traction stresses that drive cell movement. We demonstrate that the traction stresses of 

wild-type Dictyostelium cells or cells lacking the SCAR/WAVE complex protein PIR121 (pirA-) or 

SCAR (scrA-) exert stresses of different strength that correlate with their levels of F-actin. By 

processing the time records of the cell length and the strain energy exerted by the cells on their 

substrate, we show that wild-type cells migrate by repeating a specific set of mechanical steps 

(motility cycle), whereby the cell length (L) and the strain energy exerted by the cells on their 

substrate (Us) vary periodically. Our analysis also revealed that scrA- cells exhibit an altered 

motility cycle with a longer period (T=160s) and a lower migration velocity (V =6μm/min) 

compared to those of wild-type cells (T=80s, V =13μm/min). In marked contrast to these strains, 

pirA- cells, although they have a higher F-actin content, migrate as slowly as scrA- cells but their 

migration occurs in a seemingly random manner in that they lack the periodic changes in 

traction stresses that are observed for the other two strains. Finally, by quantifying the level of 

F-actin in the leading edge in combination with the traction stresses, we demonstrated that the 

level of leading edge, SCAR/WAVE complex-mediated F-actin polymerizations is critical for the 

level and spatiotemporal control of the traction stresses, cell-substrate interactions, and the 

motility cycle. 

2048 

Self-Generated EGF Gradients Guide Epithelial Cell Migration. 

D. Irimia 1 , A. J. Aranyosi 1 , B. Kulemann 1 , S. Thayer 1 , M. Toner 1 , O. Illiopoulos 1 , C. Scherber 1 ; 

1 BioMEMS Resource Center, Massachusetts General Hospital/Harvard Medical School, Boston, 

MA 

Migrating epithelial cells can contribute to wound healing processes, or can trigger lethal 

complications during cancer through invasion and distant metastasis. During their migration, 

cells must be able to orient accurately and it is generally assumed that gradients of chemokines 

or growth factors are required to guide epithelial cells towards their target location. 

Unexpectedly, we uncovered a novel strategy for epithelial cell orientation during migration that 

does not require pre-existent chemical gradients. Using microscale engineering techniques, we 

demonstrate that the strategy is dependent on the competition between epidermal growth factor 

(EGF) uptake by the cells and the restricted diffusion of EGF from surrounding 

microenvironment. Both normal and malignant epithelial cells are capable of EGF uptake and 

can use this strategy when placed in confined environment. The strategy has some surprising 

consequences for epithelial cells, like the ability to efficiently disperse through channels, or to 

navigate along the shortest path to exit from various microscopic mazes. Inhibition of EGFreceptor 

but not the inhibition of chemokine receptors, reduces the ability of the cells to orient, 

without altering their velocity of migration. Better understanding of the epithelial cells guidance 

strategy by EGF uptake-dependent self-generated gradients could lead to approaches for


restricting the migration of malignant cells to delay local invasion and distant metastases, or 

enhance the migration of normal cells to promote wound healing. 

2049 

Three dimensional traction forces exerted by migrating amoeboid cells.* 

R. Meili 1 , B. Alvarez-Gonzalez 2 , J. C. del Alamo 2 , R. A. Firtel 1 , J. C. Lasheras 2 ; 1 Division of Cell 

and Developmental Biology, University of California, San Diego, La Jolla, CA, 2 MAE 

Department, University of California, San Diego 

Cell migration is essential for many physiological processes in multicellular organisms including 

embryonic development, wound healing and cancer metastasis. Amoeboid motility requires the 

spatiotemporal coordination of forces generated by biochemical processes. This results in 

directional motility by repeated protrusion of the front and retraction of the rear driven by actin 

polymerization and actomyosin contraction. 

Mechanically, movement requires the modulation of the cell’s interactions with the environment, 

either with surrounding cells or with extracellular matrix. For quantitative analysis, these 

interactions have so far typically been characterized as tangential traction stresses imposed by 

the cell on a flat substrate. 

We have expanded the method for measuring traction stresses to determine the stresses 

exerted perpendicular to the substrate in addition to the in-plane stresses. 

We find that the deformation perpendicular to the substrate is significant and that calculating the 

stresses using one of the existing two-dimensional traction cytometry methods misses an 

important aspect of cellular mechanics. 

We used our new method to better understand the mechanical role of individual cytoskeletal 

components by comparing traction stress maps and dynamics of wild type cells moving over flat 

substrates with measurements of Dictyostelium cells with mutations in cytoskeletal proteins. We 

focused our study on mutant cell lines with crosslinking defects, such as myosin II-null cells and 

cortexilin-null cells. 

We find that cells from all cell lines studied push downward on the substrate near the center of 

the cell and pull up near the periphery. The magnitude of the perpendicular forces is 

comparable to the magnitude of the tangential forces exerted on the substrate; therefore the 

perpendicular component is expected to play a significant role in the cell behavior and cannot 

be neglected. Our initial findings show that the effects of the crosslinking mutations on the 

parallel forces do not track with the effects on the perpendicular forces. For example, myosin IInull 

cells show a significant reduction of the front to back organization of the parallel traction 

forces while the push pull distribution of forces remains unaffected. Our observations suggest 

that the generation of the stresses perpendicular and tangential to the substrate is possibly 

controlled independently. 

*Work supported by NIH grant 1RO1 GM084227. 

2050 

Profilin-1: a biomarker for breast cancer malignancy? 

Z. Ding 1 , R. Bhargava 2 , N. Lakshman 3 , M. Petroll 4 , P. Roy 5 ; 1 Bioengineering, University of 

Pittsburgh, Pittsburgh, PA, 2 University of Pittsburgh, Pittsburgh, PA, 3 University of Texas 

Southwestern Medical Center Dallas, Dallas, TX, 4 Ophthalmology, University of Texas 

Southwestern Medical Center Dallas, Dallas, TX, 5 Bioengineering and Pathology, University of 

Pittsburgh, Pittsburgh, PA 

This study unexpectedly reveals that stable loss of expression of profilin1 (Pfn1), an actinbinding 

protein that has been traditionally shown to be required for cell proliferation and 

migration, in fact promotes tumor growth and overall distant metastasis of breast cancer


xenografts in mice. Comparative studies in spontaneous vs experimental metastasis models 

suggest that loss of Pfn1 expression likely promotes metastasis-promoting events at the primary 

tumor site. ECM invasion (an important step for tumor metastasis) of breast cancer cells is 

increased upon loss of Pfn1 expression. Experiments involving selective perturbation of 

signaling pathway downstream of PI3-Kinase (PI3K) further revealed that hyper-invasiveness of 

Pfn1-depleted cells is mediated by a PI3K-PI(3,4)P2-lamellipodin signaling axis. Finally, as a 

clinical correlate of these in vitro and in vivo findings, analyses of Pfn1 expression in primary 

tumors revealed that Pfn1 downregulation occurs in all molecular subtypes of breast cancer, 

somewhat more prominently in estrogen receptor-negative tumors. Furthermore, tumors 

involving significant lymph node infiltration and/or distant metastasis present the most dramatic 

downregulation in Pfn1 expression. These findings imply that Pfn1 could serve as a new 

molecular marker for breast cancer malignancy. 

2051 

Reestablishing the polarity of Dictyostelium cells to understand the organization of the 

cytoskeleton and signaling complexes during chemotaxis. 

D. Jowhar 1 , G. Wright 1 , C. Janetopoulos 1,2 ; 

1 Biological Sciences, Vanderbilt University, Nashville, TN, 2 Cell and Developmental Biology, 

Vanderbilt University, Nashville, TN 

Cells migrating in a chemical gradient typically have a polarized morphology, where they display 

a distinct front and back. Cells can also have different localized sensitivities to chemoattractants 

depending on their degree and type of polarity. To better understand these phenomena, we 

used the social amoeba Dictyostelium discoideum that expressed GFP-tagged proteins known 

to localize to distinct regions of the cell during cell migration. Dictyostelium cells were lured into 

a specialized microfluidic device developed in our laboratory which confined the cells in narrow 

3-dimensional Polydimethylsiloxane (PDMS) channels. The chemoattractant gradient in this 

experimental platform could be reversed so 

that the rear of the cell was exposed to a higher concentration of chemoattractant than the front 

of the cell. Cells in the channels would freeze, become unpolarized, and were then capable of 

forming a new front at the former rear of the cell. We have observed the temporal and spatial 

loss and gain of localization of various signaling and cytoskeletal molecules at both the rear and 

front of the cell as polarity is broken down and re-established. We monitored the localization of 

PHcrac (a biosensor for PI(3,4)P2 and PI(3,4,5)P3), RBD (a biosensor for Ras activity), PTEN- 

GFP (a phosphatase which elevates levels of PI(4,5)P2), LimE (a marker for F-actin), and 

TACC-GFP (a microtubule centrosome and plus-end 

marker.) The signaling markers showed a re-distribution to opposite sides of the cell during the 

reversals, as did LimE. We also monitored the microtubules and centrosomes and tracked their 

redistribution during polarity re-establishment using TACC. Further experiments were performed 

in the absence of an actin cytoskeleton and in a variety of signaling mutants. These experiments 

have helped elucidate the molecular mechanisms that contribute to the establishment and 

maintenance of cell polarity. 

2052 

Alteration of α6 integrin expression and chemotactic motility of microglia upon ADP 

stimulation. 

S-H. Lee 1 , C. Y. Chung 2 ; 1 Pharmacology, Vanderbilt University Medical Center, Nashville, TN, 

2 Vanderbilt University Medical Center, Nashville, TN 

Microglia are the immune effector cells that are rapidly activated in response to even minor 

pathological changes in the central nervous system. It has been demonstrated that microglia


attach well to fibronectin and vitronectin, but only weakly to laminin, and that laminin exerts a 

dominant anti-adhesive effect on microglial adhesion. Microglial activation is reported to be 

accompanied by the alteration of integrin expression. Inflammatory cytokines increased 

expression of α4β1, α5β1, and Mac-1 integrins on microglia but changes of integrin expression 

upon ADP (a chemoattractant for microglia) stimulation remain unknown. We, in this study, 

investigated if ADP can induce the alteration of integrin species on cell surface, leading to 

changes in chemotactic ability on different ECM. FACS scans and surface biotinylation assays 

showed that ADP stimulation induced a significant increase in the expression of α6 integrin, but 

not α5 on the surface of microglia cells. Examination of microglia motility on different ECM 

revealed that cells have greater motility on laminin than fibronectin upon ADP stimulation, 

presumably due to the increased expression of α6 integrin. The alteration of integrin-mediated 

adhesion may regulate the extent of microglial infiltration into the site of damage by controlling 

their chemotactic ability. 

2053 

Directing cell migration with nano-ridges/grooves. 

X. Sun 1 , M. Driscoll 2 , J. Fourkas 1 , W. Losert 2 ; 1 Chemistry and Biochemistry, University of 

Maryland, College Park, MD, 2 Physics, University of Maryland, College Park, MD 

We evaluated the contact guidance of nano-ridges/grooves on the amoeba Dictyostelium 

discoideum, a model system for studying cell migration. Nano-ridges were fabricated with 

Multiphoton Absorption Polymerization (MAP). These nano-topographies, as well as 

complementary nano-grooves, were replicated with high fidelity from an improved composite 

polydimethylsiloxane (PDMS) mold. By analyzing the cellular velocity, orientation, boundary 

curvature and shape dynamics, we found that the ridges and grooves exert bidirectional 

guidance on migrating cells. More cells migrate in the direction of the nano-ridges/grooves than 

perpendicular to them. We also found that cells aligning in the direction of the nanoridges/grooves 

are more elongated and that cells migrating in the direction of the nanoridges/grooves 

migrate faster. Contact guidance is only achieved with topotraphies of proper 

pitch. Nano-ridges/grooves with a pitch smaller than 0.2 μm or greater than 10 μm do not exhibit 

effective guidance. This study may offer insights into fabricating complex 3D scaffolds for tissue 

engineering with MAP. 

2054 

The N-formylpeptide Receptor FPR1 Is Functionally Expressed in Lens Epithelial Cells 

and is Critical for Lens Maintenance in Mice. 

J-L. Gao 1 , J. Tuo 1 , E. H. Schneider 1 , D. Despres 1 , M. Lizak 1 , A. Maminishkis 1 , C. C. Chan 1 , P. 

M. Murphy 1 ; 1 National Institutes of Health, Bethesda, MD 

Most mouse knockouts for inflammatory chemoattractants and their receptors lack spontaneous 

phenotypes. We considered whether an aging study might identify slowly developing 

phenotypes in such mice. Here we show that mice lacking Fpr1 gene (Fpr1-/- mice) have severe 

cataracts at an early age. FPR1 is known to mediate pro-inflammatory chemotactic responses 

by phagocytic leukocytes to N-formylpeptides produced by bacteria or mitochondria and plays a 

role in host defense against certain bacteria. Fpr1-/- mice developed small eyes starting at 3 

month of age because of lens degeneration and rupture. Histopathological analysis did not 

show any infection or inflammation in these eyes, but showed abnormal lens cell migration. 

Consistent with this, we detected FPR1 mRNA in both mouse and human lens epithelial cells, 

and FPR1 proteins on primary human fetal lens cells with the level compatible to that detected 

on human neutrophils. Stimulation of lens epithelial cells with FPR1 ligands induced intracellular


calcium release and actin polymerization. Taken together, the data implicate FPR1 in mediating 

lens cell migration, a critical event for lens development and maintenance. 

2055 

PRL-3 promotes migration and invasion by up-regulating MMP-7 in human colorectal 

cancer cells. 

Y-M. Han 1 , S-K. Lee 2 , D-S. Sin 2 , Y-R. Ha 2 , J. Kim 2 , C-W. Lee 3 ; 1 University of Science and 

Technology, Daejeon, Korea, 2 Laboratory of Chemical Biology and Genomics, Korea Research 

Institute of Bioscience and Biotechnology, Daejeon, Korea, 3 Bio-Evaluation Center, Korea 

Research Institute of Bioscience and Biotechnology, Chungbuk, Korea 

PRL-3, a member of a subgroup of protein tyrosine phosphatases (PTPs) that can stimulate the 

degradation of the extracellular matrix, is over-expressed in metastatic colorectal cancer relative 

to primary tumors. To determine whether PRL-3-induced enhancement of migration and 

invasion is dependent on the expression of matrix metalloproteases (MMPs), PRL-3 was 

expressed in DLD-1 human colorectal cancer cells. The motility, migration and invasion 

characteristics of the cells were examined and metastasis to the lung was confirmed in a nude 

mouse using PRL-3-overexpressing DLD-1 cells [DLD-1 (PRL-3)]. Migration and invasion of the 

cells were inhibited by phosphatase and farnesyl transferase inhibitors. Expression of MMPs 

was enhanced 3- to 10-fold in comparison to control cells, and migration and invasion were 

partially inhibited by siRNA knockdown of MMP-2, -13, or -14. Importantly, siRNA knockdown of 

MMP-7 completely inhibited the migration and invasion of DLD-1 (PRL-3) cells, while 

overexpression of MMP-7 increased migration. The expression of MMP-7 was also downregulated 

by phosphatase and farnesyl transferase inhibitors. It was found that PRL-3 induced 

MMP-7 through oncogenic pathways including PI3K/AKT and ERK, and that there is a 

relationship between the expression of PRL-3 and MMP-7 in human tumor cell lines. The 

expression of MMP-13 and MMP-14 was very sensitive to the inhibition of farnesyl transferase; 

however, the migration and invasion of DLD-1 (PRL-3) cells did not strongly depend on the 

expression of MMP-13 or -14. These results suggest that the migration and invasion of PRL-3expressing 

colorectal cancer cells depends primarily on the expression of MMP-7. 

2056 

Serine phosphorylation in paxillin is important for focal adhesion formation during cell 

adhesion onto collagen type I. 

T. Kwak 1 , J. W. Lee 1 ; 1 Department of Pharmacy, Research Institute of Pharmaceutical 

Sciences, College of Pharmacy, Seoul National University, Seoul, Korea 

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during 

morphological change and cell migration. Upon cell adhesion, protein-protein interaction at focal 

adhesions consisting of FAK, talin, paxillin, and others, plays major roles in regulation of cell 

morphogenesis and migration. Although phosphorylation at tyrosines of paxillin has been well 

known to be critically involved in adhesion-mediated signaling, little is known about the 

significance of paxillin phosphorylation at serine 85 in cell migration. Here in this study, we 

provide evidences that paxillin phosphorylation at serine 85 occurred during HeLa cell adhesion 

onto collagen I, being concomitant with FAK and talin phosphorylations. However, 

unphosphorylatable mutant S85A paxillin impaired cell spreading, focal adhesion turnover, and 

migration toward collagen I but not to serum components. Further, the physical interaction 

between paxillin and talin was through the C-terminal tail of talin leading to focal adhesions at 

the cell boundary, but S85A paxillin did not bind talin and caused random focal adhesion 

formation. Together, these observations suggest that paxillin phosphorylation at Serine 85 

depending on cell adhesion is important for interaction with talin and for cell morphological and


migratory ability. [This work was supported by NRF by senior researchers program (Leap 

research, 2011-0001160) and Global Frontier Project grant (NRF-M1AXA002-2010-0029778), 

and a grant of the Korean Health Technology R&D Project (A100727), MHWFA, Korea to JW 

Lee]. 

Key words: focal adhesion, paxillin, talin, morphology, migration 

2057 

Nck Is Required for Focal Adhesion Maturation and Polarized Cell Motility. 

S. P. Chaki 1 , R. Barhoumi 2 , R. Yog 1 , M. E. Berginski 3 , S. M. Gomez 3 , G. M. Rivera 1 ; 1 Veterinary 

Pathobiology, Texas A&M University, College Station, TX, 2 Veterinary Integrative Biosciences, 

Texas A&M University, College Station, TX, 3 University of North Carolina at Chapel Hill, NC 

The establishment of cell polarity is essential for directional migration of endothelial cells and 

the organization and function of endothelial layers. The SH2/SH3 domain-containing adaptors 

Nck (Nck1 and Nck2) link signaling by tyrosine phosphorylation with effectors that mediate 

remodeling of the actin cytoskeleton. We previously determined an important role for Nck 

adaptors in endothelial cell migration, invasion, and in vitro morphogenesis. To understand the 

mechanisms by which Nck promotes these processes, we used differential interference contrast 

(DIC) and total internal reflection fluorescence (TIRF) microscopy to compare membrane and 

adhesion dynamics of human umbilical vein endothelial cells (HUVEC) with or without 

perturbation of Nck signaling. Expression of specific short hairpin RNAs induced > 90% and 

50% reduction in Nck1 and Nck2 levels, respectively. Double knockdown cells (Nck1 and Nck2) 

were rescued by expression of a siRNA-resistant Nck2 cDNA. Kymographs derived from timelapse 

DIC images showed a significant (p


2058 

The F-BAR domain protein PACSIN2 associates to Rac1 and regulates cell spreading and 

migration. 

B-J. de Kreuk 1 , M. Nethe 1,2 , M. Fernandez-Borja 1 , E. Anthony 1 , P. Hensbergen 3 , A. Deelder 3 , M. 

Plomann 4 , P. Hordijk 1 ; 1 Molecular Cell Biology, Sanquin Research, University of Amsterdam, 

Amsterdam, Netherlands, 2 University of Virginia, Charlottesville, VA, 3 Leiden University Medical 

Center, Leiden, Netherlands,, 4 Institute for Biochemistry 2, University of Cologne, Germany 

The Rac1 GTPase controls cytoskeletal dynamics and is a key regulator of cell spreading and 

migration via signaling through effector proteins such as the PAK kinases and the Scar/WAVE 

proteins. We previously identified a series of regulatory proteins that associate with Rac1 

through its hypervariable C-terminal domain. These include the Rac1 activator β-Pix, which 

recruits Rac1 to focal adhesions, and the membrane adapter Caveolin1, which regulates the 

poly-ubiquitylation and degradation of activated Rac1. Here, we show that Rac1 associates with 

the F-BAR domain protein PACSIN2, an inducer of membrane tubulation and a regulator of 

endocytosis. We show that loss of PACSIN2 increases Rac1-GTP loading and promotes cell 

spreading and –migration. Conversely, expression of PACSIN2 reduces Rac1-GTP levels in a 

fashion which is dependent on the PACSIN2-Rac1 interaction, on the membrane-tubulating 

capacity of PACSIN2, and on Dynamin. In addition, we show that GEF-mediated Rac1-GTP 

loading is unaffected when PACSIN2 is expressed. These results demonstrate that PACSIN2 is 

an important regulator of the small GTPase Rac1 and that PACSIN2 limits Rac1-GTP signaling 

by promoting Rac1 inactivation. 

2059 

Control over acquisition of cell motility and cell migration by the RNA binding protein 

Dead end. 

M. Goudarzi 1 , T. U. Banisch 1 , E. Raz 1 ; 1 Cell Biology, ZMBE, Muenster, Germany 

Specification of zebrafish primordial germ cells (PGCs) depends on maternally inherited 

determinants. These factors, collectively termed the germ-plasm direct cells to enter the 

germline fate and to acquire proper cell behavior. An important such maternally-provided germplasm 

component in zebrafish is encoded by the dead end (dnd) gene. The Dnd protein 

contains an RNA recognition motif and is specifically expressed in PGCs, where it is localized to 

the perinuclear granules. Knockdown of dead end results in severe defects in cell behavior (e.g. 

the acquisition of motility), cell fate maintenance and survival. 

We could previously show that Dnd functions by protecting specific mRNAs (e.g. tdrd7, nanos1 

and hub) from micro RNA–mediated translational inhibition and degradation in the germ cells, a 

process by which the same RNAs are repressed and degraded in somatic cells. To determine 

the molecular basis of the dramatic changes in PGC properties upon loss of Dnd, we identified 

RNAs regulated by miRNAs and the Dnd protein. The functional role the proteins encoded by 

these mRNAs play in controlling cell shape and behavior will be discussed. 

2060 

Nonmuscle Myosin II Isoforms regulate microtubule dynamics in three-dimensional cell 

migration through the distinct MLC phosphorylations. 

S. Komatsu 1 , M. Ikebe 2 ; 1 Microbiology and Physiological Systems, UMass Medical School, 

Worcester, MA, 2 UMass Medical School, Worcester, MA 

Cell migration is an extremely complex process which is controlled by the rearrangement of 

cytoskeletal systems. Most studies of cell migration have been focused on controlling the 

actomyosin and microtubule networks, and studied a migrating cell on two-dimensional (2D)


surfaces such as plastic or glass. However, cells under physiological environment interact with 

extracellular matrices on all surfaces, not just on the basal surface. This raises an idea that cells 

showing rounded spindle shape in 3D matrices migrate with the different mechanism from flatshaped 

cells in 2D surface. To address this question, we use a 3D migration in collagen gel as 

an in vitro model system for identifying the mechanisms that regulate cell migration under 

physiological conditions. 

First, we observed distribution of nonmuscle myosin IIA (NMIIA) and IIB (NMIIB) isoforms in 3D 

migrating cells by using NMII isoforms specific antibodies and fluorescent protein-fusion NMII 

isoforms, mCherry-NMIIA and GFP-NMIIB. Although fluorescent signals of the NMIIA and the 

NMIIB were observed at both anterior and posterior regions in the 3D migrating cells, the 

detailed distribution of both isoforms were different from each other. The strong signals of NMIIA 

were particularly detected at the tips of cell extensions, while the signals of NMIIB were 

accumulated at the cortex around the spindle-shaped cell body. Inhibition of either actomyosin 

or microtubule networks by blebbistatin or nocodazole resulted in a decrease in 3D cell 

migration. Isoform-specific knockdowns by recombinant adenovirus siRNA for NMIIA and NMIIB 

reveal that NMII isoforms are respectively involved in the regulation of the microtubule dynamics 

in the 3D migrating cells. We also studied the localization of myosin II isoforms phosphorylated 

at functionally distinct sites of myosin light chain (MLC) in the 3D migrating cells. Interestingly, 

our results showed that NMIIA phosphorylated at Ser19 localized at the anterior tip of cell 

protrusion, while NMIIB phosphorylated at Ser1 localized at posterior region of the protrusion. 

On the other hand, di-phosphorylated myosin II at Thr18 and Ser19 colocalized with cortical 

actin structure. 

Our results suggest that the functionally distinct MLC phosphorylation plays an important role in 

the function of NMII isoforms in 3D cell migration. Further studies are in progress to elucidate 

the role of the functionally distinct MLC phosphorylation in the 3D cell migration. 

2061 

Wound healing: The combination use of regenerative agents and bioavailable antioxidant 

constituents of vitamin E and vitamin D in a model in vitro system. 

A. Bettica 1 , N. Christensen 2 , T. Ragasha 2 ; 1 Biology, Manhattanville College, Purchase, NY, 

2 Manhattanville College, Purchase, NY 

Wound healing models have fostered a greater understanding of the cell types involved and the 

many complex series of events from the site of trauma through the phases of inflammation, 

proliferation, and remodeling. Many in vitro studies have concentrated on the proliferation 

stages to elucidate agents that may aid migrating fibroblasts in producing growth factors, laying 

down fibers and ground substance, and attracting epithelial cells to the site. Fat-soluble vitamins 

such as vitamin D3 and vitamin E are potent antioxidant and anti-inflammatory agents and have 

been shown to be effective in ameliorating dental and surgical wounds, burns, and scars. To 

determine the optimal effectiveness of these properties, appropriate formulations of the isomeric 

constituents must be tested. The combination of these vitamin species may show additive 

effects and enhanced efficacy under in vitro wound healing conditions. In dental wound healing, 

liners and sealers containing varying amounts of calcium hydroxide can trigger a cascade of 

events which include increased cell proliferation, growth factor synthesis, and cell differentiation. 

Skin cells, keratinocytes, and fibroblasts are susceptible to a range of UVA and UVB 

wavelengths that cause cellular and DNA damage, including the formation of thymine dimers. 

Avobenzone and zinc oxide (ZnO) provide true broad-spectrum protection against UVA 

wavelengths above 360 nm and are the only two sunscreen active ingredients approved in the 

US. Studies suggest that proper formulation strategies are necessary for superior attenuation of 

UVA wavelengths and may be achievable at 3% avobenzone or 5% ZnO. An in vitro wound 

healing model was standardized to test the combination of vitamin D3 and vitamin E isomers for


additive effectiveness with proven or suggested regenerative agents. WS1 human skin 

fibroblasts are seeded at a density of 105 cells/well on a 6-well culture plate. Using a 4mm 

sterile biopsy punch, three wound areas are created in each well at confluency. WS1 cell 

wounds are exposed to 10-fold serial dilutions of calcium hydroxide, avobenzene, or ZnO in 

appropriate solvents for 24 hours in control wells, or before cells are exposed to UVA. Effective 

concentrations are selected from standard dose response curves for the various regenerative 

reagents and are used in combination with the vitamin D3 and E isomers. During the test 

periods, wound areas are digitally recorded beginning at day 0, for every 12 hours over a four 

day period. Measurements of the radius (rw - radius of the minimal diameter of the wound), the 

wound area (Aw) are taken using Motic 2.0 software and compared to day 0 (r0 and A0). 

Triplicate data sets are statistically analyzed using a one-way ANOVA with Tukey’s post hoc 

tests. Effective concentrations of calcium/ D3 and cholcalciferol alone are seen at 10-4 mg/ml. 

Vitamin E isomers did improve cell proliferation and overall wound healing. The most effective 

doses were 10-7 mg/ml for alpha-tocopherol and 10-8 mg/ml for delta-tocotrienol, showing 

complete wound healing between 48 and 60 hours. It is suggested that agents with greater 

antioxidant activities may shorten healing times, reduce the incidence of wound infection and, in 

the most serious wound cases, lower the mortality rate in burn patients. 

2062 

Arp 2/3 Plays a Crucial Role in Focal Adhesion Initiation at the Leading Edge. 

R. J. Vasquez 1 , K. Sayegh 2 , J. Stricker 3 , Y. Beckham 3 , M. Gardel 3 ; 1 Department of Pediatrics, 

Section of Hematology, Oncology and Stem Cell Transplantation, University of Chicago, 

Chicago, IL, 2 University of Chicago, Chicago, IL, 3 Department of Physics, University of Chicago, 

Chicago, IL 

The lamellipodia is a dense, Arp-2/3 mediated actin network at the leading edge of migrating 

cells that generates forces sufficient to generate sheet-like protrusions of the cell edge. In 

addition to this force generating role, the initiation of focal adhesions also occurs within the 

lamellipodia. However, the role of lamellipodial actin in focal adhesion initiation is not well 

understood. We used a recently identified inhibitor of the ARP 2/3 complex, CK 869, to 

investigate the role of the lamellipodia in leading edge protrusion and focal adhesion formation 

in cell motility and adhesion in two cell types- MCF10A cells (a human breast epithelial cell line) 

and U20S cells (a human osteosarcoma cell line). We find that treatment with CK 869 disrupts 

or inhibits the formation of an organized lamellipod, as demonstrated by phalloidin staining and 

immunofluoresce with cortactin, a protein enriched in the lamellipod. Motility assays of single 

cells and scratch assays with MCF-10A cells demonstrate that treatment with CK-869 reduced 

the number of motile cells and reduced the motility rate, and in the case of the scratch assay, 

greatly increased the time to closure of the cell sheet. Despite these effects on cell migration, 

protrusions of the cell edge persist, suggesting that ARP 2/3 mediated actin assembly is not the 

only mechanism through which to generate sheet-like membrane protrusions. We hypothesized 

that these membrane protrusions might differ from lamellipodial protrusions in their ability to 

form new focal adhesions. Immuno-staining of the focal adhesion proteins paxillin and vinculin 

demonstrated that the ARP 2/3 inhibited cells had fewer but larger peripheral adhesions and 

fewer central focal adhesions. Live cell imaging with GFP-paxillin in U2OS cells revealed that 

the protrusions formed in the presence of the ARP-inhibitor revealed reduced focal adhesion 

assembly. Collectively, these results indicate that ARP 2/3 is not necessary for sheet-like 

protrusions of the cell edge but plays a crucial role in focal adhesion formation.

2063 

Quantitative Analysis of Protein Binding Dynamics in Live Cells with Photoswitchable 

Fluorescent Tags. 

M. Rubashkin 1 , M. J. Paszek 1 , C. C. Dufort 1 , P. Kumar 2 , T. Wittmann 2 , V. M. Weaver 1,3 ; 

1 Surgery and Center for Bioengineering and Tissue Regeneration, University of California at 

San Francisco, San Francisco, CA, 2 Cell and Tissue Biology, University of California at San 

Francisco, San Francisco, CA, 3 Anatomy, Bioengineering and Therapeutic Sciences, Eli and 

Edythe Broad Center of Regeneration Med., University of California at San Francisco, San 

Francisco, CA 


The kinetics of individual proteins can dictate focal adhesion dynamics and cellular behaviors 

such as polarity, motility and migration. We developed a new quantitative fluorescence 

microscopy technique, RAPS (Regression After Photo-Switching), to study the binding state and 

kinetics of proteins in live cells. In brief, RAPS involves tagging a protein with a photoswitchable 

fluorescent protein, such as mEOS2. Initially, an image is taken of the protein in the unswitched 

green state, 488nm, to identify focal adhesions of interest. Then, a digital micro-mirror device 

concurrently illuminates all adhesions of interest at the activation wavelength, 405nm, 

converting a subpopulation of the protein to a stable red fluorescent state. The cell is imaged in 

the converted state, 568nm, to measure the dynamics of the protein. Lastly, kinetic modeling 

analysis is implemented to determine the Koff and mobile fraction of the protein. In comparison 

to existing numerical fluorescence photobleaching techniques, RAPS reduces the time and 

intensity of light needed for activation, enables imaging in a secondary wavelength to increase 

contrast and minimize noise, permits diffusion kinetics to be ignored in analysis, and facilitates 

simultaneous investigation of multiple cell structures within a single cell. In order to verify that 

RAPS can be used to quantify changes in protein binding kinetics, we studied focal adhesion 

proteins in MCF10a breast cancer epithelial cells. In the study of talin, we observed a Koff rate of 

0.024 ± 0.007 (1/s) and a bound protein fraction of 63 ± 12%. Furthermore, we detected that the 

mobile fraction of talin significantly increased after inducing expression of the glycoprotein 

Mucin1, a cancer oncogene associated with changes in cell motility. 

Development and Morphogenesis I 

2064 

Expression of new growth factor of the VEGF family in sea urchin development. 

Y. O. Kipryushina 1,2 , K. V. Yakovlev 1 , N. A. Odintsova 1,2 ; 1 A.V. Zhirmunsky Institute of Marine 

Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia, 2 Far 

Eastern Federal University, Vladivostok, Russia 

Growth factors regulate many processes, including cell proliferation, differentiation, migration 

and apoptosis, and play an important role in embryonic development. Among them vascular 

endothelial growth factors (VEGF), of the VEGF/PDGF superfamily, are important signaling 

proteins involved in induction of angiogenesis and playing a central role in the regulation of 

vasculogenesis in mammals. In invertebrates, such as insects and nematodes, VEGF/PDGF 

superfamily members guide cell migration and cell differentiation during development. The aim 

of our study is to investigate the role of VEGFs during development of the sea urchin 

Strongylocentrotus intermedius. Three Sp-Vegf genes have been predicted in the genome of a 

closely related sea urchin S. purpuratus. One of them, Sp-Vegf3, has been reported to play an 

essential role in sea urchin development (Duloquin et al., 2007). In this study, we investigated 

the expression of a new gene of the VEGF family, Si-Vegf2, and expression of the Si- 

Pdgfr/vegfrL gene in sea urchin development by RT-PCR. We demonstrated a high similarity of


the central part of the new gene, which we found in the sea urchin S. intermedius, with that of 

the predicted gene Sp-Vegf2 in S. purpuratus. The Si-Vegf2 was found to be expressed from 

the zygote stage, whereas Si-Pdgfr/vegfrL expression began later at the blastula stage. Our 

data have shown that a nonspecific inhibitor of heparine-binding GF receptors, suramin, results 

in significant disturbances in embryonic development: archenteron formation is suppressed, and 

spicule growth is inhibited. An addition of human PDGF-AB to embryonic culture leads to partial 

recovery of archenteron growth. These results match the findings on another sea urchin species 

(Katow and Aizu, 2002). In summary, we have discovered the expression of a new VEGF family 

member in sea urchin development and shown that temporal expression profiles of the Si-Vegf2 

and Si-Pdgfr/vegfrL are not similar in sea urchin development, but signaling through 

VEGF/PDGF pathway is essential for embryonic development. (This work was supported by 

Presidium of the FEB RAS (grants 09-I-P22-04, 09-II-SB-06-001) and Program at the Far 

Eastern Federal University (grant 11 G34.31.0010)). 

2065 

How “acellular” embryos gastrulate: Investigation of cytoplasm movements during 

gastrulation in developing Drosophila melanogaster embryos. 

B. He 1 , O. Polyakov 2 , E. Wieschaus 1,3 ; 1 Department of Molecular Biology, Princeton University, 

Princeton, NJ, 2 Department of Physics, Princeton University, Princeton, NJ, 3 Howard Hughes 

Medical Institute 

During Drosophila gastrulation, the mesoderm precursor cells are internalized from the ventral 

surface of the embryo and form a ventral furrow. The invagination of these cells is accompanied 

by apical constriction, a process driven by cycles of pulsed contraction and stabilization of the 

actin-myosin network underneath the apical membrane. Previous studies have shown that in 

response to individual constriction pulses, the ventral cells elongate along their apical-basal axis 

and their nuclei move basally in an incremental manner. However, it is unclear how contractile 

forces constrained to the vicinity of the apical surface drives cell shape change along the entire 

cell length. We recently identified a cellularization mutant in which the periplasm of the syncytial 

blastoderm is never partitioned into individual cells. Intriguingly, these “acellular” embryos still 

gastrulate, and exhibit initial gastrulation movements largely reminiscent of those in normal 

cellular embryos. In particular, a contractile actin-myosin network forms on the ventral cortex 

and drives apical constriction, which leads to formation of a pseudo-ventral-furrow. The 

observation suggests that the cytoplasm can respond to cellular forces and undergo 

morphogenetic movements even in the absence of cell membrane. To test this, we investigated 

the micromechanical property of the cytoplasm and its response to constriction pulses by 

tracking injected fluorescent polystyrene microspheres. Constriction pulses were identified by 

simultaneously imaging a GFP-tagged membrane marker or myosin subunit. We found that the 

periplasm in both cellular and acellular embryos was highly viscous (~3 orders magnitude 

higher than water) but on the other hand did not show measurable elasticity over 

developmentally relevant timescales. In the cellular embryos, upon gastrulation, beads within 

the ventral cells moved basally in an incremental manner that largely correlated with pulsed 

constriction. Interestingly, beads initially localized to the apical region above the nucleus were 

never observed to flow past the nucleus upon apical constriction. Furthermore, the distance 

between beads localized in the same cell remained correlated during cell lengthening and did 

not change abruptly over time, as if the cytoplasm was a gel that underwent gradual 

deformation under force. Strikingly, this behavior does not appear to rely on the presence of cell 

membrane, as in the acellular embryos the beads within the ventral periplasm display similar 

global movements as in the cellular embryos. Based on these observations we propose that 

during Drosophila ventral furrow formation the cytoplasm responds to cellular forces in a more 

“gel-like”, rather than a “liquid-like”, manner. This “gel-like” property would allow for an


instantaneous transmittance of apically generated force throughout the entire tissue to drive cell 

shape change and their corresponding invagination. Our study also implicates that cellular 

components that determine the viscoelastic property of the cytoplasm are potential targets of 

regulation during tissue morphogenesis. 

2066 

Probing tension and dynamics in actomyosin mediated cell shape change. 

C. D. Higgins 1 , U. S. Tulu 2 , L. Gao 3 , E. Betzig 3 , D. P. Kiehart 2 , B. Goldstein 1 ; 1 Biology, UNC 

Chapel Hill, Chapel Hill, NC, 2 Biology, Duke University, Durham, NC, 3 Janelia Farm Research 

Campus, Howard Hughes Medical Institute, Ashburn, VA 

Apical constriction is a cell shape change critical for morphogenesis in diverse organisms. 

Apical constriction requires the activity of a dynamic, contractile actomyosin meshwork that is 

enriched at the apical cortex of the cell. In the 26-cell C. elegans embryo, two cells apically 

constrict and internalize. We have found previously that contractility in the apical actomyosin 

meshwork of these cells is initially uncoupled from apical constriction, and embryos deficient for 

RacGEF ced-5 and classical cadherin hmr-1 display normal myosin dynamics that fail to drive 

apical constriction. These findings suggest a model where the contractile actomyosin meshwork 

must become coupled to apical cell-cell contacts to drive cell shape change. Here, we use 

particle image velocimetry (PIV) to map the movements of the actomyosin meshwork and cellcell 

contacts over time. An unbiased PIV algorithm confirms our manual measurements 

indicating that actomyosin contractility precedes cell shape change. Furthermore, we estimate 

the tensile properties of the meshwork by cortical laser ablation and show that contractile forces 

are established prior to cell shape change. We also show that tension in the apical meshwork is 

higher in apically constricting cells than neighboring cells. To examine actomyosin dynamics 

and cell shape change with improved axial resolution, we have also taken advantage of Bessel 

beam structured plane illumination microscopy. Together, our results suggest that both apical 

actomyosin contractility and coupling to cell-cell contacts are required to facilitate cell shape 

change. 

2067 

Imaging Assays for High-throughput Quantification of Synaptic Formation and Neurite 

Dynamics in Human iPSC-derived Neurons. 

J. A. Hesley 1 , L. Chase 2 , M. Strathman 2 , O. Sirenko 1 , P. Tavormina 1 ; 1 Molecular Devices, LLC, 

Sunnyvale, CA, 2 Cellular Dynamics International, Inc., Madison, WI 

Central nervous system (CNS) disorders affect millions of people worldwide. Considerable effort 

has been made in an attempt to develop relevant in vitro human model systems for studying 

degenerative (i.e. Alzheimer’s and Parkinson’s disease) and genetic (i.e. Huntington's disease 

and muscular dystrophy) disorders as well as neurotoxicity. Induced pluripotent stem cells 

(iPSCs) of human origin provide an ideal model for scientists both within the academic and 

pharmaceutical settings. By pairing these cells with an automated high content imaging system, 

endpoint and live-cell assays can be used to determine whether pharmaceutical drug 

candidates or environmental contaminants exert neurotrophic, neuroprotective, or neurotoxic 

effects. High content imaging of neurons allows scientists to both characterize and measure 

changes in neuronal networks such as neurite number, length, and branching, as well as to 

quantitate synaptogenesis. 

iPSC-derived iCell® Neurons were treated with neurite modulating compounds at varying doses 

and images were acquired over time using the ImageXpress® Micro High Content System. 

These experiments were done using time-lapse imaging of live cells with transmitted light or 

fluorescence as well as using immunofluorescence staining of fixed cells with the neuronal


marker class III beta tubulin (TuJ1). Images of the neural networks were analyzed with 

MetaXpress® Software to identify numerous aspects of the cells and their outgrowths. The data 

shows over a doubling of neurite outgrowths over a 12 hour period without stimulation and a 

clear response to drug doses as well. In addition, emergence of GABAergic and glutamatergic 

synaptic phenotypes was seen by tracking the number of vGAT+ and vGLUT2+ puncta within a 

1-3 week post-thaw timecourse. 

These results demonstrate that human iPSC-derived neurons paired with high content imaging 

techniques provide valuable information for assessing the effects of compounds on neuronal 

networks and can be applied to various academic and pharmaceutical applications. 

2068 

Growth Hormone Regulates Survival of Primary Cultures of Embryonic Neural Precursor 

Cells. 

C. Regalado Santiago 1 , M. L. Lopez Meraz 2 , E. Juarez Aguilar 3 ; 1 Doctorado en Ciencias 

Biomedicas, Instituto de Ciencias de la Salud Universidad Veracruzana, Xalapa, Mexico, 

2 Centro de Investigaciones Cerebrales Universidad Veracruzana, Xalapa, Mexico, 

3 Departamendo de Biomedicina, Instituto de Ciencias de la Salud Universidad Veracruzana, 

Xalapa, Mexico 

During central nervous system development, cellular proliferation and cellular survival play a 

critical role. It has been suggested that these biological processes are regulated in an 

orchestrated way by numerous neurotrophic factors. In addition to the well known effects of 

growth hormone (GH) on growth and metabolism regulation, there is a growing body of 

evidence that suggest a central role of this hormone in the development and function of the 

brain. The presence of the GH and its receptor has been widely reported in different regions of 

the brain. In spite of these findings, the biological role of GH in the CNS is not completely 

understood. In order to analyze the effect of GH during CNS development, we performed 

studies on primary cultures of embryonic (~E14) neural precursor cells from striatum. In 

presence of the epidermal growth factor (EGF), this population gives rise to neurospheres of 

undifferentiated cells that can generate neurons, oligodendrocytes and glia. In a first approach, 

we showed that GH was unable to stimulate the formation of neurospheres in absence of EGF. 

Secondly, we incubated primary cultures of striatum with different concentrations of GH (1, 10, 

50, 100, 500 and 1000 ng/ml) in presence of EGF and determined the total number of 

neurospheres produced. The results showed a typical “bell shaped” curve with an increase 

since 1 ng/ml of GH and a maximal response at 10 ng/ml of hormone. In order to discriminate 

whether the increase in the number of neurospheres stimulated by GH was due to a mitogenic 

effect, primary cultures of striatum were incubated with different doses of GH and the analogue 

of thymidine BrdU (10 µM), during 24 hours. Counting of BrdU positive cells showed no 

difference between control and cultures treated with different doses of GH (10 ng/ml - 100 

ng/ml), while this hormone inhibited BrdU incorporation at doses of 500 ng/ml and 1000 ng/ml. 

Interestingly, GH stimulated a proliferative effect on primary cultures of striatum in absence of 

EGF. Next, we evaluated if GH has a neuroprotective action on the primary cultures using an 

apoptosis assay (TUNEL). Results showed a decrease in the apoptosis rate on cultures treated 

with GH at 10 ng/ml. Altogether, these results suggest that GH could be considered a survival 

factor rather than a mitogenic factor during embryonic development of the CNS at the E14 

stage.


2069 

Endothelium effects on the differentiation of iPSCs to insulin-producing cells. 

D. Talavera 1 , S. Kurtovic 1 , D. C. Dafoe 1 ; 1 Regenerative Medicine Institute, Cedars-Sinai Medical 

Center, Los Angeles, CA 

Formation of fully differentiated insulin-producing beta cells depends of the interaction between 

endothelial cells (ECs) and pre-patterned endodermal cells in vivo. It has been described that 

endothelial-derived bone morphogenetic proteins (BMPs) play an important role in this 

differentiation process. However, such EC signals are not fully characterized. Therefore, our 

objective is to study the effects of ECs in the differentiation of induced pluripotent stem cells 

(iPSCs) to insulin-producing cells and the role of EC-BMPs in this process. To accomplish this 

objective, we generated human embryoid bodies (EBs) from iPSCs. Different groups of EBs 

were cultured in a three-dimensional system of collagen-laminin gels that allowed EB-EC 

interaction for longer periods. After four weeks, the differentiated cells from EBs were analyzed 

by immunocytochemistry (ICC), immunohistochemistry (IHC), qRT-PCR, and Western blot (WB) 

looking for the expression of specific beta-cell, progenitor-cell, and islet-cell markers. To study 

the role of BMP-2/-4 and BMPR1A in the differentiation process, expression of BMP-2/-4, and 

BMPR1A as well as co-expression of C-peptide and phosphorylated Smad1/5/8 (pSmad1/5/8) 

were analyzed in all EB groups. Our results showed a significant increase in the expression of 

specific beta-cell markers (insulin, PDX-1, GLUT2, Nkx6.1, Kir6.2, SUR1, GKS, PC1/3, and 

PC2), progenitor-cell markers (Isl-1, Ngn3, Pax4, Pax6, Nkx2.2, Nkx6.1, and Ptf1a), and isletcell 

markers (GCG and STT) in those EBs co-cultured with ECs in comparison to EBs cultured 

alone. Islet-like structures composed of cells that co-expressed C-peptide and PDX-1 or Cpeptide 

and pSmad1/5/8 were identified exclusively in co-cultured EBs. In addition, higher 

expression of BMP-2, BMP-4, and BMPR1A was found in these EBs in contrast to EBs cultured 

alone. Our results indicate that ECs promote the differentiation of iPSCs to insulin-producing 

cells and that the BMP pathway activation plays a key role in this process. 

2070 

Myogenic progenitors regulating skeletal muscle homeostasis through Bmpr1a 

signaling. 

P. Huang 1 , T. J. Schulz 2 , A. Beauvais 1 , Y-H. Tseng 2 , E. Gussoni 1 ; 1 Children's Hospital Boston, 

Harvard Medical School, Boston, MA, 2 Joslin Diabetes Center, Harvard Medical School, Boston, 

MA 

Skeletal muscle maintenance and regeneration require finely coordinated interaction of multiple 

cell types. The molecular mechanism regulating this coordination is not clear. Bmp-signaling is 

suggested to balance proliferation and differentiation of myogenic cells in vitro. Here, we ablated 

expression of Bmpr1a in Myf5+ and MyoD+ progenitors in vivo (Myf5+/cre:Bmpr1af/f and 

MyoD+/cre:Bmpr1af/f ). Mutant Myf5+/cre:Bmpr1af/f mice were born runted and remained 

significantly smaller in size throughout life. Ablation of Bmpr1a resulted in increased fat 

infiltration and smaller regenerated myofibers in acutely injured Myf5+/cre:Bmpr1af/f and 

MyoD+/cre:Bmpr1af/f muscles. Interstitial myogenic cells (IMCs, Sca-1posCD45neg 

CD31posPDGFRaneg) isolated from mutant mice proliferated more slowly as compared to wild 

type cells. Following culture and differentiation in vitro, Sca-1posCD45neg cells from wildtype 

mice gave rise to myogenic and adipogenic progenitors, whereas Sca-1posCD45neg cells from 

mutant mice generated, nearly exclusively, adipocytes. Ablation of Bmpr1a expression in 

Bmpr1a f/f Sca-1posCD45neg progenitors also resulted in adipocyte differentiation, as opposed 

to myogenic cells and adipocyte when Bmpr1a is expressed. Altogether, these results show that 

Bmpr1a signaling is necessary for the commitment and proliferation of interstitial progenitors 

towards the myogenic lineage, and its presence might regulate intramuscular adipogenesis.


2071 

Human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is 

regulated by AMP-activated protein kinase. 

E-K. Kim 1 , S. Lim 1 , J-M. Park 1 , J. Seo 1 , J. Kim 2 , K. Kim 3 , S. Ryu 3 , P-G. Suh 1 ; 1 School of Nano- 

Bioscience & Chemical Engineering, UNIST, Ulsan, Korea, 2 Physiology, Pusan National 

University, Busan, Korea, 3 Division of Molecular and Life Sciences, POSTECH, Pohang, Korea 

AMP-activated protein kinase (AMPK) is an energy-sensing kinase that has recently been 

shown to regulate the differentiation of preadipocytes and osteoblasts. However, the role of 

AMPK in stem cell differentiation is largely unknown. Using in vitro culture models, the present 

study demonstrates that AMPK is a critical regulatory factor for osteogenic differentiation. We 

observed that expression and phosphorylation of AMPK were increased during osteogenesis in 

human adipose tissue–derived mesenchymal stem cells (hAMSC). To elucidate the role of 

AMPK in osteogenic differentiation, we investigated the effect of AMPK inhibition or knockdown 

on mineralization of hAMSC. Compound C, an AMPK inhibitor, reduced mineralized matrix 

deposition and suppressed the expression of osteoblast-specific genes, including alkaline 

phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). 

Knockdown of AMPK by shRNA-lentivirus infection also reduced osteogenesis. In addition, 

inhibition or knockdown of AMPK during osteogenesis inhibited ERK phosphorylation, which is 

required for osteogenesis. Interestingly, inhibition of AMPK induced adipogenic differentiation of 

hAMSC, even in osteogenic induction medium (OIM). These results provide a potential 

mechanism involving AMPK activation in osteogenic differentiation of hAMSC and suggest that 

commitment of hAMSC to osteogenic or adipogenic lineage is governed by activation or 

inhibition of AMPK, respectively. 

2072 

Thymic nurse cells express signature markers consistent with epithelial progenitor 

phenotype. 

R. V. Chilukuri 1 , V. K. Patel 1 , M. D. Samms 2 , J. C. Guyden 1 ; 1 Biology, The City College of New 

York, New York, NY, 2 The City College of New York, The City College of New York, New York, 

NY 

Thymic nurse cells (TNCs) represent a subset of thymic cortical epithelial cell with the unique 

ability to entose other cell types. Our earlier studies demonstrated that TNCs bind and 

internalize immature thymocytes bearing the αβ TCR lo CD4 + CD8 + phenotype. Subsequent 

studies showed that the binding and internalization of thymocytes by TNCs is mediated by a 

TNC-specific antigen that is recognized by the monoclonal antibody, pH91. Internalized 

thymocyte subset received life or death signals during the process of MHC restriction, resulting 

in survival and release of some thymocytes or the induction of apoposis in the thymocyte subset 

that remained trapped within specialized TNC compartments. In this study, we examined the 

ontogenesis of the TNC-specific antigen. Using confocal microscopic analyses, the onset 

expression of the pH91-specific antigen occurs as early as E11.5 stage of development and 

persists throughout embryological development as well as postnatally in mice. Further analyses 

of PH91 + cells show the co-expression of K5, K8, Foxn1 and P63. At E17.5 stage of 

development, we observe the formation of the multicellular, lympho-epithelial complexes that 

exhibit the pH91 + K8 + K5Foxn1 + P63 + phenotype. These markers have been implicated in the 

identification of thymic epithelial progenitors elsewhere. Interestingly, in a previous report, we 

showed pH91 + K5 + K8 TNCs to be strategically localized within the cortico-medullary junction of 

the thymus, thus possibly allowing for differentiation of K8 + cells to proceed toward cortex and 

K5 + cells to the medulla. By E18.5 stage of development, the pH91 + K8 + K5 + Foxn1 + P63 + TNCs 

express elevated levels of MHC-class II antigens on their cell surfaces. In addition, confocal


analyses show that in post-natal mice 26% of pH91 + TNCs also express K8 and K5, whereas 

76% of the K8 + K5 + cells in the thymus are TNCs. Taken together our data suggest that 

pH91 + K8 + K5 + Foxn1 + P63 + TNCs have thymic epithelial progenitor potential. 

2073 

The Role of Endogenous FGF-2 in heterogeneity and pluripotency of human embryonic 

stem cells. 

K. Matulka 1 , L. Eiselleova 1 , M. Kunova 1 , A. Hampl 2,3 , Y-M. Sun 1 , P. Dvorak 1,3 ; 1 Department of 

Biology, Masaryk University, Brno, Czech Republic, 2 Department of Histology, Masaryk 

University, Brno, Czech Republic, 3 st. Anne´s University Hospital, International Clinical 

Research Center, Brno, Czech Republic 

Exogenous fibroblast growth factor-2 (FGF-2) is one of crucial supplements in media for human 

embryonic stem (ES) cells. In our systematic work, we investigate its roles in plating efficiency 

and survival rate under stressed conditions. We show that exogenous FGF-2 enhances the 

expression of high molecular mass (HMM) isoforms of FGF-2, which have been implicated in 

promoting proliferation and survival in most cell types. Stably over expression of HMM FGF-2 

isoforms has been shown to link to malignant phenotypes. 

We found that all isoforms of FGF-2 are expressed in undifferentiated human ES cells, whereas 

the low molecular mass FGF-2 retains its expression when cells undergo differentiation. The 

withdrawal of exogenous FGF-2 results in cell differentiation depending upon the size of 

colonies. Our data suggest that exogenous FGF-2 is involved in the early establishment of 

heterogeneous population. Furthermore, FGF-2 knockdown human ES cells using shRNA are 

predisposed to differentiate, suggesting that FGF2 plays a pivotal role in pluripotency. 

We hypothesise that FGF-2 signalling maintains the undifferentiated growth of hESCs. This 

action is enhanced by exogenous FGF-2. Both, extrinsic and intrinsic, activities may synergize 

to regulate transition between self-renewal and differentiation of hESCs. 

2074 

Mutant frequency increases with the length of cultivation in human embryonic stem cells. 

M. Kruta 1 , L. Balek 1 , R. Hejnova 1 , M. Seneklova 1 , A. Hampl 2 , P. Dvorak 1 , V. Rotrekl 1 ; 

1 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 

2 Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, 

Czech Republic 

Human embryonic stem cells (hESCs) derived from blastocyst of early embryo are capable of 

differentiation into all three germ layers and can produce all cell types of human body. Although 

hESCs immortality and their differenciation potential make them promising candidates to 

become powerful tool of regenerative medicine, their long-term propagation in vitro induces 

accumulation of genetic changes as demonstrated by karyotyping, SNP analysis or analysis of 

copy number variation. These changes might render the cells useless in regenerative medicine. 

We have shown one of the mechanisms contributing to these changes, Our data demonstrate 

that decreased efficiency of base excision repair (BER) in late passage hESCs contributes to 

the genome instability. Decrease of apurinic/apyrimidinic endonuclease APE1, a key enzyme of 

BER, with increasing time in culture leads to changes in DNA repair dynamics as well as to 

changes in DNA damage signaling. To dissect the role of such changes in DNA repair and 

damage signaling during prolonged hESC cultivation we have designed specific mutation assay. 

It is a method based on mutagenesis of hypoxanthine-phosphoribosyltransferase (HPRT) gene. 

Despite the current thinking based on computer models, that time in culture does not increase 

the mutagenic pressure, we have shown significantly increased both spontaneous and IRinduced 

frequency of mutants in late passage hESCs in comparison with early passage.


However the higher doses of IR (above 1Gy) induce similar effect in early and late passage, 

suggesting that the most dramatic effect happens under non stressing conditions. Taken 

together, our results suggest that the changes in the APE1 level during long-term cultivation of 

hESCs play an important role in their response to acquired DNA damage and that the long-term 

propagation increases the mutagenic burden and that these events present a chain of events 

from DNA repair failure all the way to genome instability, presented as increased mutation 

burden. 

Supported by Ministry of Education, Youth and Sports the Czech Republic (LC06077) and by 

the MZ CR (grant NS10237-3) 

2075 

Primary Cilia of Proliferating Cells in Rodent Dorsal Root Ganglia. 

M. V. Coronel 1 , D. Hao 1 , W. Huang 1 , H. D. Schwark 1 ; 1 Biology, University of North Texas, 

Denton, TX 

Primary cilia are non-motile, microtubule based organelles that arise from the membrane of 

most vertebrate cells. Mutations in primary cilia genes can lead to severe developmental 

abnormalities. In the nervous system these include hydrocephaly, neural tube patterning defects 

and sensory deficits that may include anosmia, hearing loss, retinal degeneration and impaired 

somatic sensory inputs. Many of the developmental defects seen in primary cilia mutants have 

been attributed to impaired sonic hedgehog signaling. The mitogen sonic hedgehog is essential 

in development of neural crest-derived structures such as the dorsal root ganglia (DRG): 

disruption of sonic hedgehog signaling results in failure of primary sensory neurons to develop. 

This essential developmental role played by primary cilia may continue into adulthood. 

Each sensory neuron in the DRG is tightly ensheathed by surrounding satellite glial cells. These 

cells arise around E9.5 (neurons) and E11 (glia) from multipotent progenitors derived from the 

neural crest. Neurons, and perhaps satellite glial cells, continue to be added to the postnatal 

DRG, but the origin of these new cells is unknown. It seems likely that primary cilia play a role in 

postnatal proliferation and maintenance of DRG homeostasis for the following reasons: primary 

cilia play an active role during the cell cycle; proliferation of stem cell niches is aberrant in 

primary cilia mutants; peripheral nerve injury induces sonic hedgehog expression in the DRG; 

and patients with abnormal primary cilia have sensory deficits. 

We have begun our investigation into the role of primary cilia in the developing and adult rodent 

DRG by determining the incidence and lengths of the cilia. In the rat DRG, primary cilia 

decrease in length from P3 (2.6 um) to P14 (2.1 um) to adult (1.9 um). A similar trend of 

decreasing cilium length with development is seen in proliferating satellite glial cells labeled by 

antibodies to the cell cycle marker Ki67. At P1 the average cilium length is 1.4 um, and at P7 

the average length is 1.2 um. The percentage of ciliated proliferating cells also decreased 

across these ages, from 35.5% to 28.5%. Finally, we also identified primary cilia in cells 

expressing the transcription factor Sox2, which is thought to maintain certain cell types in an 

undifferentiated state. Cilia characteristics in this small sample of cells were not quantified. The 

decreasing lengths and incidence of primary cilia with development may underlie a decreased 

sensitivity to sonic hedgehog signaling.

2076 

HNF-4α control of pancreatic endocrine differentiation from pluripotent stem cells. 

E. C. Moorefield 1 , X. Guan 1 , M. E. Furth 1 , C. E. Bishop 1 ; 1 Wake Forest University, Winston- 

Salem, NC 


The HNF4A gene codes for hepatocyte nuclear factor (HNF)-4α, which has an important role in 

pancreatic development and maintenance of β-cell function. Haplo-insufficiency of this 

transcription factor causes a form of monogenic type 2 diabetes (T2D), maturity-onset diabetes 

of the young 1 (MODY1). Recently, the advancement of embryonic stem (ES) cell and induced 

pluripotent stem (iPS) cell technology makes study of the genetic basis of the disease 

achievable. Pluripotent stem cells can be differentiated into insulin producing β-cells using stepwise 

protocols that recapitulate signaling pathways necessary for endocrine pancreas 

development in vivo. Growth factors and small molecules are applied in a step-wise fashion to 

replicate the stages in β-cell development which include endoderm formation, pancreas 

specification, endocrine specification and β-cell maturation. By applying this protocol to normal 

human ES cells we have shown that HNF-4α is expressed at high levels during pancreas 

specification and continues to be expressed at lower levels in throughout the differentiation 

process. To investigate the effect of HNF-4α deficiency on pancreatic β-cell development and 

function we have examined two systems. First, we have generated iPS cells from MODY1 

patient fibroblasts using a polycistronic human lentivirus carrying Oct4, Sox2, Klf4 and c-myc. In 

addition, we have created dox-inducible HNF-4α knockdown hES cells. The stemness of both 

systems has been confirmed by analysis of a panel of pluripotency markers. However, the 

MODY1 iPS showed a large clone to clone variation in differentiation capability toward insulinproducing 

cells. On the contrary, we have confirmed that HNF-4α expression can be efficiently 

knocked down throughout in vitro differentiation to endocrine pancreas by shRNA delivery. 

Thus, the HNF-4α KD hES system will allow us to elucidate the impact of genetic variation on 

the development and function of pancreatic β-cells which may lead to identification of novel 

therapeutic targets for treatment of T2D. 

2077 

RNAi screen of predicted E3 ubiquitin ligases that may mark MEX-3 for degradation in 

the Caenorhabditis elegans embryo. 

A. Lin 1 , K. Reichard 1 , A. Desai 2 , P. Heng 2 , K. Schwartz 2 , N. Huang 1 ; 1 Biology, Colorado College, 

Colorado Springs, CO, 2 Colorado College, Colorado Springs, CO 

Early embryonic development in C. elegans depends on precise timing of division and 

differentiation, which requires precise regulation of maternally supplied cell fate determinants. 

MEX-3, a maternally supplied RNA binding protein, is present throughout the oocyte, 1-cell, and 

2-cell embryo. By the 4-cell stage, MEX-3 becomes depleted in the posterior blastomeres. By 

binding to the 3’ UTR of pal-1 mRNA, MEX-3 prevents translation of PAL-1 in anterior 

blastomeres. PAL-1 is a homeodomain transcription factor that promotes posterior 

development, including muscle development. In mex-3 mutants, PAL-1 is expressed throughout 

the early embryo, leading to muscle excess and embryonic lethality. After the 4-cell stage of 

embryogenesis, MEX-3 is rapidly broken down. The E3 ubiquitin ligase component of the 

ubiquitin-proteosome system determines protein specificity of ubiquitination, which leads to 

degradation. There are numerous predicted E3 ubiquitin ligases in the C. elegans genome. 

The goal of this project is to use RNA inhibition (RNAi) to knock out predicted E3 ubiquitin 

ligases, and determine which one(s) mark MEX-3 for degradation in the C. elegans embryo. 

Genes encoding predicted E3 ubiquitin ligases that were previously shown to be maternally 

expressed and/or that produced embryonic lethality in previous RNAi experiments were chosen


as candidate genes to knock out. We first determined if knockout of the candidate genes 

resulted in embryonic lethality higher than 20%. Then, fluorescence microscopy was used to 

determine whether RNAi effected the degradation of a GFP::MEX-3 fusion protein. If an E3 

ubiquitin ligase contributing to MEX-3 degradation was successfully knocked out, GFP::MEX-3 

would remain present throughout the embryo. RNAi was induced by feeding young adults with 

bacteria expressing dsRNA, or by directly injecting the dsRNA. To date, numerous predicted 

HECT-domain, U-box, and monomeric RING finger E3 ubiquitin ligases have been knocked out 

by RNAi. Depletion of five of these genes resulted in high levels of embryonic lethality, with two 

resulting in abnormal patterns of GFP::MEX-3 expression. However, mis-expression of MEX-3 

does not appear to be the primary defect in these RNAi embryos. The screen is being continued 

with additional predicted E3 ubiquitin ligases. 

2078 

Osteoblast response to a nanostructured titanium surface. 

M. M. Beloti 1 , R. B. Kato 1 , A. Nanci 2 , P. T. de Oliveira 1 , A. L. Rosa 1 ; 1 School of Dentistry of 

Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil, 2 Faculté de Médecine Dentaire, 

Université de Montréal, Canada 

Titanium (Ti) surface nanotopography obtained by chemical deoxidation and controlled 

reoxidation affects early events of osteoblast phenotype development in cultures derived from 

rat calvaria. The aim of our study was to evaluate the effect of the nanostructured Ti surface on 

the responses of human osteoblastic cells. Machined Ti discs, 12 mm in diameter, were 

chemically treated with H2SO4/H2O2 for 2 hours. Osteoblastic cells were obtained by enzymatic 

digestion of human alveolar bone and cultured under osteogenic condition until subconfluence. 

First passage cells were cultured (2�10 4 cells/disc) on nanostructured and machined Ti discs for 

periods of up to 21 days. For cell attachment assay, adherent cells were counted at 24 hours 

and data were expressed as a percentage of the initial cell number. For proliferation, cells were 

counted at 1 and 7 days and data were expressed as doubling time in hours. At day 7, cell 

viability was evaluated using trypan blue and expressed as a percentage of viable cells. At days 

1, 3, and 7, cell morphology was evaluated with phalloidin and DAPI; alkaline phosphatase 

(ALP) labeling was performed using a primary mouse anti-human ALP antibody. Extracellular 

matrix mineralization was detected by Alizarin red staining at 21 days and data were expressed 

as a percentage of the total disc area. Quantitative data were compared by Mann-Whitney test 

(n=3). There were no statistically significant differences between nanostructured and machined 

Ti in terms of cell attachment (34.7±6.4 and 40.3±10.5; p=0.70), proliferation (50.4±2.7 and 

48.1±2.1; p=0.28), and viability (92.1±2.8 and 93.1±1; p=0.83). At day 1, cells were spread and 

exhibited an elongated polygonal shape on machined Ti whereas on nanostructured Ti they 

were mostly stellate-shaped. At day 7, both Ti surfaces exhibited areas of cell multilayering and 

cells were predominantly elongated. ALP positive cells were observed at all time points with a 

similar labeling pattern on both Ti surfaces. It was noticed five times more extracellular matrix 

mineralization (2.7±0.2 and 15.2±4.6; p=0.01) on nanostructured compared with machined Ti. In 

conclusion, our results indicate that nanotopography induces a significant increase in the late 

key event of in vitro osteogenesis, i.e.: extracellular matrix mineralization and this surface 

modification may, therefore, be considered a useful strategy to accelerate/enhance the process 

of Ti implant osseointegration. 

Acknowledgements: FAPESP (Grant # 2010/18395-3, 2010/19280-5)

2079 

Effects of hedgehog signaling on human differentiated osteoblasts. 

F. S. Oliveira 1 , G. B. Junqueira 1 , M. M. Beloti 1 , A. L. Rosa 1 ; 1 School of Dentistry of Ribeirao 

Preto, University of Sao Paulo, Ribeirao Preto, Brazil 


Hedgehog (Hh) signaling is involved in many developmental processes including osteoblast 

differentiation of mesenchymal stem cells. Here, we aimed at determining the effect of Hh 

signaling modulation on phenotype expression of differentiated osteoblasts. Cells harvested 

from human dental alveolar bone were cultured in osteogenic media under four conditions: nonsupplemented 

(control), supplemented with the Hh agonist purmorphamine (2 �M), 

supplemented with the Hh antagonist KAAD-cyclopamine (100 nM), and supplemented with 

both purmorphamine and KAAD-cyclopamine. Hh signaling was evaluated by gene expression 

of Gli1 at day 7. Osteoblast phenotype expression was evaluated by activity and gene 

expression of alkaline phosphatase (ALP) at day 7, and extracellular matrix mineralization at 

day 21. Data (n=3) were compared by Kruskall-Wallis test followed by Fisher’s test when 

appropriated. Hh pathway was stimulated by purmorphamine as revealed by an increase of 

almost four times in the expression of Gli1 compared to control (p=0.001). This increase was 

only partially reversed by KAAD-cyclopamine (p=0.05) that by itself did not present any effect 

when compared to control (p>0.05). Despite this, ALP activity (p>0.05), ALP gene expression 

(p>0.05) and matrix mineralization (p>0.05) were not modified by any culture condition. These 

results suggest that the modulation of Hh signaling does not affect the phenotypic expression of 

human differentiated osteoblasts. 

Acknowledgments: FAPESP and CNPq 

2080 

Bavachinin A modulates the differentiation of hADMSCs on adipogenesis and 

osteogenesis. 

H-J. Jang 1,2 , S. Lim 1 , J-M. Kim 1,2 , S. Ryu 2 , P-G. Suh 1 ; 1 School of Nano-Bioscience and Chemical 

Engineering, UNIST, Ulsan Metropolitan, Korea, 2 Division of Molecular and Life Science, 

POSTECH, POHANG, Korea 

Human mesenchymal stem cells are an attractive model not only to investigate molecular 

mechanisms of cell growth and differentiation under normal and pathological conditions but also 

to use cell-based therapies for regenerative medicine. Therefore, it is important to understand 

how their differentiation is regulated. We screened the molecules which could regulate 

adipogenesis using natural compound library. As a screening result, we found that bavachinin A 

enhanced adipogenesis of human adipose-derived mesenchymal stem cells(hADMSCs). 

Bavachinin A also increased the mRNA level of adipocyte markers, such as CCAAT-enhancerbinding 

proteins α, peroxisome proliferator-activated receptor γ(PPAR-γ) and adipocyte fatty 

acid–binding protein during adipocyte differentiation. Furthermore, PPARγ activity was 

increased by treatment of bavachinin A. To evaluate the effect of bavachinin A in vivo, we used 

implanted de novo adipose tissue formation system. It showed that intraperitoneal 

administration of bavachinin A in mice, implanted with mass of 3t3-f442A cells, increased de 

novo adipogenesis and weight of inborn adipose tissues. Whereas bavachinin A inhibited 

osteogenesis of hADMSCs. Our results suggest that bavachinin A is a potent activator of 

adipogenesis and a inhibitor of osteogenesis via activation of PPAR-γ.

2081 

Directed Stem Cell Differentiation Using PEG/α-CD-derived Biomaterials. 

A. Singh 1 , J. Zhan 1 , J. Elisseeff 1,2 ; 1 BME, Johns Hopkins University, Baltimore, MD, 

2 Ophthalmology, Wilmer Eye Institute, Baltimore, MD 


Adult stem cells (ASCs), e.g., mesenchymal and adipose-derived (MSCs and ADSCs) hold 

great promise in tissue engineering because of their proliferative capacity and ability to 

differentiate into several phenotypes, and ultimately built new tissues. However, to enable 

translation of stem cell therapies, there is a significant need to generate materials that can be 

used to study fundamentals in biology and modulate or enhance differentiation. Poly(ethylene 

glycol) (PEG)-based hydrogels are frequently utilized to encapsulate cells in a 3D environment. 

Unfortunately, PEG based biomaterials lack functionality to incorporate multiple chemical and 

biological moieties as signals for stem cells to probe behavior and guide development. 

Conventional methods to modify PEG hydrogels include copolymerization and chain extension, 

which leads to changes in mechanical properties of the material. Therefore, we developed an 

advanced multifunctional hydrogel by combining PEG- and α-Cyclodextrin (α-CD). α-CD is a six 

membered oligosaccharide ring, which threads onto the PEG chains. [1] The hydroxyl groups of 

α-CD provide necessary functional sites to introduce various moieties, such as hydrophobic 

groups (CH3), hydrophilic groups (PO4 - ) and integrin-binding peptide RGD by chemical 

modifications. This simple strategy provides us a unique ability to independently control 

chemical functionality, mechanical and bioadhesive properties of the hydrogels to probe stem 

cell behavior. 

ASCs were either seeded onto or encapsulated in UV-photopolymerizable PEG-α-CD hydrogels 

with varying stiffness (0.5 kPa to 30 kPa), adhesive peptide concentrations (0% to 10% w/v) and 

different functional groups. In an initial experiment, the enhanced chondrogenic differentiation of 

gMSCs in 10% PEGDA/1% α-CD hydrogel was evident from increased gene expression for Col 

II and Aggrecan, and Safranin O staining for GAG deposition. At the similar mechanical 

properties of the hydrogel, introducing a hydrophobic group (CH3) in α-CD enhanced adipogenic 

differentiation, while a hydrophilic group (PO4 - ) increased osteogenic differentiation of hADSCs. 

We also found that cell spreading and morphology are dependent on both stiffness and 

concentrations of integrin-binding peptide. On the stiffer substrate, hMSCs adopted elongated 

morphology at all concentrations of RGD. Interestingly, on the softer substrate hMSCs adopted 

elongated morphology at higher concentrations of RGD. RT-PCR experiments showed that 

softer matrix upregulates gene-expression markers for adipogenesis at both lower and higher 

cell-binding peptide concentrations, while moderately stiffer substrate with lower cell-binding 

peptide concentration upregulates markers for myogenesis. The stiffer substrate enhances both 

chondrogenesis and osteogenesis at higher concentrations of integrin-binding peptide. This 

indicates that both substrate-stiffness[2] and concentration of cell-binding peptide sites dictate 

stem cell differentiation. In summary, this biomaterial enables us to probe stem cell behavior, 

such as proliferation and lineage specific differentiation by independently controlling the 

functionality, mechanical and bioadhesive properties of the material. 

References: 

1. A. Harada, J. Li, M. Kamachi, Nature, 1992, 356, 325-327. 

2. A.J. Engler, S. Sen, H.L. Sweeny, D.E. Discher, Cell, 2006, 126(4), 677-689. 

Acknowledgements: Maryland Stem Cell (TEDCO) fellowship, Dr. O. Manuel Uy, and Cindy 

Berlinicke, and JHE group.

Cell Biology of the Neuron 

2082 

PINK1 and Parkin Target Miro for Phosphorylation and Degradation to Arrest 

Mitochondrial Motility. 

X. Wang 1 , D. Winter 2 , G. Ashrafi 2 , J. Schlehe 3 , Y. Wong 4 , D. Selkoe 3 , S. Rice 4 , J. Steen 2 , M. 

LaVoie 3 , T. Schwarz 3 ; 1 Stanford University/Harvard Medical School, Boston, MA, 2 Children's 

Hospital Boston, MA, 3 Harvard Medical School, Boston, MA, 4 Northwestern University, 

Evanston, IL 


Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial 

movement, distribution, and clearance. We report here that two Parkinson's disease proteins, 

the Ser/Thr-kinase PINK1 and ubiquitin-ligase Parkin, participate in this regulation by arresting 

mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary 

motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation 

of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of 

Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial 

movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their 

clearance. PINK1 has been shown to act upstream of Parkin but the mechanism corresponding 

to this relationship has not been known. We propose that PINK1 phosphorylation of substrates 

is necessary for the subsequent action of Parkin and the proteasome. 

2083 

Essential Role of a Conserved Homophilic Interaction Domain of SYD-2/Liprin-alpha 

Protein in Presynaptic Assembly in C.elegans. 

H. Taru 1 ; 1 Creative Research Institution, Hokkaido University, Sapporo, Japan 

Background 

Synapses are asymmetric structures that are specialized for neuronal signal transduction. A 

unique set of proteins is present at the presynaptic active zone, which is a core structure 

essential for neurotransmitter release. C. elegans SYD-2, a Liprin-α family protein, is localized 

at the center of presynaptic active zones and acts together with a GAP protein SYD-1 to 

promote active zone organization in various types of neurons. Liprin-α proteins are scaffolding 

proteins and composed of coiled-coil rich regions in the N-terminal halves, which mediate 

interactions with adapter proteins at the presynaptic active zone, and three SAM domains in the 

C-termini, which bind proteins such as LAR receptor tyrosine phosphatase. 

Objective 

To address the molecular mechanism by which SYD-2 activity in presynaptic assembly is 

regulated, core functional domains of SYD-2 were dissected out by transgenic structure-function 

analyses and characterized biochemically. 

Methods 

A series of SYD-2 deletion constructs were transgenically expressed in C.elegans, and were 

analyzed for their abilities to rescue presynaptic assembly defects in HSN neuron in loss-offunction 

mutants of syd-2 or syd-1. Recovery of presynaptic assembly was evaluated by 

restored egg-laying behavior and localization of fluorescent tagged presynaptic protein reporters 

such as synaptobrevin::YFP. Biochemical property of recombinant SYD-2 coiled-coil region was 

analyzed by pull-down, crosslinking and gel-filtration assay. Clustering of human Liprin-α was 

analyzed by Blue Native-PAGE and live-cell imaging of GFP in cultured cells. 

Results 

Transgenic structure-function studies of SYD-2 revealed that the N-terminal half of SYD-2 is 

minimally required for rescuing presynaptic defects in syd-2 mutants. A highly conserved short


coiled-coil segment named Liprin Homology 1 (LH1) domain is both necessary and sufficient to 

suppress presynaptic defects in syd-1 mutants. Biochemical analyses revealed that the LH1 

domains of SYD-2 and Liprin-α form dimer in vitro. The role of SYD-2 LH1 domain in 

presynaptic assembly can be partially complemented by artificial dimerization. In human Liprin-α 

deletion of the LH1 domain impaired proper clustering and/or complex formation in cells. 

Conclusions 

These findings suggest a model by which the self-assembly of SYD-2/Liprin-α proteins mediated 

by the evolutionally conserved coiled-coil LH1 domain is one of the key steps to the 

accumulation of presynaptic components at nascent synaptic junctions. 

2084 

Quantum-dot based presynaptic probe reveals dynamic modulation of synaptic vesicle 

trafficking behaviors by synaptic activity. 

S. Lee 1 , S. Chang 1 ; 1 Biomedical science, SNU, Seoul, Korea 

Presynaptic contribution during synaptic plasticity is a matter of debate. A small nature of 

presynaptic bouton and a lack of suitable technique, however, hamper detailed analysis of 

synaptic vesicle (SV) behaviors at synapse. Here, we created a quantum dot-based presynaptic 

probe and characterized dynamic behaviors of individual SVs in CA3-CA1 synapse of cultured 

neurons. As previously reported, SVs show multiple exchanges between neighboring boutons. 

Actin disruption induces a dramatic decrease in diffusive behaviors of SVs at synapse while 

microtubule disruption only reduce extrasynaptic mobility. Chemical LTP induces NMDA 

receptor-dependent increase in synaptic mobility of SVs and in inter-boutonal traffickings of SVs 

between neighboring boutons, causing a bias to incorporation of SVs into synapses. NMDA 

induced-LTD decreased diffusion coefficient of SVs at synapse. Together, our results suggest 

that a dynamic modulation of SV diffusive behaviors could be the one of key mechanisms of 

regulating presynaptic efficacy during presynaptically expressed long-term synaptic plasticity. 

2085 

Characterization of the DOPA transporter in an in vitro cellular model of dopaminergic 

neurons. 

E. Moura 1,2 , E. Magalhães Ribeiro 1 , E. Silva 3,4 , M. P. Serrão 1 , J. Guimarães 5 , M. A. Vieira 

Coelho 1,2 ; 1 Pharmacology and Therapeutics, Faculty of Medicine University of Porto, Porto, 

Portugal, 2 Neuropharmacology, Institute for Molecular and Cell Biology, Porto, Portugal, 

3 Laboratório de Biologia Celular e Molecular, Faculty of Medicine University of Porto, Porto, 

Portugal, 4 Departamento de Stress in Animals, Instituto de Biologia Celular e Molecular, Porto, 

Portugal, Portugal, 5 Neurology Department, Faculty of Medicine University of Porto & Hospital 

de São João 

L-3,4-dihydroxyphenylalanine (DOPA) is the immediate product of the rate-limiting step in 

catecholamine biosynthesis and the precursor of all the endogenous catecholamines Treatment 

with DOPA remains to date the most effective treatment of the slowness of movement, 

increased muscle tone, and tremor that are typical of Parkinson’s disease (PD). The human 

neuroblastoma cell line SH-SY5Y has been widely used as a cellular model of dopaminergic 

neurons for PD research. In this study we investigated the transporter involved in DOPA uptake 

in SH-SY5Y cells. 

DOPA levels in cells were evaluated by high performance liquid chromatography with 

electrochemical detection. Results are presented as arithmetic mean ± standard error mean. 

SH-SY5Y cells take up DOPA in a time dependent (linear until 6 minutes) and concentration 

dependent (2.5-2500 µM) manner. Non-linear analysis of the saturation curves revealed for 

DOPA a KM (in µM) of 570±97 and a Vmax (in nmol/mg protein/6 min) of 611±34. The uptake of


DOPA (2.5 µM) was reduced by the inhibitor of the L-type amino acid transporters 2aminobicyclo-(2,2,1)-heptane-2-carboxylic 

acid (BCH, 0.1-1000 µM) (IC50 = 47 ± 2 nM; Emax = 

24 ± 10 % control uptake) and neutral amino acids (1mM), but no by the inhibitor of the A-type 

amino acid transporters N-(methylamino)-isobutyric acid (MeAIB, 0.1-1000 µM) nor by the acidic 

and basic amino acids (1mM). DOPA uptake (2.5 µM) was unaltered by lowering the pH from 

7.4 to 6.2. In the absence of Na + there was a 20% reduction in the Vmax values for DOPA 

uptake. Accumulation of DOPA in SH-SY5Y cells was largely inhibited by the L-isomers of the 

small and large neutral amino acids (alanine, serine, threonine, cysteine, leucine, isoleucine, 

phenylalanine, methionine, and tyrosine), histidine, tryptophan, valine, asparagine and 

glutamine. Whereas the amino acids glycine, proline and the basic amino acid arginine also 

produced an inhibition of DOPA uptake, albeit minor, the basic amino acids lysine and cystine, 

and acidic amino acids aspartate and glutamate did not inhibit the uptake of L-DOPA. 

In conclusion, DOPA uptake in SH-SY5Y cells was sensitive to inhibition by BCH, but not to 

MeAIB, and was more sensitive to inhibition by neutral than to basic and acidic amino acids. 

Although most of DOPA was entering the cells in a Na + -independent manner, a minor 

component of DOPA uptake (25%) was found to require extracellular Na + . In general, these 

findings support the view that DOPA may be transported by systems B 0 (Na + -dependent) and L 

(Na + -independent). The fraction of DOPA that is handled through system L is through a highaffinity 

(KM values in the micromolar range) and pH-insensitive transport, which are 

characteristic of the LAT1. 

2086 

Deciphering the molecular interaction between sodium channel Nav1.8 and ankyrin G. 

F. Castets 1,2 , A. Montersino 1,2 , M-P. Fache 1,2 , B. Dargent 1,2 ; 1 INSERM U641, Marseille, France, 

2 Aix-Marseille University, Marseille, France 

In the central nervous system (CNS), the accumulation of the voltage-gated sodium channels, 

Nav1 (Nav1-2 and Nav1.6) at the axon initial segment (AIS) results from a direct interaction 

between the ankyrin-binding motif of Nav1 and ankyrin G. This interaction is regulated in a 

phospho-dependent fashion by protein caseine kinase 2 (CK2). The peripheral channel Nav1.8 

is predominantly expressed in dorsal root ganglion neurons. However, Nav1.8 is ectopically 

expressed in Purkinje cells in EAE animal model and in human multiple sclerosis. Interestingly, 

this channel possesses a motif close to the conserved ankyrin-binding motif found in the Nav1 

expressed in CNS. 

This study was aimed at deciphering the molecular interaction between Nav1.8 and ankyrin G. 

We observed by GST pull-downs that Nav1.8 associates with endogenous ankyrin G from rat 

brain membranes. Using surface plasmon resonance, we found that Nav1.8 constitutively 

interacts with ankyrin G 190 kDa in contrast to Nav1.2, which requires the phosphorylation of 

CK2 to bind to ankyrin G 190 kDa. Furthermore when the ankyrin-binding motif of Nav1.8 was 

fused to the somatodendritic channel Kv2.1, the resulting chimeric protein was not only 

clustered at the AIS of transfected primary hippocampal neurons but also acted as a dominant 

negative displacing the endogenous Nav1 from AIS of hippocampal neurons. 

Altogether, these results indicate that Nav1.8 carries all the information required for clustering at 

the AIS and that its accumulation is not regulated by protein kinase CK2, unlike CNS Nav1. Our 

work shows a new kind of interaction between sodium channel and the scaffold protein ankyrin 

G, which is likely to contribute to the pathological aspects of multiple sclerosis.


2087 

N-cadherin induces synapse formation in heterologous cultures of brainstem cholinergic 

neurons and CHO cells. 

R. Flannery 1 , J. L. Brusés 2 ; 1 Department of Anatomy and Cell Biology, University of Kansas 

School of Medicine, Kansas City, KS, 2 Department of Anatomy and Cell Biology & Department 

of Psychiatry and Behavioral Sciences, University of Kansas School of Medicine, Kansas City, 

KS 

N-cadherin is a cell adhesion molecule that contributes to synaptic differentiation, synaptic 

vesicle recycling, and synaptic plasticity. It promotes the contact between pre and postsynaptic 

membranes and acts co-operatively with synaptogenic proteins to induce synapse development. 

Much of what is known about the regulation of synapse formation and function by N-cadherin 

has been elucidated from experiments with glutamatergic synapses. Here we have used 

heterologous cell cultures with primary cholinergic neurons and transfected Chinese Hamster 

Ovary (CHO) cells to determine whether N- cadherin is sufficient to initiate formation of 

functional cholinergic synapses. Central cholinergic neurons were cultured from brainstem 

nuclei explants of transgenic mice expressing EGFP under the control of choline 

acetyltransferase (ChAT) transcriptional regulatory elements, and cultured with CHO cells 

(which do not express endogenous N-cadherin) transfected with N-cadherin or a fluorescent 

protein (FP) as control. CHO cells were co-transfected with nicotinic acetylcholine receptor 

(nAChR) subunits alpha 3 and beta 4 forming functional AChRs that are activated by 

acetylcholine. Immunostaining for synapsin I and SV2 demonstrated that synaptic vesicle 

proteins accumulate at contact sites between cholinergic axons and N-cadherin expressing 

CHO cells. There was a ~3-fold increase in the total area of SV2 immunolabeling and a ~2-fold 

increase in the amount of synapsin I immuno-puncta accumulation per axon length on Ncadherin 

expressing CHO cells as compared to FP expressing CHO cells. N-cadherin 

expression in CHO cells in contact with axons extending from cholinergic neurons resulted in 

the appearance of excitatory postsynaptic potentials (EPSPs), as measured by whole cell 

recordings of the CHO transfected cells. While 67% of N-cadherin transfected CHO cells 

displayed synaptic events (6 out of 9 cells) only 11% of control cells (1 out of 9 cells) showed 

activity. The mean EPSP amplitude for heterologous synapses was 66.7 +/- 13.0 pA (227.7 +/- 

36.7 pA after normalizing current density). While the mean decay time constant for heterologous 

cholinergic synapses was 10.1 +/- 1.0 msec, events in one cell had a biphasic decay with time 

constants tau1 = 42.4 +/- 10.4 msec and tau2 = 5.2 +/- 0.6 msec, suggesting the presence of 

immature synaptic release sites. Miniature EPSPs were not detected in control (n=7) or Ncadherin 

transfected CHO cells (n=12), which we attribute to a low release probability intrinsic to 

brainstem cholinergic neurons. These results indicate that expression of N-cadherin in nonneuronal 

cells is sufficient to initiate formation of functional synapses with brainstem cholinergic 

neurons; however, complete development or maturation of release sites may require expression 

of additional synaptogenic proteins. 

2088 

Fast-scanning AFM analysis of agonist-induced height transition of the NMDA receptor. 

Y. Suzuki 1 , T. A. Goetze 2 , D. Stroebel 3 , S. H. Yoshimura 1 , P. Paoletti 3 , R. M. Henderson 2 , K. 

Takeyasu 1 , J. M. Edwardson 2 ; 1 Laboratory of Plasma Membrane and Nuclear Signaling, 

Graduate School of Biostudies, Kyoto University, Kyoto, Japan, 2 Department of Pharmacology, 

University of Cambridge, Cambridge, United Kingdom, 3 Institut de Biologie de l'Ecole Normale 

Supérieure (IBENS), Ecole Normale Supérieure, Paris, France 

NMDA receptors (NMDARs) are widely expressed in the central nervous system, and play a 

major role in excitatory synaptic transmission. Activation of NMDARs requires the binding of two


agonists, glutamate and glycine (or D-serine). Here, we used fast-scanning atomic force 

microscopy (AFM) to analyze agonist-induced structural changes in the extracellular domain of 

the heterotetrameric GluN1/GluN2A receptor integrated into lipid bilayers. In the absence of 

agonist, the frequency distribution of heights of the receptors above the bilayer surface had two 

peaks, at 3.5 nm and 8.5 nm. The taller particles were specifically recognized by an anti-GluN1 

antibody that recognizes the S2 segment of the agonist-binding domain, suggesting that these 

particles represent the extracellular region of the receptor. Receptor activation was triggered by 

UV photolysis of caged glutamate. Release of glutamate into a glycine-containing buffer 

resulted in a significant structural change, with the height of the receptor decreasing from 8.5 

nm to 7.2 nm. This height shift did not occur in the absence of glycine or in the presence of the 

selective NMDAR antagonist, D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5). Hence, the 

height of the extracellular domain of the receptor is reduced upon agonist activation. Such a 

conformational change could affect interactions that NMDARs engage with synaptic partners. 

2089 

Potential of neuronal progenitor stem cell therapy for Alzheimer's disease. 

D. Raman 1 , M. Choudhary 1 , J. P. Cleary 1 , R. R. Punzalan 1 , C. V. Sharma 1 , M. V. Sharma 1 , R. 

Balmiki 2 , N. Chopra 2 , D. K. Lahiri 2 , J. P. Sharma 3 ; 1 Stem Cell Biology, Celprogen Inc, San 

Pedro, CA, 2 Institute of Psychiatric Research, Indiana University School of Medicine, 

Indianapolis, IN, 3 Celprogen Inc, San Pedro, CA 

Alzheimer's disease (AD) is a progressive neurodegenerative disorder which impairs the 

memory and intellectual abilities of the affected individuals.The basal forebrain cholinergic 

system is the population of neurons most affected by the neurodegenerative process. 

Depositions of beta-amyloid or Abeta (Abeta) protein, neurofibrillary tangles and neuronal loss 

are the neuropathological hallmarks of AD. The goal of our labs is to unveil the mechanism of 

neurodegeneration and devise ways to prevent /reverse neurodegeneration by various 

approaches in primary brain cultures and AD animal models. For example, we have recently 

shown that rivastigmine can preserve or enhance neuronal and synaptic terminal 

markers in degenerating primary embryonic cerebrocortical cultures (Bailey et al, PLOS One, 

2011). Further, we demonstrated neuroprotective and neurorescue effects of a novel polymeric 

nanoparticle in neuronal cultures and AD animal model (Ray et al- JAD-2011). Similarly the role 

of specific micro-RNA species is being studied (Long & Lahiri-CMC-2011). From the 

pharmacological -chemical perspective to si RNA approach, here we report of testing a novel 

cell replacement therapy based on the hypothesis that cell replacement therapy would provide 

cure by compensating the lost neuronal systems. Neuronal progenitor stem cells (NPSCs) 

obtained from adult human post mortem brains from transplant rejects were grafted into the 

intact brain of mice (10) with matching controls (10), which was then followed by their 

incorporation into the host parenchyma and differentiation into functional neurons. In the 

lesioned mouse brains (SCID), NPSCs exhibited a significant targeted migration towards the 

damaged regions of the brain, where they engrafted, proliferated and matured into functional 

neurons. These NPSCs were intravenously administered and migrated into brain damaged 

areas and induce functional recovery. These results in animal models of AD suggest that 

transplanted NPSCs survive, migrate, and differentiate into cholinergic neurons, astrocytes, and 

oligodendrocytes with possible amelioration of the learning/memory deficits. In addition to 

replenish lost or damaged cells, NPSCs most likely stimulate endogenous neural precursors, 

enhance structural neuroplasticity, and down regulate proinflammatorycytokines and neuronal 

apoptotic death. Further, the NPSCs could be genetically modified to express growth factors 

into the brain. An attempt at mobilizing this endogenous pool of resident neuronal progenitor 

stem cell-like cells would provide another attractive approach for the treatment of AD. This 

paved the way towards exploring the possible cell based therapy of neurogenesis

which would offer an alternative approach for the treatment of AD. 


2090 

DSCAM Associates with UNC5C in Netrin-1-Mediated Growth Cone Collapse. 

A. Purohit 1 , C. Qu 1 , W. Li 2 , K-L. Guan 3 , G. Liu 1 ; 1 Department of Biological Sciences, University 

of Toledo, Toledo, OH, 2 University of Michigan Health Systems, University of Michigan, Ann 

Arbor, MI, 3 Department of Pharmacology, University of California San Diego, La Jolla, CA 

In the developing nervous system, neuronal growth cones explore the extracellular environment 

for guidance cues, which can attract or repel axons and guide them along specific trajectories 

towards their targets. Netrin-1, a bifunctional guidance cue, binds to receptors deleted in 

colorectal cancer (DCC) and Down syndrome cell adhesion molecule (DSCAM) mediating axon 

attraction, and uncoordinated-5 (UNC5) mediating axon repulsion. Here, we show that DSCAM 

interacts with UNC5C and netrin-1 stimulation increases this interaction in primary cortical 

neurons and postnatal cerebellar granule cells. Netrin-1 induces mouse cerebellum external 

granule layer (EGL) cell growth cone collapse and knocking-down DSCAM or UNC5C, by 

specific shRNAs or blocking their signaling by over-expressing mutants lacking the intracellular 

domains, eliminates netrin-1-induced growth cone collapse. Simultaneous knockdown of 

DSCAM and UNC5C blocks netrin-1 induced growth cone collapse in EGL cells as well. Netrin- 

1 increases the tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and 

PAK1, and promotes the formation of a protein-protein interaction complex of DSCAM with 

these signaling molecules in primary postnatal cerebellar neurons. PP2, a Src family kinase 

inhibitor, efficiently inhibits the tyrosine phosphorylation of these molecules and netrin-1-induced 

mouse EGL cell growth cone collapse. These data present the first evidence that DSCAM 

coordinates with UNC5C in netrin-1 repulsion. 

2091 

p75 neurotrophin receptor mediates ephrin-B3 signaling for axonal growth inhibition. 

N. Uesugi 1 , T. Yamashita 1 ; 1 Osaka University, Suita, Japan 

p75 neurotrophin receptor, a member of the TNF (tumor necrosis factor) receptor family, is 

known as a key molecule for axonal growth inhibition in the central nervous system (CNS). Axon 

elongation in the CNS is inhibited by molecules expressed in oligodendrocytes, e.g. MAG, 

Nogo, OMgp. These inhibitory molecules bind to p75 receptor indirectly, activate RhoA, known 

as a cytoskeletal regulator, thus suppressing axonal elongation. It has been reported that 

ephrin-B3, a member of the ephrin family, is also expressed in oligodendrocytes in the CNS and 

inhibits axonal elongation in vitro. Here we report that p75 neurotrophin receptor mediates the 

signal of ephrin-B3 for axonal growth inhibition. The axonal growth of cortical neurons was 

inhibited on ephrin-B3 coated dishes. This inhibitory effect of ephrin-B3 was reversed by 

treatment with Pep5, an inhibitory peptide of the signal transduction of p75 receptor. 

Furthermore, knockdown of p75 receptor by siRNA also silenced the inhibitory effect of ephrin- 

B3. Our results reveal a novel signaling cascade of ephrin-B3-induced axonal growth inhibition 

via p75 receptor. Moreover, in this study, we assessed if regeneration of optic nerve was 

induced by Pep5 application after crush injury.

2092 

Maintenance of Dendritic Spine Morphology by PAR1b through Regulation of 

Microtubule Growth. 

K. Hayashi 1 , A. Suzuki 1 , C. C. Hoogenraad 2 , S. Ohno 1 ; 1 Molecular and Cellullar Biology, 

Yokohama City University Graduate School of Medical Science, Yokohama, Japan, 2 Cell 

Biology, Faculty of Science, Utrecht University, The Netherlands 


Dendritic spines are postsynaptic structures that receive excitatory synaptic input from 

presynaptic terminals. Actin and its regulatory proteins play a central role in morphogenesis of 

dendritic spines. In addition, recent studies have revealed that microtubules are indispensable 

for the maintenance of mature dendritic spine morphology by stochastically invading dendritic 

spines and regulating dendritic localization of p140Cap, which is required for actin 

reorganization. However, the regulatory mechanisms of microtubule dynamics remains poorly 

understood. 

PAR1b, a cell polarity-regulating serine/threonine protein kinase, is thought to regulate 

microtubule dynamics by inhibiting microtubule binding of microtubule-associated proteins. 

Results from the present study demonstrated that PAR1b participates in the maintenance of 

mature dendritic spine morphology. Immunofluorescent analysis revealed PAR1b localization in 

the dendrites, which was concentrated in dendritic spines of mature neurons. PAR1b 

knockdown cells exhibited decreased mushroom-like dendritic spines, as well as increased 

filopodia-like dendritic protrusions, with no effect on the number of protrusions. Live imaging of 

microtubule plus-end tracking proteins directly revealed decreases in distance and duration of 

microtubule growth following PAR1b knockdown in a neuroblastoma cell line and in dendrites of 

hippocampal neurons. In addition, reduced accumulation of GFP-p140Cap in dendritic 

protrusions was confirmed in PAR1b knockdown neurons. In conclusion, the present results 

suggested a novel function for PAR1b in the maintenance of mature dendritic spine morphology 

by regulating microtubule growth and the accumulation of p140Cap in dendritic spines. 

2093 

PDZ-RhoGEF ubiquitination by Cullin3-KLHL20 controls neurotrophin-induced neurite 

outgrowth. 

M. Y. Lin 1,2 , Y. M. Lin 1,3 , T. C. Kao 1,3 , H. H. Chuang 1 , R. H. Chen 1,3 ; 1 Institute of Biological 

Chemistry, Academia Sinica, Taipei, Taiwan, 2 Institute of Molecular Medicine, College of 

Medicien, National Taiwan University, Taipei, Taiwan, 3 Institute of Biochemical Science, 

National Taiwan University, Taipei, Taiwan 

The induction of neurite outgrowth and arborization is critical for developmental and 

regenerative processes. Here we report that the BTB-kelch protein KLHL20 promoted neurite 

outgrowth and arborization in hippocampal and cortical neurons through its interaction with 

Cullin3 to form a ubiquitin ligase complex. This complex targeted PDZ-RhoGEF, a protein 

abundantly expressed in brain, for ubiquitin-dependent proteolysis, thereby restricting RhoA 

activity and facilitating growth cone spreading and neurite outgrowth. Importantly, targeting 

PDZ-RhoGEF to KLHL20 required PDZ-RhoGEF phosphorylation by p38 MAPK. In response to 

p38-activating neurotrophins, such as brain-derived neurotrophic factor and neurotrophin-3, 

KLHL20-mediated PDZ-RhoGEF destruction was potentiated, leading to neurotrophin-induced 

neurite outgrowth. Our study identified a ubiquitin-dependent pathway that targets PDZ- 

RhoGEF destruction to facilitate neurite outgrowth, and indicates a key role of this pathway in 

neurotrophin-induced neuronal morphogenesis.


2094 

c-Abl inhibition decreases the levels of Amyloid beta peptide in plasma of Alzheimer 

transgenic mice. 

L. D. Estrada 1 , D. chamorro 1 , N. C. inestrosa 2 , A. R. alvarez 1 ; 1 Lab. Señalizacion celular, Depto 

Biología Celular y Molecular, P. University of Catolica De Chile, Santiago, Chile, 2 Centro de 

Envejecimiento y Regeneración, P. Universidad Catolica de Chile, Santiago, Chile 

Introduction: AD is a progressive degenerative disorder characterized by memory loss, 

confusion and a variety of cognitive disabilities. The major neuropathological features of AD are 

the presence of amyloid plaques and neurofibrillary tangles. The main component of amyloid 

plaques is the amyloid-beta peptide (Aβ), which is a 39-42 amino acid peptide part of a much 

larger transmembrane protein; the amyloid precursor protein (APP). Accumulation of toxic Aβoligomers 

in the brain of the AD patients appears to be central in the pathogenesis of AD. 

Our group previously report a dramatic clearance of amyloid plaques in old AD transgenic mice 

treated with the c-Abl kinase inhibitor; STI571. 

Objective: 

The aim of this work was to investigated the effect of STI571 on peripheral blood and evaluate 

the cellular mechanisms involved in this clearance. 

Material and Methods: 

APPsw/PSEN1ΔE9 mice (6-months) were treated with imatinib (STI571, Novartis) or vehicle 

every other day for 14-days by intraperitoneal injection. Plasma fractions were isolated using a 

Ficoll gradient and amyloid beta was measured using an anti-A� ELISA. Brains were removed 

and fixed in paraformaldehide for histological analysis. In addition, primary hippocampal 

neurons and HT22-hippocampal cells lines expressing APP-GFP were treated with STI571 and 

APP localization and processing was evaluated. 

Results: The inhibition of c-abl by peripheral administration of the inhibitor STI571 decreased 

both, cerebral and plasmatic levels of A�-oligomers in APPswe/PSEN1ΔE9 transgenic mice. In 

addition, hippocampal neurons and HT22-APP(swe) cells treated with STI571 show differences 

in the distribution of APP and decreased levels of A� in comparison with control cells. 

Conclusion: It has been shown that c-Abl participates in endosomal regulation and since APP 

amyloidogenic proteolytic cleavage occurs in endosomal compartments, we believe that this 

processing may be regulated by inhibiting c-abl by STI571, thus decreasing A� production. 

Besides, the fact that the administration of STI571, a drug that poorly cross the blood–brain 

barrier, reduced accumulation of Aβ in both peripherical blood and the brain, suggest a 

peripheral clearance of A�. The findings of these experiments support a therapeutic value for 

STI571 and related compounds in Alzheimer's disease. 

Fondecyt-Postoctorado-3110052/Fondecyt-1080221 

2095 

Septin-dependent microtubule control during mammalian neurite outgrowth. 

N. Ageta-Ishihara 1 , M. Kinoshita 1 ; 1 Graduate School of Science, Nagoya University, Nagoya, 

Japan 

The outgrowth, arborization, connection and functional organization of neurites (axons and 

dendrites) are prerequisites for the development of the nervous system. Previous studies 

revealed the implications of mammalian septins in multiple steps in neural development 

including neuritogenesis and synaptogenesis. However, the mechanism of action remains 

unclear due to the functional redundancy and pleiotropic roles of this cytoskeletal/scaffold 

protein family. Here we focus on the role of septins in neurite outgrowth of mouse cerebral 

cortical neurons. Quantitative morphometric analysis demonstrated that shRNA-mediated 

depletion of the pivotal subunit SEPT7 inhibited the outgrowth of both axons and dendrites in


vitro, which was recapitulated in vivo. Septin-depleted neurons contained more acetylated αtubulin, 

indicating hyperstabilized microtubules are responsible for the phenotype at least in 

part. Tubacin, a small molecule inhibitor of the major tubulin deacetylase HDAC6 gave a 

phenocopy of septin depletion, i.e., neurite outgrowth and hyperacetylated α-tubulin. 

Biochemical analyses indicated that septins, HDAC6 and acetylated α-tubulin can physically 

interact and that septin depletion can reduce the interaction between HDAC6 and acetylated αtubulin. 

These and other data indicate that septins serve as a scaffold that facilitates the 

interaction between HDAC6 and acetylated α/β-tubulin heterodimer, which ensures microtubule 

remodeling for neurite outgrowth by optimizing the acetylation level of α-tubulin. 

2096 

Neuroproteomic Analysis of Homer2 Knockout and Wildtype Mice Reveals Novel Gene 

Products Contributing to Alcohol Aversion Behavior. 

S. P. Goulding 1,2 , N. Shulman 3 , N. W. Bateman 2 , M. J. MacCoss 3 , K. K. Szumlinski 4 , C. C. Wu 2 ; 

1 Neuroscience Program, University of Colorado Anschutz Medical Campus, Aurora, CO, 

2 Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 

Pittsburgh, PA, 3 Department of Genome Sciences, University of Washington, Seattle, WA, 

4 Department of Psychology and the Neuroscience Research Institute, University of California, 

Santa Barbara, Santa Barbara, CA 

In excitatory synapses of the mammalian central nervous system, the Homer family of proteins 

mediates excitatory signal transduction and receptor plasticity. The product of three genes, 

Homer1/2/3, this family shares domain features that include a conserved amino-terminal 

Ena/Vasp Homology 1 (EVH1) domain and two leucine zippers that form the hydrophobic core 

of a coiled-coil motif unique to the carboxyl-terminal of long, but not short, Homer isoforms. 

EVH1 containing Homers bind proline-rich sequences found in numerous postsynaptic 

glutamatergic signaling proteins such as group 1 metabotropic glutamate receptors (mGluRs) 

and the N-methyl D-aspartate (NMDA) receptor adaptor protein Shank. Coiled-coil, or long 

Homer isoforms, oligomerize into scaffolds and regulate signaling through interactions with 

cytoskeletal constituents like Drebrin, an actin-binding protein. Consistent with structural 

observations, Homer2 knockout mice present with numerous molecular, neurochemical and 

behavioral phenotypes in response to acute and chronic alcohol administration. Since ethanol 

alters glutamate neurotransmission in parts of the brain implicated in alcohol addiction, such as 

the hippocampus, and Homers regulate excitatory neural signaling through interactions with 

binding partners in the postsynaptic density (PSD), we set out to investigate alcohol avoiding 

behavior of the Homer2 knockout mouse at the neuroproteomic level. To better understand 

molecular mechanisms contributing to alcohol aversion, we studied differentially expressed 

peptides in the hippocampus of wildtype and Homer2 knockout mice using label-free mass 

spectrometry. By combining liquid chromatography-tandem mass spectrometry (LC-MS/MS) 

and a suite of software tools capable of detecting chromatographic features of differential 

peptide peak areas between samples, we demonstrate that Homer2 deletion alters global gene 

products between wildtype and knockout mice. While Homer2 deletion is necessary and 

sufficient for alcohol aversion in mice, these data suggest that differentially abundant gene 

products also contribute to the alcohol aversive phenotype and provide a readout of functional 

protein effectors of the Homer2 protein.


2097 

Seizure susceptibility in mice lacking somatostatin type 3 receptors. 

S. K. Evans 1 , S. Lewis 1 , J. L. Fuchs 1 ; 1 Biological Sciences, University of North Texas, Denton, 

TX 

The role of primary cilia as essential mediators of cell proliferation in the developing brain is well 

established, but the function of cilia in mature, postmitotic neurons is just beginning to be 

investigated. The somatostatin type 3 receptor (Sst3R) is concentrated mainly on neuronal 

primary cilia in most brain regions. We found that this receptor was typically absent in cilia of 

neural progenitors and immature neurons, but was present in cilia of more mature neurons in 

postnatal mice. Given the generally homeostatic, inhibitory, anti-mitotic effects of the hormone 

somatostatin, we hypothesized that Sst3R on neuronal cilia serves to protect neurons against 

excitotoxicity, thus enhancing neuron survival in the face of stressful conditions. We compared 

seizure-related behaviors in C57BL/6 wildtype versus SstR3 knockout mice, in response to 

intranasal kainic acid (KA) administered under isoflurane anesthesia. Mice were observed 

periodically for 24 hours. The mutant mice showed behavioral symptoms of seizures at level 4-5 

on a standard seizure scale, where 0 is no symptoms and 5 is the most severe. In contrast, 

wildtypes scored only 0-1, even with up to two additional doses of KA (0.3 mg total per mouse). 

After a 3-day post-KA survival period, the brains of control and KA-treated mice were 

immunostained for GFAP to visualize fibrous astrocytes and adenylate cyclase type 3 to 

visualize primary cilia. KA-treated mutants showed evident astrogliosis, a hallmark of epilepsy, 

whereas KA-treated wildtypes showed only mild astrogliosis. We examined cilia in the hilus of 

the dentate gyrus in all four groups of mice and found that KA administration was associated 

with reductions in the numerical density of immunostained cilia in the mutants. These 

observations support the hypothesis that ciliary Sst3R has a neuroprotective role in mature, 

postmitotic neurons. 

2098 

SNX26, a GAP for Cdc42, Interacts with PSD-95 and Regulates Dendritic Spine Formation 

in Neurons. 

Y. Kim 1 , S. Chang 1 ; 1 Department of Physiology and Biomedical Sciences, Seoul National 

University College of Medicine, Seoul, Korea 

Dendritic spines are tiny protrusions composed of highly specialized actin microfilaments that 

receive the majority of excitatory glutamatergic synaptic inputs in the CNS. SNX26 (Sorting 

nexin 26) is a brain-enriched Rho GTPase-activating protein known to be localized in spines, 

though little is known about its neuronal function. Here, we report that SNX26 interacts with 

PSD-95, a well-known glutamatergic postsynaptic scaffolding protein and regulates spine 

formation in cultured hippocampal neurons. SNX26/TCGAP is present in dendritic spines where 

it colocalizes with PSD-95. We found that C-terminal domain of SNX26 interacts with SH3 

domain of PSD-95. Overexpression of SNX26 decreased the number of dendritic spines while it 

increased the number of filopodial form of protrusions. RhoGAP domain is responsible for its 

effect on spines to filopodia transition. Accordingly R350I, a GAP defective mutant form of 

SNX26/TCGAP, has no effect on spines to filopodia transition. We further found that expression 

of SNX26 largely decreases the F-actin contents in COS-7 cells as well as in neurons, 

suggesting that SNX26 as a RhoGAP destabilizes F-actin, which may mediate spines to 

filopodia transition in neurons. Evidently, knocking-down endogenous expression of SNX26 by 

shRNA affects the number and morphology of dendritic spines. Taken together, our results 

indicate that SNX26 acts as a GAP for Cdc42, interacts with PSD-95 in dendritic spines, and 

regulates spine formation. Our results also raise the possibility that coordinated interaction of


Cdc42 with SNX26 and other GEF proteins during dendritic spinogenesis in developing neurons 

may also play a pivotal role during synaptic plasticity in mature neurons.

MONDAY, DECEMBER 5- Late Abstracts 2 

Science Education 

MONDAY 

2099 

Industry Engagement in STEM Education. 

S. P. Denker 1 , L. Lindburg 2 , G. Maderis 3 ; 1 BayBio Institute, South San Francisco, CA, 2 BayBio 

Institute, 3 BayBio 

Bio-Community: Building the Next Generation of Life Science Professionals is the BayBio 

Institute′s Science Education program aimed at bringing quality science, technology, 

engineering and math (STEM) educational experiences to students of diverse socio-economic 

and ethnic backgrounds. It is a hands-on program designed to connect students and educators 

to life science professionals, and to bring these professionals as volunteers into middle/high 

school and community college classrooms. Members of the Bio-Community are Northern 

California life science companies and educators committed to improving STEM education. The 

program and network provide a mechanism to establish the critical partnerships between 

industry, community based organizations and schools necessary to improve STEM education, 

teacher impact and student motivation. The objectives of Bio-Community are to 1) enhance 

STEM education in the region′s middle, high schools, and community colleges, 2) engage life 

science employees and companies in STEM-related activities and 3) increase student interest in 

STEM subjects, STEM-related majors, and awareness of the variety of careers available within 

the life science industry. The program was developed and launched in Fall, 2010 and has 

reached nearly 900 students thus far. 

2100 

Case-based learning to understand Cell Communication. 

S. W. Gorsich 1 ; 1 Biology, Central Michigan University, Mount Pleasant, MI 

Cell Communication is an advanced biology course. The goal of this course is for students to 

learn various cell communication mechanisms, become familiar with critically evaluating the 

primary literature, and to improve critical thinking skills. Thirteen students were enrolled in the 

class and they were divided into five groups with each group having a lap top computer. During 

the first week of the course an introduction to how cells communicate was given in a more 

traditional lecture style. In addition during this first week I introduced the Osternsky family to the 

class. Beginning in the second week we began a 3-4 day cycle of 1) case presentation, 2) 

discussion of cell communication topic, and 3) discussion/presentation of primary literature. 

During the case presentation a member of the Osternsky family is presented, which often 

centers on a medical concern. As the case unfolds students are provided leading information for 

them to think about and make predictions at each stage. At the end of the case students will 

diagnose what’s wrong with the family member and then link it to a cell communication problem. 

For instance in week one the class diagnosed E. Osternsky as having diabetes. They then 

found a primary paper connecting G-protein coupled receptors to diabetes. During the next 

class I provide an interactive lecture introducing the specific cell communication pathway the 

students previously identified (e.g. G-protein coupled receptors). On the final day of the cycle 

we critically discuss the paper. Everyone in the class is expected to read the entire paper, but 

each group has one figure they present. To assure students are reading the paper before class, 

individual and group quizzes are given over the main take home points of the paper and the 

communication pathway being discussed. The three-part cycle is then repeated with a different 

Osternsky family member. Some outcomes that have resulted include: 1) students came more 

prepared to class, 2) students became proficient in critically evaluating and presenting the

MONDAY 

primary literature, 3) improved attendance, 4) retention of material improved, and 5) at a 

qualitative level student engagement, critical thinking, curiosity, and enthusiasm were high, 6) 

improved confidence, and 7) student’s became invested in the individual family members. 

Within this course additional learning strategies were used. They include: multiple quizzing, 

research ethic case studies, and oral midterm and final exams. With the success of this course I 

plan to implement a similar strategy in my larger classes. 

2101 

Go Figure: Comparing Graphics in Biology Textbooks. 

K. Chiem 1 , N. K. Dave 1 , R. Ghasemian 1 , E. J. Ortiz 1 , D. R. Perusse 1 , J. T. Sandu 1 , I. M. Tabel 1 , 

B. A. Velasco 1 , T. Vu 1 , M. L. Casem 1 , S. E. Walker 1 ; 1 Biological Science, California State 

University, Fullerton, Fullerton, CA 

The quality of a biology textbook can immensely affect a student’s ability to learn, comprehend 

and integrate concepts. Figures, drawings or photographs, are often used in textbooks to 

convey large and complex concepts that would be otherwise difficult to follow. We explored the 

quality and quantity of figures in majors, non-majors, and mixed biology textbooks. A rubric was 

developed to measure the quality of figures. Figures were evaluated for three different topics 

typically taught in a general biology course: meiosis, alternation of generations, and 

endosymbiosis. We hypothesized that major’s textbooks would provide greater quality and 

quantity of figures than non-majors/mixed textbooks. On average the majors textbooks scored 

higher in all quality categories, except for figure location and number of figures when compared 

to non-majors. Major’s textbooks tend to include entire complex processes in one large figure 

versus a non-majors text that might break down a complicated, lengthy concept (e.g. meiosis) 

into smaller more easily understandable pieces. 

2102 

An interdisciplinary team approach in undergraduate biological instruction and research. 

V. C. Ware 1 , N. G. Simon 1 ; 1 Biological Sciences, Lehigh University, Bethlehem, PA 

Lehigh University has an undergraduate science education award from the Howard Hughes 

Medical Institute (HHMI) that prepares students to integrate perspectives from multiple 

disciplines to address complex questions in the biosciences. Lehigh is also a member of the 

HHMI-sponsored Science Education Alliance (SEA), where students and faculty nationwide use 

multidisciplinary approaches in mycobacteriophage research. A curricular model around the 

theme of “biosystems dynamics” begins in the first semester and provides continuing access to 

courses and research opportunities that emphasize multidisciplinary approaches. Key 

programmatic elements are a “Bioscience in the 21st Century” survey course and the 

“Biosystems Dynamics Summer Institute (BDSI),” a 10-week, research-intensive experience for 

interdisciplinary teams of undergraduates, graduate students, and faculty. The survey course 

introduces students to diverse perspectives on bioscience issues through lectures from faculty 

spanning science, engineering, and other disciplines to imbue a broad, trans-disciplinary 

perspective as a core scientific philosophy. The BDSI has supported 23 research teams, 

spanning projects in cell and molecular biology, bioengineering, and neuroscience with faculty 

from mathematics, electrical engineering, chemistry, mechanical engineering, chemical 

engineering, computer science and biology. Student team members (which have included some 

SEA alumni) are drawn from different disciplines to reinforce a multidisciplinary 

conceptualization to problem-solving. Participation in BDSI has had several positive effects: 

publications and presentations that include student authors; grant proposals to support 

continued collaboration; undergraduate participation in research laboratories by most students 

after the BDSI experience; and several graduates of the BDSI program altered their post-

MONDAY 

graduate plans to include graduate programs that offered interdisciplinary opportunities or job 

opportunities in a discipline outside of their major. BDSI support has generated a “ripple effect” 

that significantly expanded collaborative, multidisciplinary research efforts on campus. As the 

HHMI-supported program has evolved over the project period, we see changes in institutional 

culture in teaching and research strategies designed to address complex problems in the life 

sciences more broadly. 

2103 

Undergraduate research experiences based on genomic, proteomic and gene expression 

libraries from Tetrahymena: launching students into cellular research on ciliated protists. 

L. A. Hufnagel 1 ; 1 Cell Mol Biology, University of Rhode Island, Kingston, RI 

To encourage undergraduate students to consider research on the cell and molecular biology of 

ciliated protists in their future career plans, I have established in my laboratory an informal 

educational program that combines training in 1) laboratory methods for Tetrahymena 

thermophila, 2) in silico gene sequence analysis and 3) immunocytochemistry. Each student 

who joins my laboratory is encouraged to choose a human disease of interest, do background 

reading to establish the current understanding of molecular and cellular events underlying the 

disease, and identify a protein of particular importance in the disease process. The student next 

learns to use BLAST-searching to identify possible T. thermophila orthologues of the relevant 

protein, as well as orthologues in other organisms, from ciliates to humans, using portals such 

as TGD-Wiki and Pubmed. The orthologues identified in this way have often not yet been 

functionally characterized, so a variety of other on-line-accessible tools (available at NCBI, EBI 

and elsewhere) are then used, to further analyze the functional properties of the Tetrahymena 

orthologues, and their evolutionary relationships with orthologues in other protists, invertebrates 

and chordates. In preparation for immunocytochemical studies to characterize the spatial and 

temporal expression of the identified proteins, sequence alignment tools are used to compare 

sequences of immunogens used in preparation of commercially available antibodies with the 

sequences of the tetrahymena orthologues. To determine whether the predicted protein may 

actually be expressed, the student learns to use the expression data available at TGED, which 

provides temporal information that also helps to predict when during the cell cycle, starvation 

and mating the cells will most likely immunolabel. Using this approach, my undergraduate 

students are 1) characterizing the cell surface distribution of a Tetrahymena ribosomal protein, 

phosphoprotein P0, identified as a possible target for vaccine development in apicomplexan 

parasites; 2) investigating the Tetrahymena orthologue of JAK2, a protein mutated in individuals 

with polycythemia vera (a rare blood disorder), and 3) analyzing the Tetrahymena orthologue of 

human FANCM, a protein implicated in Fanconi anemia and cancer. 

2104 

Converting a senior-level cell biology course to a fully active learning format. 

D. J. Matthes 1 ; 1 Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, 

MN 

While there have been efforts nationwide to convert traditional introductory college biology 

courses to an active learning format, there have been few reports of shifting high-enrollment 

senior-level cell biology courses to incorporate such innovative instructional strategies. Here I 

report such a course conversion, where a modified Team Based Learning approach has been 

adopted for cell biology courses with approximately 100 students meeting in a SCALE-UP style 

active learning classroom at a large public university. Particular strategies (cell biology concept 

inventories, learning readiness quizzes, team challenges, and a team project to design 

nanoparticle-based cell-targeting therapies) will be described. Learning retention as assessed

MONDAY 

by pre- and post-test concept inventories has improved significantly with this course format 

compared to the same material presented by the same instructor in similar lecture format 

courses. 

Microtubule Cytoskeleton 

2105 

Analysis of the expression of microtubule plus-end tracking proteins (+TIPs) during 

Xenopus development. 

E. C. Park 1,2 , H. Lee 3 , M. Kim 3 , J-K. Han 3 , G-H. Kim 1,2 ; 1 Division of Life Science, Korea Basic 

Science Institute, Daejeon, Korea, 2 Pioneer Research Center for Protein Network Exploration, 

Korea Basic Science Institute, Daejeon, Korea, 3 Division of Molecular and Life Sciences, 

Pohang University of Science and Technology, Pohang, Korea 

Microtubules are a component of the cytoskeleton and are important for maintaining cell 

structure and providing platforms for intracelluar transport, as well as other cellular processes. 

Microtubule plus-end tracking proteins (+TIPs) are a structurally and functionally diverse group 

of proteins. +TIPs are specifically accumulated in microtubule plus end and regulate dynamic 

microtubules behavior. We characterized the +TIPs, XClip-170, Xp150glued, XClasp1b, XLis1, 

XStim1 and XStim2, in Xenopus laevis and report the expression patterns during 

embryogenesis. All six Xenopus +TIP genes are maternally expressed and show similar 

expression patterns during Xenopus embryo development. The expressions of Xenopus +TIPs 

are localized in the animal hemisphere and ectoderm region at early stages. As embryos 

develop to later stages, the ectodermal expression of Xenopus +TIPs persists in head and 

neural tube structures. XClasp1b and Xp150glued,especially, shows ectopic expressions in the 

cranial ganglia. Remarkably, Xenopus +TIPs are also expressed in the involuting mesoderm 

during gastrulation. This is the first study of developmental expression patterns of +TIPs and our 

analysis highlights the future insight of research fields of microtubules in vertebrate 

development, gastrulation cell movements and neurogenesis. 

2106 

Nuclear gamma-tubulin associates with nucleoli and interacts with tumor suppressor 

protein C53. 

B. Horejší 1 , S. Vinopal 1 , E. Dráberová 1 , V. Sulimenko 1 , T. Sulimenko 1 , A. Philimonenko 2 , P. 

Hozák 2 , P. Dráber 1 ; 1 Dept. Biology. Cytoskeleton, Inst. Mol. Genet AS CR, Prague, Czech 

Republic, 2 Dept. Cell Nucleus, Inst. Mol. Genet AS CR, Prague 

γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules 

from microtubule organizing centers. Using immunolocalization and cell fractionation techniques 

in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained 

evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse 

cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar 

centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with 

nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli 

during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γtubulin, 

making it unlikely that rapid active transport via nuclear pores participates in the 

transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and 

time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. 

Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an 

association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular

MONDAY 

compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was 

corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γtubulin 

antagonized the inhibitory effect of C53 on DNA damage G2/M checkpoint activation. 

The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin 

may also have nuclear-specific function(s). 

2107 

Cap-gly proteins at microtule plus ends: is EB1 detyrosination involved? 

A. Bosson 1 , J-M. Soleilhac 1 , O. Valiron 1 , D. Job 1 , A. Andrieux 1 , M-J. Moutin 1 ; Grenoble Institut 

Des Neurosciences, Grenoble, France 

Localization of CAP-Gly proteins such as CLIP170 at microtubule + ends results from their dual 

interaction with α-tubulin and EB1 through their C-terminal amino acids -EEY. Detyrosination of 

α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect 

EB1 as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated 

forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. 

Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal 

amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination 

regulates CLIPs interaction with tubulin, but not with EB1. They highlight the specificity of 

carboxypeptidase toward tubulin. 

2108 

Tubulin Acetylation Is Required for the Maintenance of Stable Microtubules Arrays 

During Cell Migration. 

Z. Xu 1,2 , M. Nachury 2 ; 1 Department of Dermotology, Stanford University School of Medicine, 

Palo Alto, CA, 2 Department of Molecular and Cellular Physiology, Stanford University School of 

Medicine, Palo Alto, CA 

Migrating cells possess a subset of polarized stable microtubules oriented towards the leading 

edge. These long lived microtubules that are highly acetylated on lysine 40 site are generally 

considered as tracks for delivery of signaling molecular for cell migration. However, the 

functional significance of tubulin acetylation is still elusive in large part because the tubulin 

acetyltransferase remains unidentified. 

Recently, in the course of proteomic study, we identified a protein with distant structural 

similarity to the GNAT clade of acetylatransferases that is exclusively and universally conserved 

in ciliated organisms. Strikingly, this putative acetyltransferase specifically acetylates a-tubulin 

at K40 in vitro and was therefore named aTAT1. 

In a variety of cell lines, overexpression of aTAT1 is sufficient to induce massive a-tubulin K40 

acetylation. Furthermore, a catalytically inactive mutant of aTAT1 fails to acetylate tubulin in 

vivo. Thus, the enzymatic activity of aTAT1 is necessary for acetylation a-tubulin at K40 in cells. 

Meanwhile, Depleting aTAT1 from hTERT-RPE cells abolishes MT acetylation and affects the 

kinetics of primary cilium assembly. Additionally, aTAT1 is required for the maintenance of 

stable microtubule arrays and for directional cell migration. Interestingly, we found that the 

median length of nocodazole-resistant MTs falls from 4 μm in control-treated cells to less than 1 

μm in αTAT1-depleted cells. This result suggests that in the absence of acetylation, stable MTs 

become highly fragile and fragmented. We conclude that aTAT1 is the major a-tubulin K40 

acetylatransferase in mammalian cells and that a-tubulin K40 aceytlation enable the assembly 

of stable microtubule tracks utilized in various cellular processes.

MONDAY 

2109 

HURP regulates chromosome congression by modulating kinesin Kif18A function. 

F. Ye 1 , L. Tan 1 , Q. Yang 1 , Y. Xia 1 , L-W. Deng 2 , M. Murata-Hori 3 , Y-C. Liou 1 ; Department of 

Biological Sciences, National University of Singapore, Singapore, 2 Department of Biochemistry, 

National University of Singapore, Singapore, 3 Temasek Life Sciences Laboratory, Singapore 

Chromosome biorientation and congression during mitosis require precise control of microtubule 

dynamics. The dynamics of kinetochore microtubules (K-MTs) are regulated by a variety of 

microtubule associated proteins (MAPs). Recently, a MAP known as HURP (hepatoma upregulated 

protein) was identified. During mitosis, RanGTP releases HURP from the Importin â 

inhibitory complex and allows it to localize to the kinetochore fiber (K-fiber). HURP bundles and 

stabilizes K-fibers and promotes chromosome congression. However, the molecular mechanism 

underlying the role of HURP in regulating chromosome congression remains elusive. Here, we 

show that overexpression of the N-terminal microtubule binding domain (1-278 aa, HURP278) 

of HURP induces a series of mitotic defects including lagging chromosomes during the late 

prometaphase to metaphase stage, prolonged mitosis and increased kinetochore oscillation 

amplitude, mimicking the effects of Kif18A depletion. In addition, co-immunoprecipitation and 

BiFC assays identify Kif18A as a novel interaction partner of HURP. Furthermore, quantitative 

results from live cell imaging analyses illustrate that HURP regulates Kif18A localization and 

dynamics at the plus-end of K-MTs. Lastly, misaligned chromosomes in HURP278overexpressing 

cells can be partially rescued by the overexpression of Kif18A, suggesting the 

specificity of Kif18A regulation by HURP. Our results demonstrate in part the regulatory 

mechanism for kinesin Kif18A at the K-MTs during chromosome congression and provide new 

insights into the mechanism of chromosome movement at the metaphase plate. 

2110 

MTWD40 is a Novel Microtubule Regulatory Protein Expressed in Neurons. 

W. Wang 1 , V. Lundin 1 , I. Millan 1 , M. Maloney 1 , M. Kapur 1 , N. Chen 1 ; 1 Neurology, Stanford 

School of Medicine, Stanford, CA 

Neurons are uniquely dependent on a highly regulated microtubule (MT) cytoskeleton. Several 

MT-associated proteins (MAPs) are involved in maintaining MT stability and dynamics, which 

support essential neuronal functions such as axonal transport. Given the critical role of MAPs in 

regulating MTs in both healthy and diseased neurons, we sought to understand how this 

regulation is achieved. Here, we identify a novel protein containing a WD40 repeat domain, 

tentatively named microtubule-related WD40 (MTWD40), as a potential regulator of MAPdependent 

MT dynamics. mRNA and protein expression analyses in mice indicate that 

MTWD40 is enriched in the nervous system. Protein expression is detected as early as E11 and 

continues throughout adulthood. At the cellular level, MTWD40 localizes as puncta along the 

MT network in both COS7 cells and primary cortical neurons. Interestingly, co-overexpression 

with MAP8 causes MTWD40 to lose its punctate pattern and instead colocalize with MAP8 

uniformly along MTs. These results suggest that MTWD40 may serve an important role in 

regulating the neuronal cytoskeleton through an interaction with MAP8.

MONDAY 

2111 

Role the Microtubule (+)-End Binding protein EB1 in differentiating osteoblasts. 

S. Pustylnik 1 , N. Nabavi 2 , R. Harrison 3 ; 1 Department of Biological Sciences, University of 

Toronto, Toronto, ON, Canada, 2 Department of Cell and Systems Biology, University of Toronto, 

3 University of Toronto, Toronto, ON, Canada 

Osteogenesis is a strictly controlled developmental process in which exogenous factors, such 

as hormones and growth factors activate osteoblast-specific signaling proteins and transcription 

factors required for osteoblast differentiation. Our microarray analysis identified the microtubule 

(+)-End-Binding protein (EB1) as an upregulated gene in 5-day ascorbate-induced 

differentiation of mouse MC3T3 mouse cells compared to undifferentiated control cells. The 

main functions of EB1 are regulation of microtubule dynamics, cell polarity, chromosome 

stability and cell migration. EB1 is known to bind to the tumor suppressor APC (Adenomatosis 

Polyposis Coli). This interaction has led to the speculation that EB1 is a component of the 

canonical APC tumor suppressor pathway, which regulates the levels of β-catenin, a key Wnt 

signaling effector. 

In this study, we examined the role of EB1 in osteoblasts during differentiation to elucidate the 

possible involvement of EB1 in Wnt signaling. Immunoblotting of extracts from undifferentiated 

and differentiated osteoblasts showed that EB1 could be detected in freshly plated cells and its 

level increases during the first 5 days of differentiation. Osteoblasts in which EB1 was knocked 

down by RNA interference showed changes in cell morphology and osteoblasts specific gene 

expression. EB1 and β-catenin co-immunoprecipitate in osteoblasts and we see an induction in 

β-catenin protein levels during differentiation using immunoblotting. Interestingly, EB1 protein 

knockdown led to a decrease of nuclear accumulation of β-catenin by immunofluorescence. 

These results suggest that EB1 is important for β-catenin degradation events that lead to its 

nuclear accumulation and potential activation of Wnt signaling genes. Variations in cell density 

significantly influenced the whole differentiation process while block of adherent junctions with 

E-cadherin antibody resulted in decreased cell-cell adhesions and altered bone matrix protein 

expression. We are now using live imaging to discern the cellular location of interactions 

between these 2 proteins to show a definitive role for EB1 in osteoblast differentiation. 

2112 

Characterization of HIV Rev and Tubulin Interactions. 

J. L. Chang 1 , B. E. Dukes 1 , M. W. Miller 1 ; 1 Biological Sciences, Wright State University, Dayton, 

OH 

The HIV Rev protein has the ability to bind tubulin heterodimers and depolymerize microtubules 

(MTs) in vitro producing bilayered rings called Rev-tubulin toroids (RTTs) (Watts et al. 2000. J. 

Cell Biol. 150: 349-360). These interactions may account for MT defects observed in HIV 

infected cells or cells that over-express Rev. Watts et al. hypothesized Rev interacts with MTs 

by a mechanism shared with Kinesin-13 (Kin13) proteins owing to the presence of a shared 

amino acid sequence. Kin13 proteins are potent MT depolymerizing agents affecting MT 

behavior during mitosis. 

To test this hypothesis, point mutations were introduced into Rev substituting amino acids 

shared with Kin13. In one set of experiments, the abilities of four mutant Rev proteins (A37D, 

R42A, E47A, and E57A) to form RTTs when mixed with colchicine-treated tubulin were 

compared to wild-type Rev. With the exception of RevR42A, wild-type and mutant Rev formed 

comparably sized RTTs at a high frequency. In contrast, mutating arginine at position 42 to 

alanine completely abrogated formation of RTTs. This observation suggests this amino acid is 

critical for Rev-tubulin interactions, consistent with our hypothesis.

MONDAY 

In a second set of experiments, wild-type and seven mutant Rev proteins (T34A, A37D, R39A, 

R42A, E47A, R50A, E57A, and the E47A/E57A double mutant E2A) fused to YFP were overexpressed 

in HeLa cells and the ability of these cells to grow in culture and elaborate MT 

cytoskeletons was measured. Previous results show over-expression of wild-type Rev slows 

growth in culture and alters cell cycle progression. If these defects are due to Rev-MT 

interactions, mutation of residues critical for these interactions should mitigate these defects. 

Several Rev mutants, notably A37D, E47A and the double mutant E47A/E57A (E2A), alter cell 

morphology and growth in culture suggesting the affected amino acids are dispensable. 

Curiously, over-expression of RevR42A also alters doubling times and increases the frequency 

of multinucleated cells. Taken together, Rev over-expression can lead to defects not directly 

attributable to Rev-MT interactions. 

2113 

Differential Interaction of Dystrophin and Utrophin with Microtubules. 

J. J. Belanto 1,2 , D. M. Henderson 1 , M. A. Jaeger 1 , J. M. Ervasti 1 ; 1 Biochemisty, Molecular 

Biology, and Biophysics, University of Minnesota, Minnapolis, MN, 2 Molecular, Cellular, 

Developmental Biology, and Genetics, University of Minnesota, Minneapolis, MN 

The dystrophin gene resides on the X chromosome and encodes a 427 kD cytoplasmic protein 

expressed in striated muscle which links the costameric actin cytoskeleton to the extracellular 

matrix via a sarcolemmal-associated glycoprotein complex. Mutations in dystrophin that abolish 

or reduce its expression lead to Duchenne muscular dystrophy (DMD) or Becker muscular 

dystrophy (BMD), respectively. Patients with DMD succumb to fatal cardiac and/or respiratory 

failure in their late teens to early twenties while BMD patients are more mildly affected. 

Currently, there is no effective treatment available for DMD or BMD. 

Our lab previously demonstrated that dystrophin directly binds and organizes microtubules into 

a rectilinear lattice beneath the sarcolemma (Prins et al., J. Cell Biol. 2009). Using an in vitro 

cosedimentation assay, we show that dystrophin binds to microtubules with high affinity 

(KD=0.20µM). We also show that transgenic expression of nearly full-length dystrophin on the 

dystrophin-deficient mdx background restores microtubule lattice organization whereas mdx 

mice present with a disorganized microtubule lattice. Based on cosedimentation assays with an 

array of short dystrophin isoforms and truncation constructs, we conclude/suggest that 

microtubule binding activity maps to a region located between spectrin-like repeats 9 and 19 of 

the large middle rod domain. 

Utrophin is the fetal homolog of dystrophin that is down-regulated in skeletal muscle at birth but 

ubiquitously expressed at low levels in all tissues, including skeletal muscle, in adults. Because 

utrophin binds to nearly all of dystrophin’s previously identified molecular partners, several 

therapies under development for DMD aim to upregulate utrophin in skeletal muscle. We show 

that utrophin binds microtubules in vitro with 10-fold lower affinity (KD=2.34µM) than dystrophin 

and that transgenic over-expression of utrophin is not sufficient to rescue the disorganized 

microtubule network in mdx muscle. Our results suggest that any deficiency in microtubule 

trafficking or function caused by loss of dystrophin may not be restored by upregulation of 

utrophin. Therefore, it remains important to determine whether any specific pathologies can be 

attributed to the microtubule disorganization associated with loss of dystrophin protein 

expression. 

This work was supported by the NIH Training Program in Muscle Research AR007612 and NIH 

RO1 AR042423.

2114 

XMAP215 uses avidity to promote microtubule growth. 

P. O. Widlund 1 , J. Howard 1 , A. A. Hyman 1 ; 1 Max Planck Inst Moleculcar and Cell 

Biolology/Genetics, Dresden, Germany 

MONDAY 

Microtubule-associated proteins adjust the dynamic properties of microtubules so that they can 

be used to carry out cellular functions such as chromosome segregation, vesicle transport, and 

cell motility. The XMAP215/Dis1 family of proteins has been shown to dramatically promote 

microtubule growth and their activities significantly contribute to the rapid growth rates of 

microtubules seen in cells. Repeats at their N-termini, called TOG domains, are important for 

this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction 

translates to polymerase activity. Understanding the functional roles of TOG domains is further 

complicated by the fact that the number of these domains present in the proteins of different 

species varies. We determined the contributions of the individual TOG domains to microtubule 

growth and looked at the affinities of the TOG domains for tubulin. Based on these experiments, 

we have developed a model where TOG domains each have low affinity for tubulin dimers, but 

their combination allows for high overall avidity. This makes it possible for TOG proteins to 

efficiently recruit tubulin dimers, but also be able to release them into the growing microtubule 

end. 

2115 

A High-Throughput and High-Content Microscopy Approach to Identify Microtubule 

Regulators. 

X. E. Studera 1,2 , V. Graml 1,2 , M. Bortfeld-Miller 2 , R. E. Carazo Salas 1,2 ; 1 The Wellcome 

Trust/Cancer Research UK Gurdon Institute, Cambridge, United Kingdom, 2 Institute of 

Biochemistry/ETH Zurich, Zurich, Switzerland 

Microtubules (MT) are key determinants of countless eukaryotic cellular features, such as cell 

shape, intracellular transport, organelle positioning and cell division, yet to this day a 

comprehensive (genomic) catalogue of microtubule regulators is lacking even for the simplest 

cells. 

We have carried out a screen for novel microtubule regulators, using a commercial genomewide 

gene-knockout (‘deletion’) collection of haploid mutant strains of the fission yeast 

(Schizosaccharomyces pombe) and high-throughput/high-content microscopy. So far, only 26 

genes are known from previous studies to be bona fide microtubule regulators in fission yeast, 

although the Gene Ontology database suggests that there could be a few hundred more. 

Using a high-throughput spinning disk confocal microscope, we imaged approximately 3 000 

non-essential gene knockout strains expressing GFP-labelled tubulin, allowing us to inspect 

their intact microtubules in live cells. The images were screened visually to look for strains 

displaying microtubule defects. Over 500 000 images were also analyzed computationally using 

custom-made, automated microtubule detection algorithms to look for knockouts with 

statistically significantly aberrant microtubule structures. 

After preliminary quality control, we have identified approximately 100 gene knockouts 

displaying a phenotypic defect in microtubule organization. About 40 % of the corresponding 

genes have homologues in other organisms and 50 % of them have been experimentally 

characterized but not reported to regulate microtubules, and are hence potentially novel 

regulators. We are in the course of carrying out further quality control and detailed strain 

analysis to confirm which of those preliminary hits are conserved, new microtubule regulators

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and to clarify their mode of action. Here we describe the strategy of our screen and our 

preliminary results. 

2116 

Calcium-Dependent Reorganization of Microtubules in Activated Mast Cells. 

V. Sulimenko 1 , Z. Hájková 1 , S. Vinopal 1 , E. Dráberová 1 , P. Dráber 1 ; 1 Institute of Molecular 

Genetics, Prague, Czech Republic 

Mast cells play a pivotal role in innate immunity, allergy and inflammation. They express plasma 

membrane-associated high-affinity IgE receptors (FcεRIs), the aggregation of which by 

multivalent antigen-IgE complexes triggers mast cell activation resulting in degranulation and 

release of inflammatory mediators such as histamine, proteases, lipid mediators, and cytokines. 

Tyrosine kinases of the Src family are crucial for triggered signaling pathways. Although FcεRI 

aggregation leads to reorganization of microtubules and their accumulation in the cell periphery 

(J. Cell Biol. 170:115,2005; J. Immunol. 176:7243,2006), the molecular mechanisms that control 

microtubule rearrangement after cell activation are poorly understood. Here we show that 

changes in cytosolic Ca 2+ concentration, controlled by store-operated Ca 2+ entry (SOCE), 

affected microtubule plus-end dynamics detected by plus-end tracking protein EB1 and led to 

generation of protrusions containing microtubules. Experiments with knock-down or 

reexpression of STIM1, the essential regulator of SOCE, confirmed the important role of STIM1 

in this process. Changes in Ca 2+ concentration also modulated interactions of γ-tubulin, the key 

player in microtubule nucleation, with γ-tubulin complex proteins (GCPs) as well as with other 

proteins that are substrates for tyrosine kinases of the Src family. Combined data suggest that 

rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and 

that Ca 2+ regulates the formation of microtubule protrusions in activated mast cells. 

2117 

Assembly of a millimeter-sized microtubule aster in a model cytoplasm. 

K. Ishihara 1 , C. M. Field 1 , A. C. Groen 1 , P. A. Nguyen 1 , T. J. Mitchison 1 ; 1 Department of 

Systems Biology, Harvard Medical School, Boston, MA 

The cytoplasms of large egg and embryonic cells are organized primarily by radial arrays of 

microtubules called asters. Prior to cell division, asters expand to cover the entire millimetersized 

cytoplasm in 20 minutes. Such rapid exploration of cell shape and size is thought to be 

crucial for positioning of centrosomes and cleavage plane determination (Wuhr et al., 2010, Curr 

Biol 20, 2040-45). It is unknown how microtubules (MTs) assemble at such a large distance 

from their presumable nucleation centers or centrosomes. We hypothesize three distinct 

processes that contribute to addition of MT at the aster periphery: (1) MT polymerization at plusends, 

(2) MT transport by minus-end directed motor proteins and (3) MT-stimulated MT 

nucleation. We used interphase Xenopus egg extract with either centrosomes or beads coated 

with Aurora A antibody (Tsai & Zheng, 2005, Curr Biol 15, 2156-63) to recapitulate aster growth 

and assembly. To quantify the MT polymerization dynamics in the interphase extract, we 

tracked single MT plus-ends using time lapse microscopy. MT transport was assayed within a 

growing aster by fluorescent speckle microscopy. We explored the consequences of noncentrosomal 

MT nucleation on aster growth by constructing a biophysical model of plus-end 

distribution. Our model suggests that MT-mediated MT nucleation, when coupled with plus-end 

dynamic instability, contribute to the aster expansion velocity depending on parameter values. In 

the depleting limits of a hypothetical MT nucleator, the model offers an explanation to how MT 

density is maintained at the aster periphery. Our work begins to explore the biophysical 

mechanisms underlying the assembly of a millimeter-sized cytoskeletal structure.

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2118 

Evaluating models of self-organisation of linear microtubule arrays by computer 

simulation. 

Y-C. Oei 1,2 , X. E. Studera 1 , R. E. Carazo Salas 1 , T. Duke 3 ; 1 The Gurdon Institute, University of 

Cambridge, Cambridge, United Kingdom, 2 Cavendish Laboratory, University of Cambridge, 

Cambridge, United Kingdom, 3 London Centre for Nanotechnology, University College London, 

London, United Kingdom 

In a wild-type fission yeast cell in interphase, microtubules (MTs) are organized in 3-4 bundles 

of 2-7 dynamic, anti-parallel MTs (interphase microtubule arrays, IMAs) whose correct number 

and organisation are essential for longitudinal cell growth and precise nuclear centring prior to 

mitosis (Sawin and Tran, Yeast 2006). Disruption of MT organisation, in cells mutant for certain 

genes or in cells treated with MT poisons, correlates with defects in cellular growth, shape 

and/or function. 

Within IMAs, MTs are thought to self-organise in a process that has been shown dependent on 

the bundling protein Ase1 and the motor protein Klp2, resulting in a configuration in which MT 

plus-tips point towards the cell periphery, with a central region where MTs overlap which is 

traditionally referred to as the microtubule organising centre (MTOC) (Janson et al, Cell 2007). 

Given the small number of constituent MTs, their continuous turnover, and the implied dynamic 

regeneration of IMAs, it is intriguing that the bundle number remains so stable at 3-4. Also, 

strikingly, cells that have had their nucleus and all IMAs removed eventually manage to reestablish 

IMAs in a configuration that resembles the wild-type (Carazo-Salas and Nurse, NCB 

2006). 

Aiming to understand how IMA number is regulated, we developed a computational Langevin 

simulation of interphase MT organisation, and use it to explore the influence of cell shape, of the 

concentrations of bundle constituents, and of the properties commonly attributed to them. 

2119 

Communication between Flagellar Outer and Inner Dynein Arms. 

T. Oda 1 , T. Yagi 1 , M. Kikkawa 1 ; 1 Department of Cell Biology, University of Tokyo Graduate 

School of Medicine, Tokyo, Japan 

The beating motion of cilia and flagella is driven by two rows of dynein arms: the outer dynein 

arms (ODA) and the inner dynein arms (IDA). The ODAs are required to generate the normal 

beat frequency and the IDAs are responsible for the amplitude of the waveform. Although 

structural connections between the ODA and the IDA have been observed by cryo-electron 

tomography of Chlamydomonas axoneme, the functional communication between the two 

species of the dynein arms has not been elucidated. In this study, we investigated the roles of 

the two intermediate chains (IC1 and IC2) of Chlamydomonas ODA. We constructed the 

expression vectors of IC1 and IC2 with biotinylation-tags added to the N- and the C-termini and 

introduced them into the Chlamydomonas strains deficient in the respective ICs. The 

transformants were selected with drug resistance and the recovery of the beat frequency. The 

ODAs containing the biotinylated ICs were extracted from the axoneme of the transformants. 

We solved the three dimensional structures of the biotin-streptavidin labeled ODA-microtubule 

complexes using cryo-electron microscopy. We located the positions of the termini of the IC1 

and IC2 near the β and the γ heavy chains, respectively. It is noteworthy that the location of the 

N-terminus of IC2 is close to the previously observed ODA-IDA linker. The biotin-tags at the Nterminus 

of IC1 and the C-terminus of IC2 did not alter the swimming phenotypes of the

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transformed Chlamydomonas cells, but the biotin-tag added to the N-terminus of IC2 reduced 

the amplitude of the beating by half while the beat frequency was not decreased. These results 

suggest the N-terminus of IC2 mediates the communication between the ODA and the IDA. 

2120 

A Gas8 Knockout Mouse Model Reveals Defects in Cilia Motility. 

W. R. Lewis 1 , E. Malarkey 1 , R. Pasek 1 , N. Berbari 1 , B. Yoder 1 ; 1 Cell Biology, University of 

Alabama-Birmingham, Birmingham, AL 

Mouse Growth Arrest Specific 8 (Gas8) has been associated with microtubules and more 

specifically the Dynein Regulatory Complex (DRC) where it is thought to play a role in cilia and 

flagella motility. While previous work on Gas8 has been done utilizing in vitro knockdown 

approaches and in model organisms such as Trypanosoma brucei and Chlamydomonas 

reinhardtii, this gene has not been studied in an in vivo mammalian genetic model. We have 

generated a Gas8 mutant mouse and found that mutants survive gestation; however they do not 

survive to sexual maturity. Consistent with a role in the DRC, it appears that the Gas8 mutant 

mice die as a result of severe hydrocephalus. Furthermore, analysis of ependymal cilia of the 

lateral ventricles of the brain indicated a severe defect in motility based on fluorescent bead 

tracking. Intriguingly, Gas8 is also expressed in cells that lack motile cilia, suggesting it has an 

additional role outside of the DRC. Recently, Gas8 has been implicated in Sonic Hedgehog 

signaling pathway using knockdown approaches where it has been proposed to be a positive 

regulator of the pathway activator Smoothened. However, our analysis of the Sonic Hedgehog 

pathway in the Gas8 genetic mutants using neural tube and limb bud patterning as readouts for 

pathway activity has yet to reveal any abnormalities consistent with a hedgehog defect. Future 

studies of this mouse model will focus on determining the precise nature of the motile cilia 

defect using high speed video waveform analysis and analyzing potential novel roles for Gas8 in 

cells with primary cilia. 

2121 

Cryo-Electron Tomography of Chlamydomonas Flagella Reveals Radial Spoke 

Heterogeneity and Connectivity. 

C. F. Barber 1 , T. Heuser 1 , B. I. Carbajal-Gonzalez 1 , V. V. Botchkarev Jr. 1 , D. Nicastro 1 ; 

1 Biology Department, Rosenstiel Center, Brandeis University, Waltham, MA 

Cilia and flagella are highly conserved cellular organelles critical to many cellular, tissue and 

developmental processes. The core structure of cilia and flagella, called the axoneme, consists 

of 96-nm long units that repeat along the axoneme length. Within each repeat are two or three 

(depending on species) radial spokes (RSs) that transiently interact with the central pair 

complex (Warner and Satir, 1974). Studies of the unicellular alga Chlamydomonas reinhardtii 

have revealed that the RSs play a critical role in transmitting regulatory signals from the central 

pair complex to the axonemal dyneins, ultimately controlling the activity of dynein proteins 

(reviewed Porter and Sale, 2000; Smith and Yang, 2004). In both Chlamydomonas and 

humans, failure to assemble RSs results in flagellar and ciliary paralysis (Sturgess et al., 1979; 

Witman et al., 1978). While several molecular components of the regulatory pathway have been 

identified, little is known about their specific locations or physical interactions. To shed further 

light on the mechanism of axonemal dynein regulation we used cryo-electron tomography and 

sub-tomogram averaging of Chlamydomonas axonemes to visualize the three-dimensional 

structure of the radial spokes in unprecedented detail. 

Unlike many other species, Chlamydomonas has only two spokes per 96-nm repeat, RS1 and 

RS2, which were till recently believed to be structurally and functionally homogenous. Our data

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reveal that while the overall structure of RS1 and RS2 is similar - including a two-pronged spoke 

base and direct connections to inner arm dynein tails - unique densities are seen in each spoke. 

The structural comparison of Chlamydomonas wild type and the mutant pf17 with head-less 

spokes, also revealed the morphology and boundaries of the RS head, including a direct RS1 to 

RS2 interaction. Finally, in place of a third radial spoke we found an uncharacterized, shorter 

density, the “Radial Spoke 3 Stand-in” (RS3S), which bears structurally no resemblance to RS1 

and RS2, and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes 

are heterogeneous in structure and may play functionally distinct roles in regulating the 

movement of cilia and flagella. 

References: 

Porter, M.E., and Sale, W.S. (2000). The 9 + 2 axoneme anchors multiple inner arm dyneins 

and a network of kinases and phosphatases that control motility. J Cell Biol 151, F37-42. 

Smith, E.F., and Yang, P. (2004). The radial spokes and central apparatus: mechano-chemical 

transducers that regulate flagellar motility. Cell Motil Cytoskeleton 57, 8-17. 

Sturgess, J.M., Chao, J., Wong, J., Aspin, N., and Turner, J.A. (1979). Cilia with defective radial 

spokes: a cause of human respiratory disease. N Engl J Med 300, 53-56. 

Warner, F.D., and Satir, P. (1974). The structural basis of ciliary bend formation. Radial spoke 

positional changes accompanying microtubule sliding. J Cell Biol 63, 35-63. 

Witman, G.B., Plummer, J., and Sander, G. (1978). Chlamydomonas flagellar mutants lacking 

radial spokes and central tubules. Structure, composition, and function of specific axonemal 

components. J Cell Biol 76, 729-747. 

2122 

How flagellum controls cell length. 

Y. Sun 1 , C. Y. He 1 ; 1 Department of Biological Sciences, National University of Singapore, 

Singapore 

Trypanosoma brucei is an extracellular parasite causing African sleeping sickness in humans 

and Nagana in cattle. This single-celled pathogen possesses a single flagellum attached to the 

cell body via flagellum adhesion proteins that link plasma membrane to flagellar membrane, and 

a complex flagellar attachment zone (FAZ) mostly located in the cell body. In addition to being a 

motility and sensory organelle, the flagellum also acts as a molecular ruler that controls cell 

morphogenesis. However, it remains to be understood how the length information contained in 

the flagellum is transmitted to the FAZ, which directly controls cellular microtubule biogenesis 

and organization. Here we focused on TbFla1, a surface glycoprotein required for adhesion 

between flagellar and cell membranes. Using immunoprecipitation of YFP tagged Fla1 and 

mass spectrometry, a Fla1 binding protein (Fla1BP) was identified and was localized to 

flagellum membrane. Procyclic T. brucei lacking Fla1BP showed no apparent growth defects, 

produced flagellum of normal length but shorter FAZ of relatively fixed length, uncoupling the 

regulation of cell length by flagellum. Our results provided a molecular basis for flagellum 

membrane adhesion to cell membrane and may explain how flagellum controls cell 

morphogenesis. 

2123 

A Neuronal Calcium Sensor Protein, Calaxin, is Essential for Modulation of Ciliary Beat 

Direction in Sea Urchin Embryos. 

K. Mizuno 1 , J. Yaguchi 1 , K. Shiba 1 , S. Yaguchi 1 , K. Inaba 1 ; 1 Shimoda Marine Research Center, 

University of Tsukuba, Shimoda, Shizuoka, Japan

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Calaxin is a neuronal calcium sensor (NCS)-family Ca 2+ -binding protein localized in cilia and 

flagella. Calaxin has three EF-hand Ca 2+ -binding motifs, and its orthologues are present in 

metazoan species, but not in yeast, green algae or plant. Previous research has shown that 

calaxin binds to outer arm dynein or β-tubulin in the presence or absence of Ca 2+ , respectively 

[Mizuno et al., Biol. Cell. 2009]. In addition, our recent studies showed that inhibiting calaxin 

function greatly reduced the duration of asymmetrical waveform, resulting in the suppression of 

turning movement during sperm chemotaxis. These results indicate that calaxin has an 

essential role in the control of flagellar waveform through the control of outer arm dynein activity 

in Ca 2+ -dependent manner. However, we still do not understand the whole mechanisms of 

calaxin function throughout the animal kingdom, especially in their cilia. Here, we tried the 

functional analysis of calaxin in somatic ciliary movement in the embryo of the sea urchin, 

Hemicentrotus pulcherrimus, because their cilia are suitable for motility analysis and it is easy to 

perform knockdown experiments using morpholino antisense oligo (MO). Immunostaining 

showed calaxin was localized to the entire region of sea urchin embryonic cilia. To study the 

calaxin function, we analyzed the motility of normal and calaxin-MO embryos using a system of 

digital-camera equipped dark field illumination. calaxin morphants swam much slower than 

normal embryos and showed abnormal swimming trajectories. Detailed analysis using a highspeed 

camera, however, revealed that cilia of calaxin morphants beat with normal frequency. 

On the other hand, beating direction of cilia in calaxin morphants was greatly disorganized as 

compared with cilia of normal embryos that show highly organized beat direction from the 

animal pole toward the vegetal pole. These results suggest that calaxin is required to coordinate 

ciliary beat direction by controlling outer arm dyneins. Furthermore, we observed that 

gastrulation was greatly inhibited in calaxin morphants. Although the mechanism how the 

gastrulation was inhibited in calaxin morphants is not clear, it is possible that calaxin is involved 

in the establishment of planer cell polarity and contributes to the convergent extension of 

embryonic cells. 

2124 

Loss of basal feet on Odf2 deletion perturbs polarization of basal bodies. 

K. Kunimoto 1 , Y. Yamazaki 1 , T. Nishida 2 , K. Shinohara 3 , H. Ishikawa 4 , T. Hasegawa 2 , T. 

Okanoue 5 , H. Hamada 3 , T. Noda 6 , A. Tamura 1 , S. Tsukita 7 , S. Tsukita 1 ; 1 Laboratory of Biological 

Science, Department of Biological Science, Osaka University, Suita, Japan, 2 Research Center 

for Ultra-high Voltage Electron Microscopy, Osaka University, Ibaraki, Japan, 3 Developmental 

Genetics Group, Department of Biological Science, Osaka University, Suita, Japan, 

4 Department of Biochemistry and Biophysics, UCSF, San Francisco, 5 Department of 

Hepatology, Saiseikai Suita Hospital, 6 Department of Cell Biology, Cancer Institute of Japanese 

Foundation for Cancer Research, Tokyo, Japan, 7 Department of Cell Biology, Kyoto University, 

Kyoto, Japan 

Synergic multiciliary beating relies on cilia generated from basal bodies, with which basal feet 

are regularly associated through molecular mechanisms that remain unknown. Here we show 

that the coordinated multiciliary action is disturbed in Odf2 mutant mice, resulting in primary 

ciliary dyskinesia and a characteristic coughing/sneezing-like phenotype. Odf2 mutant depleted 

basal feet from basal bodies to perturb the planar cell polarity (PCP) of basal bodies, as shown 

by ultra-high voltage electron microscopic tomography of wild and Odf2 mutant tracheas. The 

apical microtubular lattice, which is organized by the keystone positioning of basal feet/basal 

bodies, was lost in Odf2-mutant animals, irrespective of normal localization of Vangl1, the PCP 

core protein. These findings demonstrate that Odf2 is required for the formation of basal feet. 

Odf2-based basal feet play a critical role in the PCP-based arrangement of the microtubular 

lattice and basal bodies, thereby enabling coordinated multiciliary beating.

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2125 

Functional Analysis of a Gliding-Associated Ciliary Protein with Putative Lipase Activity. 

E. Betleja 1 , T. Birch 1 , R. A. Bloodgood 2 , J. L. Watts 3 , D. G. Cole 1 ; 1 Dept of Biological Sciences, 

University of Idaho, Moscow, ID, 2 Dept of Cell Biology, University of Virginia School of Medicine, 

Charlottesville, VA, 3 School of Molecular Biosciences, Washington State University, Pullman, 

WA 

The unicellular green algae Chlamydomonas reinhardtii exhibits two independent forms of 

whole cell locomotion, both utilizing the flagellum: swimming through a liquid medium and 

gliding across a solid substrate. Essential for the assembly and function of these ciliary 

organelles is the bidirectional transport of protein particles known as intraflagellar transport 

(IFT). In a search for IFT-associated cargo, pull down assays of Chlamydomonas flagellar 

extract with the retrograde IFT motor, cytoplasmic dynein 1b/2, yielded a set of proteins, among 

which the most prominent were the major flagellar surface glycoprotein, FMG-1B and a flagellarassociated 

protein of unknown function, which we have named GAP12 (gliding-associated 

protein 12). This finding suggests that the retrograde IFT motor may play a role in ciliarydependent 

gliding motility. In contrast to a well established role for FMG-1B which acts as a 

transmembrane gliding receptor that contacts substrate during gliding motility, the biological 

function of GAP12 is unknown. To address the functional roles of GAP12, an artificial micro 

RNA interference was used to reduce expression of the GAP12 gene. Western blot analysis of 

whole cellular extracts from RNAi transformants showed that in 14 out of 28 knockdown lines 

GAP12 protein level was significantly reduced. Selected GAP12-RNAi lines were assayed for 

gliding phenotype. Some of the knockdown strains had difficulty attaching to the glass slides 

and when they did attach, they would glide for only short distances of a few microns before 

detaching. The observation that GAP12-deficient cells are blocked at the stage of adherence 

suggests that GAP12 is involved in an early stage of gliding motility. Because GAP12 contains a 

putative lipase domain, we also looked for effects on lipid metabolism. By shifting cells into a 

nitrogen-deficient medium, Chlamydomonas is forced to accumulate greater levels of 

triglycerides in specialized storage cytosolic organelles known as lipid bodies. When GAP12deficient 

strains were examined after 72 hrs of nitrogen starvation, the lipid-soluble fluorescent 

Bodipy dye revealed enlarged lipid bodies, which would be consistent with a possible role for 

GAP12 lipase activity in the degradation or hydrolysis of lipid body triglycerides. We are 

currently testing, at the biochemical level, if GAP12-RNAi knockdown strains accumulate 

specific fatty acids when starved in N-free medium. Any further analysis of GAP12 will need to 

consider this potential role in lipid metabolism, which may or may not be independent of gliding 

motility. 

2126 

Monte Carlo simulation of centrosomal self-centering due to pushing by microtubules in 

large cells. 

S. K. Tang 1,2 , B. T. Castle 3,2 , D. J. Odde 2,3 ; 1 Department of Mechanical Engineering, Stanford 

University, Stanford, CA, 2 Physiology Course 2011, Woods Hole Marine Biological Laboratory, 

Woods Hole, MA, 3 Department of Biomedical Engineering, University of Minnesota, 

Minneapolis, MN 

The overarching questions of our work are: what drives self-centering in cells, and if cells with 

different sizes use different mechanisms for centering? Finding the center in a cell is critical for 

many processes such as cell division. Various models for self-centering exist. Here we 

implement a 1D Monte Carlo model to simulate the centering of centrosome (or other 

microtubule-nucleating site) due to the pushing forces generated when microtubules reach the 

boundary of the cell. Our model consists of one centrosome that is originally positioned close to

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the cell boundary. Microtubules nucleate at this site, and grow/shrink in length governed by 

experimentally measured dynamic instability parameters. Our model assumes that the cell 

boundary is rigid, and that microtubules push the whole length of the microtubule backwards, 

causing the nucleating site to move. 

Our model shows that the growth and pushing of multiple microtubules (up to 1000 in number) 

in opposing directions can center the centrosome in cells smaller than 500-600 microns in 

diameter. However, with the experimental dynamic instability parameters, microtubules do not 

grow longer than 250-300 microns within ~90 min, a typical time scale for first division in 

Xenopus eggs (diameter ~ 1.2 mm). As microtubules do not reach the cell boundary, the 

pushing model alone cannot explain centering of centrosomes for cells with diameters larger 

than 600 microns. In addition, the centering time scale is highly sensitive to small variations in 

microtubule dynamics. We conclude that the pushing model is not a robust centering 

mechanism in large cells and that other mechanisms must play a role in driving centering in 

these cells. 

2127 

A Sas-4/tubulin module regulates the formation of centrosomal complexes and 

centrosome biogenesis. 

J. Gopalakrishnan 1 , Y-C. F. Chim 1 , A. Ha 1 , M. Basiri 1 , T. Avidor-Reiss 1 ; 1 Harvard Medical 

School, Boston, MA 

Centrosomes are conserved organelles that are essential for cilium formation and accurate cell 

division, and centrosome abnormality can result in genomic instability. A centrosome consists of 

a pair of centrioles surrounded by a protein network of pericentriolar material that is essential for 

centrosome function. Pericentriolar material assembly is a tightly regulated, critical step in 

centrosome biogenesis that determines a centrosome’s size and capability. Here we show that 

a Sas-4/tubulin module regulates the formation of centrosomal complexes and centrosome 

biogenesis. Using a combination of genetic and biochemical approaches in Drosophila, we 

found that Sas 4 and tubulin regulate the formation of several cytoplasmic complexes, which 

include CNN, Asl and D-PLP (named S-CAP complexes) and γ-tubulin ring proteins (named S-γ 

complexes). The proteins of these complexes are components of the pericentriolar material and 

are essential for pericentriolar material assembly and for the centrosome’s capacity to nucleate 

microtubule asters. Interestingly, the S-CAP complex contains several microcephaly-linked 

proteins and disturbing this complex causes abnormal centrosome formation. The finding that 

microcephaly-linked proteins form a common complex may explain why mutations in any of 

these lead to the same disorder. Tubulin (α-and β-tubulin heterodimer) is known to be a building 

block of microtubules. Our data points to a new and novel function for tubulin as a regulator of 

centrosome complex formation, binding to Sas-4 and regulating the interaction of Sas-4 with 

other centrosomal proteins. By regulating centrosome complex formation, the Sas-4/tubulin 

module regulates centrosome biogenesis. 

2128 

Mechanisms of Centrosome Separation. 

B. R. Mardin 1 , F. G. Agircan 1 , C. Lange 1 , A. M. Fry 2 , E. Schiebel 1 ; 1 ZMBH Uni Heidelberg, 

Heidelberg, Germany, 2 University of Leicester, United Kingdom 

In human cells, duplicated centrosomes are first held together by a proteinaceous linker that 

extends between the proximal ends of the two mother centrioles. To facilitate centrosome 

separation and bipolar spindle formation at mitotic entry, this linker is disassembled in G2/M in a 

process known as centrosome disjunction. Centrosome disjunction is driven by the Nek2A

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kinase, which phosphorylates the linker proteins, C-Nap1 and rootletin. Previously we 

demonstrated that the ability of Nek2A to initiate centrosome disjunction is controlled by two 

components of the Hippo pathway, the mammalian sterile 20-like kinase 2 (Mst2) and the 

scaffold protein Salvador (hSav1). Conventionally, Hippo pathway proteins are tumor 

suppressors that are well known for their function in tissue growth and apoptosis. By forming a 

separate module, hSav1 and Mst2 associate with and regulate the localized activity of Nek2A by 

phosphorylation. Our recent data revealed that the Mst2-Nek2A kinase module is regulated by 

the Polo-like kinase 1 (Plk1). Plk1 selectively binds to and phosphorylates Mst2, which controls 

the binding of Nek2A to the phosphatase PP1γ providing a balance between the counteracting 

Nek2A and PP1γ activities on the centrosome linker. 

Importantly, we provided the first evidence that the Mst2-Nek2A module has overlapping 

functions with the kinesin motor protein Eg5 in centrosome separation and bipolar spindle 

formation. In fact, in cells with reduced Eg5 motor activity, the hSav1-Mst1/2-Nek2A pathway 

becomes essential for bipolar spindle formation. Plk1 is crucial for the targeting of Eg5 to the 

centrosomes and this process is supported by the microtubule cytoskeleton. Taken together, 

our data significantly contribute to the understanding of how centrosome disjunction and spindle 

formation are regulated on the molecular level. 

2129 

Differentiating Naegleria assemble two basal bodies de novo by counting from 0 to 1 to 2. 

L. Fritz-Laylin 1 , Y. Levy 2 , E. Levitan, S. Chen, Z. Cande 3 , E. Lai 4 , C. Fulton 4 ; 1 University of 

California, San Francsico, San Francisco, CA, 2 CLARIFY Scientific and Medical 

Communications, 3 Department of Molecular and Cell Biology, University of California, Berkeley, 

4 Department of Biology, Brandeis University, Waltham, MA 

Naegleria gruberi differentiate from amoebae to flagellates in about an hour. This phenotypic 

transformation requires the de novo assembly of an entire microtubule cytoskeleton, including 

two basal bodies (centrioles) and flagella. The formation of new basal bodies occurs about 10 

minutes before the appearance of visible flagella. Here we use antibodies against tubulin, 

centrin, and SAS-6 to reveal that the sequence of basal body formation proceeds in two distinct 

steps: first one basal body forms de novo, after which a second basal body assembles adjacent 

to the first. This second round of basal body assembly is similar to the centriole assembly 

typically seen in human cells. Interestingly, when microtubule assembly is temporarily blocked 

using a pulse treatment of oryzalin, followed by inhibition of protein synthesis with 

cycloheximide, cells form a single flagellum. Because these cells successfully assemble two 

basal bodies but only a single flagellum, this demonstrates that there is a moment in 

differentiation when one basal body is competent to template flagellar assembly while the other 

is not. Naegleria utilizes both commonly studied modes of centriole assembly, de novo and 

template-associated, and forms two basal bodies in a 0 to 1 to 2 sequence rather than 

simultaneously. 

2130 

Microtubule anchoring is facilitated by the shoulder/sidearm subunits of the dynactin 

complex. 

A. G. Le 1 , R. L. Schneider 1 , L. M. McCullough 2 , N. J. Quintyne 2 ; 1 Florida Atlantic University, 

Jupiter, FL, 2 Harriet L. Wilkes Honors College, Florida Atlantic University, Jupiter, FL 

Dynactin is a multifunctional protein complex identified as a required cofactor for cytoplasmic 

dynein. Dynactin also plays a dynein-independent role in anchoring microtubules to the 

centrosome during interphase. Dynactin is the only component of the microtubule anchoring 

complex that has been shown to directly bind to microtubules. Previous studies have shown that

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the shoulder/sidearm region of dynactin, consisting of p150Glued, dynamitin and p24 are 

integral to this anchoring process. The p150Glued subunit of dynactin contains two microtubule 

binding domains of different affinity. The CAP-Gly domain binds microtubules tightly, whereas 

the Basic domain has a much weaker affinity for the microtubule. The function of p24 remains 

largely uncharacterized. Using overexpression and siRNA-mediated knockdown, we have 

examined the contribution of two of the three shoulder/sidearm subunits to microtubule 

anchoring in more detail. We find that anchoring is abrogated when either of these subunits 

expression levels are changed. We observe both microtubule organization and regrowth 

patterns to vary when comparing knockdown and overexpression. Our data suggest a model 

where the different microtubule-binding domains of p150Glued contribute unequally to 

microtubule anchoring and p24 serves as reinforcement for maintaining shoulder/sidearm 

rigidity at the centrosome. 

2131 

Centrosome inactivation in Drosophila oogenesis. 

I. Bento 1 , M. Bettencourt-Dias 1 ; 1 Instituto Gulbenkian de Ciência, Oeiras, Portugal 

Centrosome is the primary microtubule-organizing center (MTOC) in animal cells, whose 

number is highly controlled. Duplication of this structure occurs in parallel with DNA replication. 

During development there are tissues where centrosome number is differently regulated. The 

zygote formation is one of them. During oogenesis, centrioles degenerate and female meiosis 

relies on an acentrosomal spindle. On the other hand, during spermatogenesis, the centriole 

that nucleates the axoneme looses pericentriolar (PCM) proteins. After fertilization, centrioles 

are paternally inherited and the PCM proteins are deposited by the mother. The absence of 

centrioles in the egg has been hypothesized as a strategy to avoid parthenogenesis. This is 

reinforced by the observations that naturally occurring parthenogenic species are able to form 

de novo centrosomes. Using Drosophila melanogaster oogenesis, we aim to understand how 

centrioles disappear during oogenesis. We are characterizing Drosophila melanogaster 

oogenesis by immunostaining. We observed that proteins such as Bld10, Asl, SAS6 or SAS4 

are present in the oocyte´s MTOC until stage 10-11 although they are absent from the meiotic 

spindle. We noticed that the isolated overexpression of proteins involved in centriole biogenesis 

does not inhibit centriole disappearance in later stages of oocyte development, suggesting 

those are not limiting factors. Interestingly, Spd2, a PCM protein, is the first protein that 

disappears from the oocyte´s MTOC. Currently we are testing if PCM maintenance is crucial to 

keep centriole stability. Oogenesis characterization leads to better understanding of centriole 

inactivation and disappearance. 

2132 

Regulation of the Formation of Microtubule Organizing Centers. 

F. Leal 1,2 , S. Kandel-Lewis 2 , E. Karsenti 2 , M. Bettencourt-Dias 1 ; 1 Instituto Gulbenkian De 

Ciência, Oeiras, Portugal, 2 European Molecular Biology Laboratory, Heidelberg, Germany 

Centrosomes were originally discovered in the late 19 th century [1] , being named after their 

central position in the cell, and characterized by Theodor Boveri [2] . They are composed by a pair 

of orthogonally oriented centrioles, microtubule barrel shaped structures organized in a nine-fold 

symmetry, and a surrounding electron-dense material composed of many proteins, named 

pericentriolar material (PCM). The centrosome is the primary microtubule-organizing centre 

(MTOC) in animal cells regulating cell motility and polarity in Interphase and facilitating the 

organization of the spindle poles during Mitosis. Also, in animals, centrioles retain the ability to 

migrate to the cell membrane and form basal bodies, thus templating the assembly of cilia and 

flagella, important structures in motility and signalling and sensory perception.

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Centrosomes undergo duplication once every cell cycle so that, like the genetic material, their 

number remains stable. Abnormalities in centrosome number and structure occur in many 

cancers and are associated with genomic instability. In 2005, two separate studies [3-4] showed 

the importance of SAK/Plk4, a kinase form the serine/threonine family of polo-like kinases, in 

centriole duplication. Depletion or overexpression of this kinase led to the depletion or 

overduplication of centrioles in both Drosophila and human cells. Although SAK/Plk4 has been 

shown to be essential for centrosome duplication and biogenesis, very little is known about its 

regulation, due to the lack of knowledge of interacting partners. In order to understand the 

regulation and activity of this important kinase we are taking advantage of the biochemical 

system per excellence, the Xenopus laevis cell free extract to approach the following questions: 

1. How is SAK/Plk4 activity regulated? 

2. How does SAK/Plk4 activity regulate centriole formation? 

3. How do centrioles regulate their own biogenesis? 

Up to now we have seen that the predicted Xenopus laevis ortholog (XPLK4) is able to 

induce the formation of MTOCs which contain centriolar markers. The number and size is 

directly proportional to the concentration of XPLK4 used, suggesting that this kinase specifically 

controls centriole biogenesis. We have also seen that addition of this kinase in different stages 

of the cell cycle leads to different outcomes in terms of centriole biogenesis. Strikingly, meiotic 

extracts are not permissive for centriole biogenesis suggesting differential regulation of the 

kinase activity or of other participants in this process. 

[1] Flemming, W. (1875). Studien uber die Entwicklungsgeschichte der Najaden. Sitzungber 

Akad Wissensch Wien; 71: 81-147; 

[2] Boveri, T. (1900). Zellen-Studien: Uber die Natur der Centrosomen. Fischer: Jena; 

[3] Bettencourt-Dias, M. et al. Curr Biol 15, 2199-207 (2005); 

[4] Habedanck, R. et al. Nat Cell Biol 7, 1140-6 (2005); 

2133 

The temperature-sensitive ipl1-2 mutation in yeast is suppressed by mutations altering 

kinetochore components Duo1 and Ndc80. 

K. Tatchell 1 , L. Brou 1 , J. Phillips 1 , E. P. Boswell 1 , L. C. Robinson 1 ; 1 Biochemistry and Molecular 

Biology, Louisiana State University Health Sci Ctr, Shreveport, LA 

Ipl1/Aurora B is the catalytic subunit of a complex that is required for chromosome segregation 

and nuclear division. Prior to anaphase, Ipl1 localizes to kinetochores, where it is required to 

establish proper kinetochore-microtubule associations and regulate the spindle assembly 

checkpoint (SAC). At anaphase, the complex migrates to the spindle, where it stabilizes the 

spindle and regulates cytokinesis. We have taken a genetic approach in budding yeast to 

investigate Ipl1/Aurora B activity by isolating and characterizing mutations that suppress the 

inviability of the temperature-sensitive ipl1-2 mutant at non-permissive temperatures. These 

suppressors include three intragenic, second site revertants in IPL1, 19 mutations in 

components of the Glc7 phosphatase previously reported to suppress ipl1 (GLC7, SDS22, 

YPI1, and SHP1), and a null mutation in TCO89, which encodes a subunit of the TORC1 

complex. Two revertants contain missense mutations in kinetochore components. rev76 

contains a missense mutation in DUO1, an essential component of the microtubule binding 

DASH/DAM1 complex. The mutant is cold sensitive and arrests in G2-M due to activation of the 

SAC. rev8 contains the missense mutation ndc80-K204E, which corresponds to K116 in the CH 

domain of human Ndc80/Hec1. In vitro biochemical studies on human Ndc80 indicate that 

Ndc80-K166E binds weakly to microtubules (Ciferri et al Cell 133, 427-439, 2008). In a wild-type 

IPL1 background, ndc80-K204E cells grow slowly and the SAC is activated, consistent with the 

possibility that kinetochores bind more weakly to microtubules in the ndc80-K204E mutant. The 

slow growth and cell cycle delay of ndc80-K204E cells are partially alleviated by the ipl1-2

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mutation. These data are consistent with the published model that the NDC80 complex makes 

electrostatic interactions between negatively charged regions of Ndc80 and a positively charged 

C-terminal domain of tubulin. Reduction in the negative charge on Ndc80 can compensate for 

reduced Ipl1 activity. 

Cell-Matrix and Cell-Cell Interactions 

2134 

A discoidin domain of the Del1 protein is essential and sufficient for its deposition in the 

extracellular matrix. 

C. Hidai 1 , H. Kitano 2 , A. Mamiya 1 , S. Kokubun 1 ; 1 Biomedical Sciences, Nihon University School 

of Medicine, Itabashi-ku, Japan, 2 Dental surgery, Nihon University School of Medicine, Itabashiku, 

Japan 

Objective: Because the organization of the ECM directly influences tissue structure and cell 

behavior, determination of how ECM organization is regulated can help clarify the mechanisms 

of development and diseases. In the present study, we analyzed how Del1, an ECM protein, is 

deposited in the ECM. Del1 consists of three epidermal growth factor repeats (E1–E3) at its Nterminus 

and two discoidin domains (C1 and C2) at its C-terminus. The aim of this study was to 

investigate which domains of Del1 mediate its deposition in the ECM. 

Materials & Methods: We prepared cDNAs corresponding to fusion proteins of heat-stable 

alkaline phosphatase (AP) and Del1 deletion mutants of various lengths. For in vitro 

experiments, Cos7 cells were transfected with these cDNAs and the ratio of AP activity in the 

ECM and the medium was measured. To evaluate deposition of Dell and its fragments into the 

ECM in vivo, the cDNAs encoding the AP fusion proteins were cloned into a non-viral vector and 

injected intravenously into mice. Exogenous AP activity in serum or in liver was measured and 

AP staining of tissue sections was performed. 

Results: In vitro experiments using AP-fusion constructs of various deletion mutants of Del1 

showed that fragments containing the C-terminus of C1, which has a lectin-like structure, are 

deposited in the ECM. Digestion of these ECM with hyaluronidase from bovine testis released 

Del1 from the ECM, suggesting that glycosaminoglycans are involved in the deposition of Del1. 

The efficiency of Del1 C1-dependent deposition varied according to the presence of other Del1 

domains. A Del1 fragment containing E3 and C1 showed the highest deposition. An 

immunoprecipitation assay suggested that C2, which is highly homologous to C1, inhibits C1 

deposition by competitive binding to E3. In vivo gene transfer experiments showed that fusion of 

exogenous proteins with the C1 deposition domain of Del1 dramatically altered the distribution 

of these proteins in mice. 

Conclusions: This study suggests that C1 is essential and sufficient for deposition of Del1 into 

the ECM in vitro and in vivo. The deposition of Del1 could be regulated intramolecularly by its 

E3 and C2 domains. 

2135 

Tissue tension controls spatially patterned invasion of mammary epithelial tissue by 

regulating EMT. 

K. Lee 1,2 , N. Gjorevski 1 , C. M. Nelson 1,2 ; 1 Chemical & Biological Engineering, Princeton 

University, Princeton, NJ, 2 Molecular Biology, Princeton University, Princeton, NJ 

Cells within tissues constantly experience physical forces and dynamically adapt to these 

mechanical cues by modifying their behavior and remodeling their environment. Here we 

characterized the mechanical stress profile of mammary epithelium and determined how tissue

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mechanics regulated tissue behavior. We found that the mechanical rigidity of the 

microenvironment was distributed non-uniformly in three-dimensional tissues and this patterned 

mechanical tone played a critical role in determining sites of mammary invasion. Cells within 

regions of high mechanical stress were surrounded by a stiff extracellular matrix (ECM), and 

were also more prone to invade. Moreover, the spatial distribution of mechanical stress was 

significantly correlated with patterned expression of genes associated with epithelialmesenchymal 

transition (EMT), which promotes the switch to a motile phenotype. Changes in 

tissue rigidity altered cellular behaviors including cell morphology and scattering, and the 

expression of EMT markers. These findings indicate that tissue mechanics plays a pivotal role in 

regulating invasion of mammary epithelial cells by promoting EMT. 

2136 

An EGF motif of Del1 induces apoptosis in vitro. 

H. Kitano 1 , A. Mamiya 1 , M. Komiya 1 , S. Kokubun 2 , C. Hidai 2 ; 1 Division of Oral Surgery, Nihon 

University School of Medicine, Tokyo, Japan, 2 Department of Biomedical Sciences, Nihon 

University School of Medicine 

Introduction: Most cells cannot survive without integrin-mediated adherence to the extracellular 

matrix (ECM). However, some ECM proteins induce cell death, such as CCN1, which induces 

apoptosis in fibroblasts via binding with integrin ?6?1 and syndecan-4. The ECM controls cell 

behavior by providing either pro-survival or pro-death signals to cells. Dell, an ECM protein, is 

known to show pro-angiogenic or anti-angiogenic activities depending on the experimental 

conditions. It consists of two discoidin domains (C1, C2), and three epidermal growth factor 

(EGF) motifs (E1, E2, E3), of which E2 is reported to contain an RGD sequence that binds to 

integrin receptors and supports cell survival. In the present study, we provide evidence that E3 

induces apoptosis in vitro. 

Materials & Methods: Several cell lines were treated with recombinant Del1 or its mutants. 

Apoptosis of the cells was evaluated with DNA laddering, chromatin condensation, annexin V 

staining and TUNEL staining. The signal transduction of apoptosis was analyzed using an 

inhibitor of caspase. 

Results: Gene transfer of Del1 induced cell death in cultured cells. DNA laddering, and the 

results of annexin V staining and TUNEL staining were consistent with apoptosis. The 

experiments with Del1 mutant proteins with deletions or point mutations revealed that apoptotic 

activity was localized in E3 (34 amino acids). Because an inhibitor of caspase suppressed E3 

and induced apoptosis, it appears that signaling by E3 is caspase dependent. 

Discussion: In the Del1 protein, there is a cell death-inducing domain, E3, adjacent to the RGD 

sequence, which supports cell viability. The presence of domains with opposing activities next to 

one another in the same protein could account for the seemingly contradictory characteristics of 

Del1 activity in various cells. Thus, it is likely that Del1functions as a balancing factor between 

life and death. In the present study, we also showed that the efficiency of cancer gene therapy 

with the cytotoxic FasL gene can be increased by the addition of the E3 domain. 

2137 

Exploring the syndecan-4 adhesome. 

M. Montesion 1 , C. Flynn 1 , J. Ye 1 , M. Bonnet 1 , M. Frigault 1 , R. Bellin 1 ; 1 Biology, Holy Cross 

College, Worcester, MA 

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that has been found in almost all 

mammalian cell types. Specifically, syndecan-4 is localized most commonly to focal adhesion 

complexes, and is thought to play a role in linking cells to matrix adhesion sites. In our recent 

work, we have demonstrated that the attachment of cells to surfaces that are engineered to

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solely support syndecan-4-based connections caused the recruitment of the focal adhesion 

complex proteins vinculin and talin to adhesion sites. In addition, we have shown that cells 

adhered solely through syndecan-4 molecules are able to transmit external mechanical strain 

resulting in activation of the MAPK signaling pathway within the cell. Currently, we are working 

to build on these findings by conducting experiments to identify the full collection of proteins that 

are recruited to sites of cell adhesion when cells are subjected to syndecan-4 specific adhesion 

conditions--i.e., the syndecan-4 adhesome. To conduct these studies, fibroblast cells are grown 

on non-specific growth surfaces, and then treated with anti-syndecan-4 core protein antibodies 

coated on magnetic core microbeads to cause the generation of new adhesion complexes at 

sites of cell attachment to the microbeads. After subjecting cells to a reversible, membranepermeable 

chemical crosslinker to stabilize protein interactions, the adhesion complexes are 

collected on a magnet after cell lysis, the cross-linker based connections are broken and 

proteomic techniques are used to probe for specific proteins of interest, or for the contents of 

the entire complex. Efforts such as these have been conducted with a focus on specific integrinbased 

adhesion conditions, and we are very interested to compare our syndecan-4 based 

findings to the integrin projects' results, especially in light of long term questions in the literature 

of the need for both syndecan-4 and integrin molecules within focal adhesion complexes. 

2138 

Inverse Regulation between Integrin α2β1 and Laminin Receptors. 

L. Dao 1 , C. M. Franz 1 ; 1 Center for Functional Nanostructures, Karlsruhe Institute of Technology, 

Karlsruhe, Germany 

Integrin α2β1, a well-characterized collagen receptor, has repeatedly been reported to function 

as a laminin receptor, but its relative contribution to collagen and laminin binding has not been 

determined. In this work, we compared α2β1-mediated adhesion to bifunctional adhesion 

substrates consisting of alternating collagen type I and laminin stripes. CHO cells which do not 

express collagen–binding integrins, showed a highly polarized morphology and adhere only on 

collagen stripes, whereas CHO cells stably expressing α2β1 (CHO-A2) polarized strongly on 

collagen, indicating a preference of α2β1 for collagen over laminin. To directly compare α2β1mediated 

adhesion strength to collagen and laminin, we quantitated adhesion forces using 

single-cell force spectroscopy (SCFS). As expected, CHO-A2 cells adhered more strongly to 

collagen than wildtype cells. Comparable results were obtained for α2β1-expressing (SAOS-A2) 

and α2β1-deficient wildtype (SAOS-WT) human osteosarcoma cells. In turn, CHO-WT and 

SAOS-WT cells showed significantly stronger adhesion to laminin than the corresponding α2β1expressing 

cells, pointing towards a suppressing effect of α2β1 expression on laminin-binding. 

In agreement, RT-qPCR and western blot analysis of α2β1-expressing cells revealed a 

downregulation of integrin subunits α6 and β4, both components of the major laminin-binding 

integrin receptors α6β1 and α6β4. In conclusion, we have shown that α2β1 is an efficient 

laminin but not collagen receptor. Instead, α2β1 expression decreased laminin binding and α6 

and β4 expression, suggesting an inverse regulation of α2β1 and laminin receptors. 

2139 

Prolines in βA-sheet of neural cadherin act as a switch to control the dynamics of the 

equilibrium between monomer and dimer. 

N. Vunnam 1 , S. Pedigo 1 ; 1 Chemistry and Biochemistry, University of Mississippi, University, MS 

Neural cadherins dimerize through the formation of calcium-dependent strand-crossover 

structures. Dimerization of cadherins leads to cell-cell adhesion in multicellular organisms. 

Strand-crossover dimer forms exclusively between the first N-terminal extracellular modules 

(EC1) of the adhesive partners via swapping of their βA-sheets and docking of tryptophan-2 in

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the hydrophobic pocket. In the apo-state wild-type cadherin is predominantly monomer, which 

indicates that the dimerization is energetically unfavorable in the absence of calcium. Addition of 

calcium favors dimer formation by creating strain in the monomer and lowering the energetic 

barrier between monomer and dimer. Dynamics of the monomer-dimer equilibrium is vital for 

plasticity of synapses. Prolines recurrently occur in proteins that form strand-crossover dimer 

and are believed to be the source of the strain in the monomer. N-cadherins have two proline 

residues in the βA-sheet. We focused our studies on the role of these two prolines in calciumdependent 

dimerization. Spectroscopic, electrophoretic, and chromatopgraphic studies showed 

that mutations of both prolines to alanines increased the dimerization affinity by ~20-fold and 

relieved the requirement of calcium in dimerization. The P5A and P6A mutant formed very 

stable dimers that required denaturation of protein to disassemble in the apo conditions. In 

summary, the proline residues act as a switch to control the dynamics of the equilibrium 

between monomer and dimer which is crucial for the plasticity of synapses. 

2140 

Calmodulin-dependent CEACAM1 monomer-dimer equilibrium of in epithelial cells. 

P. C. Patel 1 , H. S. Lee 1 , S. D. Gray-owen 1 ; 1 Department of Molecular Genetics, University of 

Toronto, Toronto, ON, Canada 

Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) can associate with 

themself and/or other CEACAM members in a manner that controls cellular growth and 

differentiation. CEACAM1 has previously been shown to exist in both a cis- (parallel) and a 

trans- (antiparallel) homophilic dimer. In this study, we used epithelial cells to determine what 

controls the homophilic dimerization of CEACAM1 and describe the relative effect of the 

monomeric versus dimeric conformation on downstream effector recruitment and cellular 

responses that are mediated by CEACAM1. To establish a causal relationship between the 

oligomeric state of CEACAM1 and its biological activities, we employed mutagenic analysis to 

identify specific residues that are important for CEACAM1 dimerization. In addition, we provide 

evidence that CEACAM1 dimerization is controlled by calmodulin activation, with increasing 

intracellular free calcium levels promoting dissociation of the oligomeric state in a process that 

requires the CEACAM1 cytoplasmic domain. This stabilization of the monomeric form has 

implications on tyrosine phosphatase SHP-1 recruitment, CEACAM1-dependent bacterial 

engulfment by epithelial cells, and cellular aggregation. 

2141 

Cell-to-Cell Fusion and Links to Cancer Cell Migration 

J. E. Boutilier 1,2 , R. Duncan 1,2 ; 1 Microbiology and Immunology, Dalhousie University, Halifax, 

NS, Canada, 2 Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada 

Growing evidence suggests that cell-cell fusion may play a role in the initiation and/or 

progression of cancer. Our lab studies a unique group of reovirus-encoded cellular fusogens 

termed FAST (fusion-associated small transmembrane) proteins, which serve as ideal models 

to study the links between cancer and cellular fusion. A genetic protein-protein interaction 

screen identified the receptor protein tyrosine phosphatase mu (PTPµ) as a binding partner of 

the p14 FAST protein. PTPµ is a member of the Ig superfamily and is inhibitory to cellular 

migration via its involvement in regulating adherens junction formation and stability. In 

glioblastoma cells, migration and dispersal is regulated by the proteolytic cleavage and downregulation 

of PTPµ. Moreover, while normal prostate cancer cells express PTPµ, invasive 

prostate cancer cells do not. Co-immunoprecipitation and truncation studies indicated that p14 

interacts with the membrane-proximal portion of the cytoplasmic tail of PTPµ, which includes the 

active phosphatase domain. Expression of a catalytically null PTPµ protein in HT1080

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fibrosarcoma cells, which lack endogenous PTPµ, increased p14-induced cell-cell fusion 1.7 

fold. Similarly, siRNA knockdown of endogenous PTPµ in A549 lung adenocarcinoma cells 

increased syncytiogenesis 2.5 fold. These results indicated that PTPµ, in addition to being 

inhibitory to cellular migration, is also inhibitory to cellular fusion. Interestingly, there was 

temporal regulation of p14-PTPµ interactions. Using several fusion-dead p14 constructs and a 

kinetic analysis of co-immunoprecipitation, we determined that p14 only interacts with PTPµ 

after cell-cell fusion occurs. Syncytium formation induced by p14 requires dynamic regulation of 

adherens junctions, with cadherin engagement being required to promote initial cell-cell 

association and membrane fusion, however, adherens junctions need to be disrupted to allow 

microfusion pores to expand to the macropores needed for syncytiogenesis. We therefore 

hypothesize that the stabilization of adherens junctions by PTPµ is inhibitory to pore expansion 

during cellular fusion, and that cell-cell fusion triggers interactions between p14 and PTPµ that 

may allow p14 to downregulate PTPµ activity to promote syncytium formation. These studies 

reveal an interesting convergence of the processes that regulate cell migration during cancer 

progression and cell-cell fusion on cellular pathways that regulate cell-cell adhesion and 

membrane-cytoskeletal dynamics. 

2142 

Establishment of a cell-based assay system to screen compounds modulating tight 

junction barrier. 

A. Watari 1 , M. Kondoh 1 , K. Yagi 1 ; 1 Graduate School of Pharmaceutical Sciences, Osaka 

University, Suita, Japan 

Objective 

The intestinal epithelial cells are a first physical barrier to prevent entry of foreign antigens, 

bacteria, toxins and allergens from the external environment. Tight junction (TJ) in the epithelial 

cells mainly maintains the barrier function. Disruption of the intestinal epithelial TJ barrier results 

in increase in intestinal paracellular permeability, and induces the paracellular permeation of 

toxic luminal substances, which leads to inflammatory bowel disease. In this study, we tried to 

find compounds modulating permeability in intestinal epithelial cell sheets from various food 

additives, which are recognized as safe, by using claudin expression monitoring system which 

based on cell stably expressing claudin promoter reporter gene. 

Methods 

The cell stably expressing luciferase gene under control of 500 bp claudin-4 promoter region 

has been prepared. To screen compound modulating reporter activity, validated 88 food 

additives were tested using the claudin-4 reporter system. After adding food additives to 

claudin-4 reporter cell for 48 hours, luciferase activities were measured. Effect of food additives 

on claudin-4 expression was verified by RT-PCR and western blotting analysis. Effect on barrier 

function of Caco-2 cells, human colorectal adenocarcinoma cells, was measured by transepithelial 

electrical resistance assay. 

Results 

Potassium carbonate decreased the claudin-4 reporter activity, whereas thiabendazol, carotene 

and curcumin induced it. In accordance with the alteration of reporter activity, potassium 

carbonate decreased claudin-4 mRNA and protein expression. Thiabendazol, carotene and 

curcumin increased the expression. Furthermore, these compounds modulated barrier functions 

of Caco-2 monolayer correlating with the reporter activity. These results show that we 

successfully identified novel intestinal epithelial barrier-modulating compounds using the 

claudin-4 reporter system. 

Conclusion 

The cell-based assay system using claudin-4 promoter reporter gene is very useful for 

identifying compounds modulating permeability in intestinal epithelial cell barrier, and the

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compounds identified in this system could apply to an absorption enhancer in intestine and drug 

for inflammatory bowel disease. 

2143 

ILDR1 and ILDR2 recruit Tricellulin to tricellular tight junctions. 

T. Higashi 1 , M. Furuse 1 ; 1 Cell Biology, Grad Sch of Med, Kobe University, Kobe, Japan 

Tricellular tight junctions (tTJs) are specialized structures to obliterate the narrow extracellular 

space at tricellular contacts, where the corners of three epithelial cells meet. LSR is a tTJassociated 

transmembrane protein containing a single Ig-like domain and recruits Tricellulin to 

tTJs to maintain a sufficient paracellular barrier function. LSR has two paralogs in mammalian 

genomes, ILDR1 and ILDR2. Here, we examined whether ILDR1 and ILDR2 have the same 

functions as LSR. Immunofluorescence microscopy of frozen sections of mouse tissues 

revealed that endogenous ILDR1 was localized at tTJs in several epithelial cell types including 

the colon and kidney. ILDR2 was localized at the tTJs of the retinal pigment epithelium and 

perineurium. Furthermore, we found that LSR, but not ILDR1 and ILDR2, was expressed in 

mouse EpH4 mammary epithelial cells. It has been reported that Tricellulin is diffusely 

distributed throughout the lateral membrane in the LSR-knockdown EpH4 cells and the 

exogenous expression of shRNA-resistant LSR rescues the tTJ-localization of Tricellulin. When 

ILDR1 or ILDR2 was exogenously expressed in the LSR-knockdown EpH4 cells, they were 

localized at the tricellular contacts as well as the lateral membrane. In these cells, Tricellulin 

was recruited to the tTJs. Taking these observations together, we conclude that LSR, ILDR1 

and ILDR2 share the common characteristic of tTJ-localization and Tricellulin-recruiting ability. 

2144 

Influence of fibroblasts on the synchronization of cardiomyocyte beating and community 

effect. 

T. Kaneko 1 , F. Nomura 1 , K. Yasuda 1 ; 1 Department of Biomedical Information, Institute of 

Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan 

In the normal heart, cardiomyocytes are the dominant cell type in view of volume, and more 

than half of the cells consist of noncardiomyocytes, mainly fibroblasts. To elucidate the role of 

fibroblasts on the synchronization of cardiomyocyte beating, we have made cardiomyocytefibroblast 

network model at the single-cell level in an agarose microchamber cultivation system. 

The synchronization phenomenon of two cardiomyocyte networks connected by fibroblasts 

showed (1) propagation velocity of electrophysiological signals decreased a magnitude 

depending on the increasing number of fibroblasts, not the lengths of fibroblasts; (2) fluctuation 

of interbeat intervals of the synchronized two cardiomyocyte network connected by fibroblasts 

did not always decreased, and was opposite from homogeneous cardiomyocyte networks; and 

(3) the synchronized cardiomyocytes connected by fibroblasts sometimes loses their 

synchronized condition and recovered to synchronized condition, in which the length of 

asynchronized period was shorter less than 30 beats and was independent to their cultivation 

time, whereas the length of synchronized period increased according to cultivation time. The 

results indicated that fibroblasts can connect cardiomyocytes electrically but do not significantly 

enhance and contribute to beating interval stability and synchronization. This might also mean 

that an increase in the number of fibroblasts in heart tissue reduces the cardiomyocyte 

"community effect", which enhances synchronization and stability of their beating rhythms.

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2145 

Determining the Molecular Link Between Adherens Junctions and Tight Junctions. 

J. L. Maiers 1 , X. Peng 1 , A. S. Fanning 2 , K. A. DeMali 1 ; 1 Biochemistry, University of Iowa, Iowa 

City, IA, 2 University of North Carolina - Chapel Hill, NC 

Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. 

Adhesion between epithelial cells is mediated by three types of junctions: tight junctions, 

adherens junctions and the desmosomes. This study will focus on the adherens junctions and 

tight junctions because these junctions are linked spatially and functionally, with the formation of 

tight junctions dependent on the assembly of adherens junctions. Numerous pieces of data 

suggest that the assembly of tight junctions is linked to the assembly of adherens junctions. 

However, the molecular mechanism by which these junctions are coupled is not known. α- 

Catenin, a cytoplasmic component of the adherens junction, has been implicated in the coupling 

process as cells lacking α-catenin exhibit impaired tight junction formation and abnormal 

localization of tight junction proteins. Further support of this notion comes from data showing 

that α-catenin binds directly to the tight junction protein ZO-1. Based on these observations, I 

tested whether α-catenin is required for linking tight junctions and adherens junctions, and if it 

serves this purpose through regulated binding to ZO-1. To test this possibility, I generated a 

powerful knockdown/addback system for studying this interaction in epithelial cells. Using this 

system, I found that deletion of the alpha catenin C-terminus containing the putative binding site 

for ZO-1 delayed tight junction assembly and impaired paracellular resistance, while leaving 

adherens junction assembly and function intact. These data indicate that the α-catenin Cterminus 

is required for proper tight junction assembly and function. To determine if the effects 

of α-catenin on tight junctions require direct binding of ZO-1, I mapped the ZO-1 binding site on 

α-catenin and generated a full length α-catenin point mutant unable to bind ZO-1. This mutant is 

being introduced into epithelial cells and tight junction assembly and integrity are being 

analyzed. Collectively, these data are consistent with the notion that ZO-1 binding to α-catenin 

is required for coupling the assembly of tight junctions to adherens junctions. 

2146 

Gap Junction Assembly: Roles for the Formation Plaque and Regulation by the C- 

Terminus of Connexin43. 

R. G. Johnson 1 , J. K. Reynhout 2 , E. M. TenBroek 1,3 , B. J. Quade 1,4 , T. Yasumura 5 , K. V. 

Davidson 5 , J. D. Sheridan 1 , J. I. Nagy 6 , J. E. Rash 5,7 ; 1 Genetics, Cell Biology and Development, 

University of Minnesota, Minneapolis, MN, 2 Biology, Bethel University, St. Paul, MN, 3 Current 

address: Therapy, Research and Development, Medtronic, Inc., Fridley, MN, 4 Current address: 

Brigham and Women’s Hospital, Boston, MA, 5 Biomedical Sciences, Colorado State University, 

Fort Collins, CO, 6 Physiology, University of Manitoba, Winnipeg, MB, 7 Program in Molecular, 

Cellular and Integrative Neurosciences, Colorado State University, Fort Collins, CO 

To expand our understanding of gap junction site designation and initial assembly, we examined 

the molecular constituents, ultrastructural features, and effects of Cx43 truncation on the 

development of “formation plaques” (FPs). FPs as apparent sites of gap junction development 

were monitored in experimentally disaggregated and reaggregated hepatoma cells or in HeLa or 

N2A cells transfected with full-length Cx43 or a C-terminal truncation of Cx43 (at residue 257). 

In freeze-fracture replicas, filipin (a probe for membrane cholesterol) labeled membrane areas 

surrounding FPs but not the FPs or gap junctions. Freeze-fracture replica immunolabeling 

(FRIL) methods detected Cx43 in dispersed 10-nm membrane particles that characterize FPs, 

providing further evidence that FP membranes are distinct membrane “domains”, and that the 

10-nm particles are gap junction precursors (i.e., connexin hemichannels) engaged in assembly. 

Analysis of FPs revealed a series of defined steps in assembly, including: a) pairing of FP

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membranes and partial narrowing of the extracellular space, b) accumulation of 10-nm particles 

within FPs, c) linkage of individual 10-nm particles, forming conical membrane attachments, d) 

aggregation of 10-nm particles within FPs, with further reduction in the separation between FP 

membranes and e) lateral fusion of connexon aggregates. These observations further advance 

current models of gap junction initiation and assembly. With longer cell reaggregation times, we 

found a significant increase in the packing density of particles in aggregates. With different 

regulatory sites residing in the C-terminus of Cx43, we tested whether the M257 truncation 

mutants had altered FP development and/or reduced assembly. FPs continued to develop in 

cells with truncated Cx43, although the density of 10-nm particles within developing gap junction 

aggregates failed to achieve control levels. Negative regulation of assembly, as measured by 

intercellular dye transfer, was evaluated by following the dramatic inhibition of gap junction 

assembly when protein kinase C (PKC) was activated with TPA treatment. This inhibition failed 

to occur in the M257 cells. The studies reported here demonstrate the importance of the Cterminus 

of Cx43 in gap junction assembly and regulation. By confocal immunofluorescence 

microscopy, we found that N-cadherin was associated with Cx36 in neuronal gap junctions of 

rodent brain. Overall, these studies validate a model for gap junction assembly that can now be 

evaluated with respect to the roles played by various connexin-interacting proteins, such as N- 

and E-cadherin, nectins, β-catenins, ZO-1, and AF6 [aka afadin] (work in progress). 

Supported by NIH GM-46277 (RGJ and JDS), NS-44395 (JER), and CIHR (JIN). 

2147 

The Role of Connexin43 in the Development of Skeletal Muscle using Cell and Animal 

Models of Oculodentodigital Dysplasia. 

P. A. Merrifield 1 , S. C. Qing 1 , P. Vecchio 1 , G. I. Fishman 2 , D. W. Laird 1 ; 1 Anatomy & Cell 

Biology, University of Western Ontario, London, ON, Canada, 2 Leon H. Charney Division of 

Cardiology, New York University School of Medicine, New York, NY 

Skeletal muscle development involves the differentiation of myoblasts into myotubes; likely 

involving connexins (Cx) and the gap junctional exchange of secondary messengers and 

metabolites. Some patients with oculodentodigital dysplasia (ODDD), a rare primarily autosomal 

dominant disease caused by mutations in the gene encoding Cx43, become less ambulatory 

with aging and exhibit symptoms that may reflect defects in skeletal muscle development, 

maintenance, and repair. In this study, the role of Cx43 in skeletal muscle development was 

examined in differentiation-competent L6 myoblasts and two ODDD-linked Cx43-mutant mouse 

lines. Undifferentiated L6 myoblasts exhibited high levels of Cx43-based gap junctions which 

were inhibited by the co-expression of I130T and G60S mutants. Upon myotube formation, 

Cx43 was rapidly down-regulated and gap junctions were lost but myotubes continued to 

enlarge. Cx43-mutant mice heterozygous for the I130T mutation exhibited similar body weight, 

tibial length and muscle fiber size as littermate controls. While mutant mice heterozygous for the 

G60S mutation also had similar tibial length their overall size and muscle fibre diameter were 

significantly smaller. Overall, Cx43 regulation may be important for initial myoblast differentiation 

but not linked to myotube growth and while our mutant mouse studies suggest that some ODDD 

patients are predicted to have smaller muscle fibres, this appears to be dependent on the site of 

the Cx43 mutation or other unknown etiologies (Supported by grants from the CIHR to DWL and 

NSERC to PAM).

2148 

C-terminal interactions of Cx35 that regulate gap junction coupling. 

Y. Wang 1 , C. K. Mitchell 1 , A. Espejo 2 , M. T. Bedford 2 , J. O'Brien 1 ; 1 Department of 

Ophthalmology & Visual Science, UTHSC Houston, Houston, TX, 2 Department of 

Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Houston, TX 

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Connexin 35/36 gap junctions in central nervous system neurons display a large degree of 

plasticity. In retinal photoreceptors and AII amacrine cells, the plasticity in coupling strength is 

directly correlated to the phosphorylation states of two regulatory residues (Ser110 and Ser276, 

using numbering for Cx35). Coupling among retinal AII amacrine cells is reduced by dopamine 

D1 receptor activation through activation of PKA, downstream activation of protein phosphatase 

2A (PP2A) and dephosphorylation of Cx36. This regulation is replicated with either Cx35 or 

Cx36 expressed in HeLa cells. In an earlier study (Ouyang at al. 2005, Mol Brain Res 135:1-11) 

we found that removal of the last 7 amino acids of the Cx35 C-terminus (Ser298ter) inverted the 

effect of PKA activity on coupling. One hypothesis that can account for this phenomenon is that 

the C-terminus of Cx35/36 is essential for an interaction with regulatory proteins, and that PP2A 

is part of a complex that does not associate with the mutant connexin. To examine this 

hypothesis, we performed a protein microarray study and peptide pulldown experiments to 

examine interactions with the Cx35 C-terminus. We also examined regulation of coupling in a 

variety of C-terminal mutants expressed in HeLa cells. A 22 amino acid peptide corresponding 

to the tip of the Cx35 C-terminus bound to several different PDZ domains, including domains 

from MUPP1, NHERF2, and PDZK1. Phosphorylation of the C-terminus at Ser298 revealed 

binding to 3 different isoforms of 14-3-3 proteins. Truncation of the C-terminus at Ser298 

eliminated all of the interactions. These binding interactions were confirmed by pulldown 

experiments with recombinant PDZ and 14-3-3 proteins. In transfected HeLa cells, mutation of 

serine 298 to alanine, which prevents S298 phosphorylation and should prevent 14-3-3 binding, 

also inverted regulation of coupling by PKA activity. In contrast, Cx35 with a C-terminal YFP tag, 

which should prevent PDZ binding, showed wild-type regulation by PKA activity. The results 

suggest that phosphorylation of Ser298 and recruitment of 14-3-3 proteins is involved in the 

regulation of Cx35 coupling. 14-3-3 binding may facilitate assembly of a signaling complex 

containing PP2A or it may alter the sensitivity of Cx35 to dephosphorylation by PP2A. 

2149 

Matrix Stiffness Dictates Cytoskeletal Organization and Nuclear Morphology in 2D and 

3D. 

F. Rehfeldt 1,2 , T. Harada 2 , A. E. Brown 2 , A. Zajac 2 , S. Cai 2 , A. Zemel 3 , D. E. Discher 2 ; 1 3rd Inst. 

Physics - Biophysics, University Goettingen, Goettingen, Germany, 2 Biophysical Engineering 

Lab, University of Pennsylvania, Philadelphia, PA, 3 Hebrew University Jerusalem, Israel 

Physical aspects of the microenvironment such as matrix elasticity E can influence morphology, 

mechanics, and even the lineage choices of stem cells. An E-series of two types of hydrogels 

are ligand-functionalized here with collagen and used to elucidate wide-ranging cell, 

cytoskeletal, and nuclear responses to E in both two and three dimensional matrix geometries. 

Cross-linked hyaluronan-based hydrogels show that, within just hours of initial plating, cell 

adhesion, shape, and cytoskeletal order depend non-linearly on E over a broad range of 

physiologically relevant elasticities. Force generation by non-muscle myosin IIs is required for 

the various cell responses. These include nuclear shape changes that can also be achieved in 

part by siRNA-knockdown of a key nuclear envelope gene, lamin-A/C, which is implicated in 

differentiation of soft tissues such as fat and stiff tissues such as bone. The results lend support 

to an important matrix-myosin-nucleus pathway.

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2150 

Characterisation of tissue microenvironment by the direct culture of mesenchymal stem 

cells on tissue sections. 

W. Shen 1 , W. Y. Tong 2 , Y. Zhao 2 , C. F. Chan 3 , P. Chu 1 , W. K. Yeung 2 , Y. W. Lam 1 ; 1 City 

University of Hong Kong, Hong Kong, 2 Department of Orthopaedics and Traumatology, The 

University of Hong Kong, Hong Kong, 3 Department of Pediatircs & Adolescent Medicine, The 

University of Hong Kong, Hong Kong 

One ultimate goal of tissue engineering is to construct functional tissues and organs from 

isolated cells and fabricated materials. To achieve this, it is important to understand how 

extracellular environments affect and control cell behaviours. Although many studies have 

characterised the contributions of physical and biochemical factors in extracellular environments 

to cell physiology, these works often fail to mimic the complexity of the native tissue 

microenvironment. We have developed a novel experimental system to evaluate the interactions 

of cells and extracellular microenvironment. We discovered that cryosections of Bovine Achilles 

tendon display intricate textural details on the surface, and hypothesised that these surfaces 

represent the internal tissue environments to which endogenous cells are exposed. By 

manipulating the orientation of sectioning, we could create section surfaces with highly different 

ultrastructures. Since these sections were carved from the same piece of tendon, their 

biochemical compositions were identical. To assess how cells responded to these surfaces, we 

directly cultured mammalian cancer cell lines and human mesenchymal stem cells (MSC), on 

these sections. Morphology, adhesion and proliferation of cells were highly dependent on the 

tissue architecture they were exposed to. In particular, cells seeded on the longitudinal sections 

(LS) of tendon, but not cross sections (CS), adopted a highly elongated and aligned 

morphology. Time-lapse microscopy revealed that MSC on both LS and CS initially projected 

filopodia to all directions, but only cells on LS spread along one orientation. Remarkably, MSC 

cultured on LS, but not CS, expressed protein biomarkers characteristic of tenocytes, 

suggesting that LS contained biological cues that instructed MSC to commit to the tenogenic 

lineage. To delineate the nature of this signal, we prepared PDMS replicas using tendon 

sections as the mould. The resulting replicas, which faithfully copied the physical shape of 

tendon sections, but not the biochemical composition, promoted a small degree of cell 

elongation, but failed to induce MSC differentiation into tenocyte-like cells. This suggests that 

although the physical structure of tissue microenvironment may be enough to modulate cell 

shape and morphology, specific biochemical molecules, when presented in a correct orientation, 

are required to drive stem cell differentiation. This study demonstrated how biophysical and 

biochemical information in the extracellular microenvironment intertwined to form a unique cell 

type specific niche that influences the fate of cells within a tissue. Direct culture of cells on 

sectioned tissues is a simple and useful model system for the study of this process. 

Regulation and Organization of the Genome 

2151 

Clusterin as a target gene of HIF-1alpha in prostate cancer cells. 

E. Shin 1 , S. Park 1 , J. Park 1 , D. Lee 1 , G. Roh 1 , H. Kim 1 , S. Kang 1 , G. Cho 1 , W. Choi 1 ; 

1 Department of Anatomy and Neurobiology, BK21 Biomedical Center, Gyeongsang National 

University, JINJU, Korea 

Clusterin (CLU) is a stress-response glycoprotein that increases expression by diverse 

apoptotic triggers in several human cancers such as prostate cancers. Previous studies showed 

that CLU expression was up-regulated in patho-physiological condition such as oxidative stress.

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HIF-1alpha (hypoxia inducible factor-1alpha) is a major regulator in oxygen homeostasis and 

transcriptional factor mediating the cellular response to hypoxia. In this study we evaluated 

whether CLU expression is regulated by HIF-1alpha in human prostate cancer cells exposed to 

Cobalt chloride (CoCl2). Interestingly, we observed that both CLU and HIF-1alpha were upregulated 

in mRNA and protein level. Also overexpression of HIF-1alpha up-regulates CLU 

expression in prostate cancer cells. Therefore, to evaluate the transcriptional regulation of CLU 

promoter by HIF-1alpha, we searched hypoxia response element (HRE) of Human CLU 

promoter sequence. Electrophoretic mobility shift assay (EMSA) and Chromatin 

Immunoprecipitation (ChIP) analyses demonstrated that HIF-1alpha binds the three putative 

HRE sites in CLU promoter. Reporter gene assay showed that three HRE sites were 

functionally active. Our results suggest that the transcription factor HIF-1alpha specifically binds 

to three HRE sites of CLU promotor and directly regulates the transcription of the human CLU 

gene in prostate cancer cells exposed to hypoxia. 

2152 

Phosphorylation of p53 by p38α regulates the expression of Gadd45β in cardiomyocytes. 

Y-A. Kim 1,2 , M-Y. Kim 1 , H. Yu 1,3 , S. Mishra 1 , J-H. Lee 2 , K. Choi 2 , J-H. Kim 2 , Y-S. Jung 1,2 ; 

1 College of Pharmacy, Ajou University, Suwon, Korea, 2 Brain Korea 21 for Molecular Science 

and Technology, Ajou University, Suwon, Korea, 3 Brain Korea 21 for Medical Sciences, School 

of Medicine, Ajou University, Suwon, Korea 

Both growth arrest and DNA damage-inducible 45β (Gadd45β) and p53 plays a critical 

mediatory role in cardiomyocyte apoptosis under ischemia/hypoxia. However, little is known 

about how Gadd45β expression is regulated under ischemia/hypoxia. In this study, we 

hypothesized that Gadd45β is a direct target of p53 under ischemia/hypoxia in cardiomyocytes. 

Using actinomycin D, it was found that the expression of Gadd45β during hypoxia is regulated 

at the transcriptional level in rat heart-derived H9c2 cells. Silencing p53 gene abrogated the 

induction of Gadd45β promoter luciferase reporter (Gadd45β-Luc) activity triggered by hypoxia, 

whereas overexpression of p53 enhanced activity of Gadd45β-Luc. Moreover, chromatin 

immunoprecipitation assay revealed that binding of p53 to Gadd45β promoter region during 

hypoxia, and direct binding between them was confirmed by surface plasmon resonance 

imaging. We also observed the necessary role of p53 in inducing Gadd45β expressions in H9c2 

cells under hypoxia and in a rat myocardium of ischemia model. Further studies showed that 

p38α was activated and responsible for the p53-dependent expressions of Gadd45β under 

hypoxia. We revealed that p38α induced the distinct temporal profiles of phosphorylation at 

Ser15 or Ser20 of p53, and mutation of either site on p53 was sufficient to block the hypoxiainduced 

Gadd45β-Luc activity and Gadd45β expressions. Finally, p53-induced apoptosis during 

hypoxia was inhibited by Gadd45β gene silencing. These results provide first evidence that 

Gadd45β is a direct transcriptional target of p53 under ischemia/hypoxia and that the hypoxiap38α-p53-Gadd45β 

signaling module serves as an essential regulator of cardiomyocyte 

apoptosis. This work was supported by Basic Science Research Program through the National 

Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and 

Technology (2011-0012659). 

2153 

Tramtrack69 Regulation of Notch Target Genes in Drosophila melanogaster. 

D. Eastman 1 , E. Hildebrand 2 ; 1 Biology, Connecticut College, New London, CT, 2 Connecticut 

College, New London, CT 

The Notch pathway is critical for determining many different cell types in developing animals. In 

Drosophila, the main targets of Notch signaling are the Enhancer of split (E(spl)) genes.

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Although all of these genes are activated by Notch, they have distinct expression patterns in 

developing embryos and larval imaginal discs. We are interested in determining the factors that 

are responsible for this differential expression. We previously identified sequences within the 

promoters of these genes that are conserved in at least 12 Drosophila species: some of these 

are unique to individual genes and some are found upstream of a sub-set of the E(spl) genes. 

One of these sequences is a putative site for the known transcriptional regulator, Tramtrack69 

(TTK69). We show that overexpression of TTK69 in eye discs down-regulates HLHmgamma 

expression. In addition, we have found that TTK69 inhibits activated Notch (ICN) induced E(spl) 

HLHmgamma expression in Drosophila tissue culture cells and that this affect is dependent on 

the presence of the BTB/POZ repressor domain at the TTK69 N-terminus. We are currently 

characterizing the interaction of TTK69 with the Notch activation complex and determining 

whether it inhibits Notch activation of other E(spl) genes. 

2154 

The transcriptional activation of melanocortin 2 receptor accessory protein by 

PPAR[gamma]2 in adipocyte. 

S. KIM 1 , S. Kwak 1 , Y-J. Kim 1 ; 1 Department of Biology, Kyung Hee University, Seoul, Korea 

Adrenocorticotropic hormone (ACTH) in rodents decreases body weight as well as the secretion 

of leptin. MC2R and Mrap are known as receptors of ACTH in adipocyte. PPARγ2 also plays 

roles in the transcriptional regulation in adipocyte such as adipogenesis and fatty acid betaoxidation. 

Nuclear receptors activated by PPARγ2 bind to target genes for their transcriptional 

activation. In the present study we investigated that the Mrap transcript is regulated by PPARγ2. 

After the differentiation of adipocyte, the expression pattern of Mrap was similar to that of 

PPARγ2. In the presence of a PPAR agonist, the level of Mrap mRNA was highly increased. In 

addition, we identified putative PPRE site in the Mrap promoter by reporter assay, chromatin 

immunoprecipitation assay and the mutagenesis study. These results suggest that PPARγ2 is 

required for the transcriptional activity of Mrap during adipogenesis, which could be contributed 

to understand the molecular mechanism of lipolysis in adipocytes. 

2155 

Role of TopBP1 partner WDR18 in DNA damage checkpoint. 

J. Willis 1 , D. DeStephanis 1 , V. Gowda 1 , S. Yan 1 ; 1 Department of Biology, University of North 

Carolina at Charlotte, Charlotte, NC 

DNA damage checkpoint has significant implications for biological and pathological processes 

such as cancer and aging. Genomes of all living organisms are exposed to a variety of threats, 

but the genome’s DNA damage checkpoint functions as a surveillance mechanism, monitoring 

damage in a genome and directing cellular responses to such damage. If failing to activate DNA 

damage checkpoint when necessary, the result is unrepaired damage that leads to genomic 

instability and tumor formation. The main barriers to understanding this process are in 

identifying how checkpoints sense DNA damage and how the proteins relay the damage signal. 

Specifically, it is not known how the activated sensor kinase ATR (ATM and Rad3-related) 

phosphorylates its downstream effector protein Chk1 (Checkpoint kinase 1). In addition to the 

critical roles in DNA replication initiation and DNA replication stress response, TopBP1 (DNA 

topoisomerase II β binding protein 1) plays an important role in DNA damage checkpoint 

signaling through its C-terminus region. However, the underlying mechanism is not known. We 

hypothesize that a TopBP1-interacting protein contributes to its role in DNA damage checkpoint 

signaling. The objective of this study is to reveal the molecular mechanism of how TopBP1 Cterminus 

contributes to DNA damage checkpoint signaling. Xenopus egg extracts, a reliable 

cell-free biochemical system, has been used for a wide variety of studies in cell cycle and

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checkpoint activation. Our data have shown that WDR18 (WD40 repeat-containing protein 18), 

a TopBP1-interacting protein, interacts with TopBP1 C-terminus, and their interaction is required 

for ATR activation of Chk1 in the response to double-stranded breaks. Furthermore, 

immunodepletion of endogenous WDR18 from Xenopus egg extract also compromises DNA 

damage checkpoint signaling. Taken together, WDR18 plays an essential role in DNA damage 

checkpoint signaling via interacting with TopBP1 C-terminus. 

This work was supported, in part, by funds provided by The University of North Carolina at 

Charlotte and Wachovia foundation fund for faculty excellence. 

2156 

A genetic screen to identify interactions between cdc13-1and yku80 mutant alleles on 

yeast telomeres. 

L. Sanchez 1 , G. Guillaume 1 , S. Flores 1 , C. J. Hengartner 1 , L. R. Vega 1 ; 1 Biology, Barry 

University, Miami Shores, FL 

Telomeres are the physical ends of eukaryotic chromosomes that function to protect DNA ends 

from degradation and from end to end fusion. Telomeres consist of stretches of repeated C/Grich 

DNA ending with 3’ single stranded G-rich overhangs. The maintenance and function of 

telomeres are facilitated by the enzyme telomerase and by accessory proteins such as Ku and 

Cdc13p. Cdc13p is an essential, G-strand binding protein that functions in telomere protection 

and in telomerase recruitment. cdc13-1 is a temperature sensitive allele of CDC13, that is 

defective for telomere end protection. Ku is a non-essential heterodimer composed of Ku70p 

and Ku80p. Ku plays multiple roles in DNA metabolism including: non-homologous end joining, 

recombination and end protection. Ku also interacts with TLC1, the RNA template of the 

telomerase enzyme and has recently been shown to exhibit end-binding activity. This study 

examines the effect of mutations in yKU80 on cdc13-1 strains. We will also determine genetic 

interactions between yku80 mutants and Pif1p, a helicase that inhibits telomerase activity. We 

previously showed that cdc13-1 strains deleted for PIF1 display hyper-elongated telomeres and 

increased temperature resistance. Telomere elongation in cdc13-1, pif1∆ strains partially 

depends on the Ku-TLC1 interaction. Using a genetic library of yku80 mutations generated in A. 

Bertuch’s laboratory, we have introduced the yku80 alleles into the cdc13-1 background. The 

goals of these experiments are to determine the effects on viability and telomere end protection 

of the various yku80 mutant alleles in cdc13-1 strains. We hope to identify a cdc13-1, yku80 

double mutant that mimics the phenotypes of cdc13-1, pif1∆ strains. 

Supported by NIH-NIGMS MBRS RISE Grant: 2R25 GM059244-10, Barry University and NIH- 

NIGMS/NCI MBRS SCORE grant, 5SC 2CA 138567 to LRV 

2157 

Function of Daxx at centromeres and pericentromeres. 

V. Morozov 1 , E. Gavrilova 2,3 , A. Ishov 1 ; 1 Anatomy & Cell biology, University of Florida, 

Gainesville, FL, 2 Institute of Cytology Russian Academy of Sciences, St. Petersburg, Russia, 

3 Faculty of Biology and Soil Sciences, St. Petersburg State University, St. Petersburg, Russia 

Heterochromatin architecture is essential for the proper orchestration of nuclear processes, 

while transcription from this part of genome is required for its own maintenance. Here we 

present first evidence that depletion of protein Daxx affects transcription of human 

heterochromatin, reducing accumulation of centromeric (CEN) RNA in normal conditions and 

pericentromeric (periCEN) RNA after heat shock (HS) application. Searching for the mechanism 

of Daxx-dependent regulation of heterochromatin transcription, we found that depletion of Daxx

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decreases incorporation of transcription-associated histone H3 variant, H3.3, into both CEN and 

periCEN. 

In normal conditions, Daxx is mostly accumulated at ND10/PML nuclear bodies, with minor 

association with CEN/periCEN in subpopulation of cells. HS changes this balance forcing very 

robust accumulation of Daxx on CEN/periCEN. Surprisingly, this transient redistribution of Daxx 

does not further elevate incorporation of H3.3 that remained steady during HS and recovery. 

Instead, depletion of Daxx leads to HS-induced changes in the balance of epigenetic 

modifications at heterochromatin, most dramatically elevating levels of H3K4Me2 at periCEN. 

We propose dualistic function of Daxx-containing complexes at CEN/periCEN: 1) regulation of 

H3.3 loading in normal conditions, and 2) protection of epigenetic status upon stress application, 

thus collectively guarding epigenetic identity of heterochromatin and genome integrity. 

2158 

Structural Requirements of the JIL-1 Tandem Kinase for Histone H3S10 Phosphorylation. 

Y. Li 1 , W. Cai 1 , C. Wang 1 , X. Bao 1 , H. Deng 1 , J. Girton 1 , J. Johansen 1 , K. M. Johansen 1 ; 

1 Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 

The JIL-1 tandem kinase in Drosophila localizes specifically to euchromatic interband regions of 

polytene chromosomes and is the predominant kinase controlling the phosphorylation state of 

histone H3S10 at interphase. JIL-1 can be divided into four main domains including a NH2terminal 

domain (NTD), two kinase domains (KDI and KDII), and a COOH-terminal domain 

(CTD). Our laboratory has previously determined that the CTD-domain of JIL-1 is sufficient for 

proper chromatin localization and for rescue of JIL- 1 null mutant chromosome morphology. 

Interestingly a JIL-1 construct without the CTD-domain has kinase activity for histone H3S10 

despite that it does not localize properly. However, the potential contributions from the NTD was 

not determined. Thus, in order to further characterize the structural requirements of the JIL-1 

kinase for H3S10 phosphorylation, we generated a series of mutated JIL-1 constructs and 

expressed them in a JIL-1 null mutant background. Our results show that a JIL-1 construct 

without the NTD localizes properly to chromatin and rescues mutant polytene chromosome 

morphology. However, in immunocytochemistry and immunoblot analyses with H3S10ph 

antibody no histone H3S10 phosphorylation could be detected, strongly suggesting that the 

NTD domain is required for H3S10 kinase activity. Furthermore, we mutated either or both 

kinase domains to render them "kinase dead" and assessed their ability to phosphorylate 

H3S10. The results indicate that both kinase domains are required for kinase activity. In 

addition, using a LacI/lacO-repeat tethering system we provide evidence that only JIL-1 

constructs with H3S10 phosphorylation activity have the capacity to induce a change in higher 

order chromatin structure from a condensed heterochromatin-like band state to a more open 

euchromatic interband state. These findings provide direct evidence that the epigenetic histone 

tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of 

chromatin structure. Supported by NIH grant GM62916. 

2159 

Epigenetic Regulation of MARCO in Tolerized Macrophages. 

J. Jing 1 , B. O’Connor 2 , B. Pederson 3 , E. Davidson 3 , I. V. Yang 3 , D. Schwartz 3 ; 

1 University of Colorado Denver, Denver, CO, 2 National Jewish Health, 3 University of Colorado 

Denver 

Macrophages play a key role in host defense against microbes through phagocytosis. MARCO 

(macrophage receptor with collagenous structure) is one of the scavenger receptors on the cell 

surface of macrophages that mediates the opsonin-independent phagocytosis. The goal of our 

study was to study the role of epigenetic regulation of MARCO in lipopolysaccharide (LPS) or

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lipotechoic acid (LTA)-induced tolerance. We first demonstrated that phagocytosis was 

increased in mouse bone marrow derived macrophage (MBMM) repeated stimulation by LPS or 

LTA. The expression of MARCO, but not other scavenger receptors, was also elevated, 

suggesting that MARCO plays a key role in the phagocytosis of tolerized macrophages. We 

comprehensively evaluated genome-wide DNA methylation by CHARM (comprehensive highthroughput 

arrays for relative methylation) and profiled the mRNA expression in tolerized 

macrophages. Although there were no methylation changes associated with MARCO in 

tolerized compared to nontolerized macrophages, several transport related genes (SNX30, 

SNX24, Kif21b, Kif5, Kif6 and Kif9) have significant methylation changes and at least two fold 

mRNA expression change, indicating these genes may be involved in regulating the transport of 

MARCO to cells surface. We are in the process of confirming these findings by examining 

expression of MARCO on the cell surface following siRNA-mediated inhibition of these 

candidate genes. In addition, we are exploring whether H3K4me3 and H3K9me2 histone 

modifications contribute to increased expression of MARCO in tolerized macrophages. 

2160 

Molecular Basis for Interaction of let-7 microRNAs with Lin28. 

Y. Nam 1 , C. Chen 1 , R. Gregory 1 , J. Chou 1 , P. Sliz 1 ; 1 Harvard Medical School, Boston, MA 

MicroRNAs are small non-coding RNA molecules that regulate gene expression. Members of 

the let-7 microRNA family control many cell fate determination genes to influence pluripotency, 

differentiation, and transformation. Lin28 is a specific, post-transcriptional inhibitor of let-7 

biogenesis. Moreover, Lin28 is a potent cell reprogramming factor, abnormally expressed in 

various cancers, and linked to various developmental traits in worms and mammals. Growing 

evidence suggests that Lin28 also binds certain mRNAs to regulate translation independent of 

let-7. 

A major question regarding Lin28 activity is how it recognizes its RNA substrates, and also how 

it inhibits microRNA processing enzymes-Drosha and Dicer-while also recruiting a modification 

enzyme, TUTase, to target let-7 precursors to degradation. We report crystal structures of Lin28 

in complex with pre-element (preE) portions, or "terminal loop regions", of let-7d, let-7-f1, and 

let-7g precursors. The two folded domains of Lin28 recognize two distinct regions of the RNA; 

the terminal regions outside of these domains are dispensable for inhibition of let-7 in vivo. We 

also show by NMR spectroscopy that the linker connecting the two folded domains is flexible, to 

accommodate Lin28 binding to diverse let-7 family members. Mutagenesis studies of protein 

and RNA components identified mutations in the binding interface that affect complex formation 

in vitro, Dicer activity in vitro, and processing of let-7 microRNAs in cultured cells. 

Our work underscores the importance of preEs in microRNA regulation, and we present a 

preferred preE sequence for binding to Lin28. Our model also provides a mechanistic 

explanation for the inhibitory effect of Lin28 on miRNA processing by Dicer; it further suggests 

that induced folding in the CCHCx2:GGAG part of the complex directly influences downstream 

factor(s) important for let-7 regulation.

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2161 

The karyopherin Sal3 is required for nuclear import of the core RNA interference pathway 

protein Rdp1. 

J. Park 1 , S. I. Freitag 2 , P. Young 2 , T. C. Hobman 1 ; 1 Cell Biology, University of Alberta, 

Edmonton, AB, Canada, 2 Biology, Queen's University, Kingston, ON, Canada 

RNA-dependent RNA polymerase activity is required for RNA interference (RNAi) in many lower 

eukaryotes including the fission yeast S. pombe. Together with Ago1 and Dcr1, the RNAdependent 

RNA polymerase Rdp1 mediates small RNA-dependent transcriptional- and 

posttranscriptional gene-silencing. Whereas the bulk of Rdp1 is localized to the nucleus, Ago1 

and Dcr1 are primarily cytoplasmic. This may reflect the fact that Rdp1 is required early in the 

RNAi pathway to generate double strand RNA from transcripts that originate from centromeric 

loci. The relatively large size of Rdp1 (139.4 kD) precludes passive diffusion of the enzyme into 

the nucleus suggesting that karyopherin-dependent transport is involved in nuclear targeting of 

this enzyme. In the present study, we report that the karyopherin Sal3 is required for nuclear 

import of Rdp1 in S. pombe. Loss of nuclear Rdp1 is associated with substantially reduced 

transcriptional gene-silencing, and surprisingly, post-transcriptional gene silencing which may 

occur in the cytoplasm, was also significantly affected. Together, these results identify Sal3 as a 

modulator of RNAi-dependent transcriptional gene silencing as well as a potential link between 

nuclear import and post-transcriptional gene-silencing. 

This work is supported by Natural Science and Engineering Research Council of Canada 

(NSERC) #rgpin 183820. 

Cell Polarity 

2162 

Siah regulation of Par3 controls neuronal cell adhesion during germinal zone exit. 

D. Solecki 1 , N. Trivedi 1 , J. Famulski 2 ; 1 St. Jude Children's Res Hosp, Memphis, TN, 2 University 

of Alberta, Edmonton, AB, Canada 

The brain’s circuitry is established by directed migration of neurons during development. 

Despite tremendous advances in our understanding of the substrates, guidance mechanisms 

and cytoskeletal elements required for neuronal migrations, remarkably little is known 

concerning the cell intrinsic machinery that initiates a neuron’s exit from their germinal zone 

(GZ) niche. We found that Seven in Absentia (Siah) is the first ubiquitin ligase to target Par3 for 

degradation and Siah-mediated proteosomal degradation of Par3 regulates the initiation of 

radial migration as cerebellar granule neurons (CGNs) leave the external germinal layer (EGL). 

Both Par3 gain of function and Seven in Absentia loss of function spur precocious radial 

migration. Time-lapse imaging using a novel probe to measure neuronal cell contacts in live 

CGNs reveals that Par3 promotes adhesive interactions needed for germinal zone exit by 

recruiting the JAM-C epithelial tight junction adhesion molecule to the neuronal cell surface. Our 

findings suggest a model in which post-translational modification of PAR complex function is a 

key mechanism regulating the exit of neuronal progenitors or immature neurons from a germinal 

zone niche. Moreover, these results identify an unexpected parallel between epithelial junction 

formation and the cell-cell interactions that occur during histogenesis in the developing 

vertebrate brain.

2163 

Cognitive deficits associated with CNS myelin dysfunction. 

A. Gow 1 , K. Meharas 1 ; 1 Genetics, Wayne State University, Detroit, MI 

MONDAY 

Cognitive dysfunction is typically considered a secondary symptom associated with mental 

illness. Studies suggest that mental illnesses themselves are the cause of cognitive dysfunction, 

manifested as impairments in attention, memory, learning and executive function and lead to 

behavioral disorders. The pathophysiological processes that underlie cognitive dysfunction 

associated with behavioral changes have been debated for decades. Most studies focus on 

disrupted neural development or aberrant neurochemistry, however, an abundance of anecdotal 

clinical data and genetic evidence support a role for myelin and myelin-related genes in the 

etiology of cognitive dysfunction and behavioral disorders. In the current study, we have 

generated a myelin mutant mouse in which expression of the tight junction protein, claudin 11, 

has been ablated in oligodendrocytes. The function of this protein in contributing to the electrical 

resistance of CNS myelin has been elucidated in molecular and mathematical detail. Most 

importantly, the phenotype of these knockout mice arises from changes to the passive 

membrane properties of small diameter myelinated fibers and not from degenerative processes. 

Accordingly, we are able to interpret behavioral changes in these mutants in terms of cognitive 

dysfunction associated with slower conduction velocity. Our analyses of behavioral 

abnormalities resulting from slowed conduction will be presented. 

2164 

Cortical clustering of cell polarity regulators. 

J. Dodgson 1 , A. Chessel 1 , F. Vaggi 2 , S. Cox 3 , E. Rosten 3 , M. Yamamoto 4 , M. Geymonat 1 , Y. 

Dong 1 , J. Ahringer 1 , D. Albrecht 5 , M. Sato 4 , A. Csikasz-Nagy 2 , R. Carazo Salas 1 ; 1 Gurdon 

Institute, University of Cambridge, Cambridge, United Kingdom, 2 The Microsoft Research- 

University of Trento Centre for Computational Systems Biology, Trento, Italy, 3 Randall Division 

of Cell and Molecular Biophysics, Kings College London, United Kingdom, 4 Department of 

Biophysics and Biochemistry, University of Tokyo, Japan, 5 Institute of Biochemistry, ETH Zurich, 

Zurich, Switzerland 

Cell polarity is a fundamental part of cell physiology. Despite the identification of many polarityregulating 

factors in diverse organisms, the microscopic structure and dynamics of the polarity 

machinery itself has been difficult to elucidate. We imaged frontally the cortical areas where the 

polarity machinery accumulates in cells of the fission yeast Schizosaccharomyces pombe, using 

time-lapse and super-resolution (OMX structured-illumination and live-cell STORM) microscopy. 

Analysis of several key polarity factors reveals that they localize to discrete cortical clusters 

('nodes'), resolvable to 50nm in diameter. Different polarity factors display different node 

distributions and dynamics at the cell cortex. Strikingly, we find that many factors thought to 

regulate polarity by interacting physically at the cortex – such as the factors Tea1p and Tea3p - 

do not necessarily co-localize there, suggesting unexpected layers of regulation. Artificial 

dimerization of Tea1p and Tea3p leads to mislocalization of these factors and cell polarity 

defects, suggesting that the segregation of polarity factors into adjacent but separate nodes 

may be crucial to their function. Similar nodes are found in Saccharomyces cerevisiae and 

Caenorhabditis elegans cells, suggesting conservation of this structural arrangement.

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2165 

Rapid cycles of phosphorylation and dephosphorylation on threonine 567 control ezrin 

localization and function in microvillus formation. 

R. Viswanatha 1 , P. Ohouo 1 , M. Smolka 1 , A. Bretscher 1 ; 1 Cornell University, Ithaca, NY 

Ezrin is a member of a group of conserved plasma membrane-actin cytoskeletal cross-linkers 

(called ERM proteins) that is negatively regulated by an intramolecular interaction between the 

N- and C-termini. Ezrin is activated by threonine-567 phosphorylation near the C-terminus. In 

placental Jeg-3 cells at steady-state, we found that 50% of cellular ezrin is phosphorylated at 

threonine 567, and this phosphorylated pool is completely turned over within just 5 minutes. The 

N-terminus of ezrin is known to mediate its localization to the plasma membrane. Wild-type 

ezrin in Jeg-3 cells, however, is enriched on the microvillus membrane and virtually 

undetectable on other regions of the plasma membrane. Upon a brief treatment with a 

phosphatase inhibitor, by expressing a T567E phosphomimetic form of ezrin, or by expressing 

other constitutively open forms of ezrin, we find that this distribution is altered: ezrin “spills out” 

of the microvillar membrane and occupies other regions of the plasma membrane. To determine 

whether changing these phospho-dynamics by stabilizing the phosphorylated form of ezrin 

would affect ezrin function, we performed siRNA-rescue experiments. Ezrin depletion in Jeg-3 

cell resulted in the loss of microvilli, and only wild-type, but neither T567A nor T567E ezrin 

restored them. Taken together, these observations hint at the presence of constant, local kinase 

activity toward ezrin countering ubiquitous phosphatase activity, and this balance is essential for 

ezrin function. To determine how phosphorylation dynamics are regulated in Jeg-3 cells, we 

conducted a proteomic screen to identify kinases associated with ezrin. We identified several 

candidate kinases previously shown to phosphorylate the C-termini of ERMs in other systems. 

We are in the process of examining the distribution of these kinases in Jeg-3 cells and their 

contribution to ezrin phospho-dynamics. 

2166 

Characterization of Banderuola, a novel regulator of asymmetric cell division in 

Drosophila melanogaster. 

F. Mauri 1 , J. L. Mummery-Widmer 1 , M. Yamazaki 1,2 , J. A. Knoblich 1 ; 1 Institute of Molecular 

Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria, 2 The Global COE 

program, Akita University School of Medicine, Akita, Japan 

Asymmetric cell division is a process whereby a progenitor cell can generate daughters with a 

different fate. The underlying molecular mechanisms have been widely studied in the Drosophila 

Sensory Organ Precursor (SOP) cells, that generate the 4 different cell types forming the 

external sensory organs (bristles) through 2 rounds of asymmetric cell division. This is achieved 

through the partitioning of the protein Numb, that represses Notch signaling in the daughter cell 

where it is segregated. 

To gain a better insight of this process, we used tissue specific RNAi to score the effects of the 

knockdown of genes on the morphology of the bristles. We have screened a library of 22,247 

transgenic Drosophila RNAi lines, and identified 130 genes potentially involved in asymmetric 

cell division. To identify the genes involved in the asymmetric localization of Numb, we 

developed a live time-lapse cell-imaging assay based on a GFP reporter. Among the 130 

candidates screened and not previously thought to play a role in asymmetric cell division, there 

were three genes whose knockdown reproducibly affected Numb localization. We have 

confirmed the observed phenotypes by independent secondary RNAi lines and have started 

investigating the function of one of the identified genes, which encodes a previously 

uncharacterized protein. Loss-of-function studies suggest that the protein, which we named 

Banderuola, specifically acts in establishing an axis of cell polarity for asymmetric cell division.

MONDAY 

Co-Immunoprecipitation experiments show that Banderuola binds to Discs-large (Dlg), a 

membrane-associated guanylate kinase acting in many cell-polarity events. 

Our study has identified Banderuola, a new component of the machinery of asymmetric cell 

division. As its binding partner Dlg acts in several polarity events, including synaptogenesis and 

tumor formation, our studies may be relevant for a variety of biological processes. 

2167 

Secretory vesicles deliver Cdc42p to sites of polarized growth in S. cerevisiae. 

S. A. Dighe 1 , K. G. Kozminski 1,2 ; 1 Biology, University of Virginia, Charlottesville, VA, 2 Cell 

Biology, University of Virginia, Charlottesville, VA 

Establishment and maintenance of cell polarity in many eukaryotes depends upon activation of 

the Rho GTPase Cdc42p. In the budding yeast S. cerevisiae, Cdc42p is essential for 

establishing and maintaining an axis of polarized growth that supports bud formation. In G1 of 

the cell cycle, Cdc42p asymmetrically localizes on the plasma membrane (PM), at the 

presumptive bud site, in an actin-independent manner. Maintenance of this axis of growth 

during bud formation, in S and G2, requires the actin-dependent localization of Cdc42p at the 

bud tip. How Cdc42p is maintained at the bud tip is unclear, leading to divergent models of 

Cdc42p and cell polarization (Wedlich-Soldner et al. 2003. Science 299:1231-1235; Layton et 

al. 2011. Current Biology 21:184-194). One model suggests that Cdc42p is delivered to the bud 

tip by secretory vesicles, replenishing Cdc42p that is lost at the bud tip through diffusion in the 

plane of the PM or by endocytosis. Consistent with this model, we show Cdc42p associated with 

secretory vesicles both in vivo and in vitro. By immunogold labeling EM thin sections of whole 

cells, both wild-type and secretory mutants, we localized Cdc42p to vesicles 80-100nm in 

diameter, the signature size of secretory vesicles in S. cerevisiae, in addition to the PM and ER. 

We also show that Cdc42p co-fractionates with secretory vesicles and the secretory vesicle 

markers Sec4p, Bgl2p, Osh4p, and invertase. These results indicate that secretory vesicles can 

serve as a vehicle for the delivery of Cdc42p to sites of polarized growth. Supported by NSF 

0723342. 

2168 

Ral GTPases regulate biogenesis of epithelial polarity. 

C. Hazelett 1 , C. Yeaman 1 ; 1 Anatomy & Cell Biology, University of Iowa, Iowa City, IA 

Tight junctions (TJs) are indispensible structures to epithelial cells, and are responsible for 

regulation of paracellular diffusion and maintenance of cellular polarity that is crucial to human 

health. A detailed mechanistic understanding of how TJs form, however, is incomplete. TJ 

proteins assemble at the interface between apical and lateral membrane domains, but factors 

that target vesicles to these sites during TJ formation have not been identified. TJ maturation is 

associated with establishment of polarity, and in vivo epithelial cells form tubes with distinct 

lumenal and basal domains. Furthermore, polarized epithelial cells generate a primary cilium on 

the apical surface. The Exocyst is required for ciliogenesis and cystogenesis, and under control 

of Ral GTPases, has been shown to direct vesicle trafficking to basolateral membranes. We 

investigated the roles of Ral GTPases in TJ assembly and ciliogenesis, and also in the more 

complex process of cystogenesis. We show that RalA and RalB serve opposing functions during 

the former two processes, and both are indispensible for the latter. Silencing expression of RalA 

or RalB altered TJ gate function in opposite ways during initial establishment of TJs in MDCK 

cells; RalA knockdown abrogated the trans-epithelial resistance (TER) overshoot while RalB 

knockdown greatly exaggerated it. However, TJ gate function, as assayed by transcellular lipid 

diffusion, was unaffected by either knockdown. Immunofluorescence and detergent solubility 

studies revealed Ral-specific differences in TJ composition. Levels of some TJ components

MONDAY 

were reduced in RalA knockdown cells when compared to controls, but were increased in TJs of 

RalB knockdown cells. Additional data showed that both Ral GTPases exert effects on TJ 

formation through binding to the Exocyst. Furthermore, RalB was necessary for endocytosis of 

the junctional protein E-cadherin. RalA knockdown also decreased ciliogenesis, while RalB 

knockdown led to an increase of ciliated cells. Finally, formation of a central lumen during 

cystogenesis in three-dimensional Matrigel cultures was dependent on expression of both 

GTPases. Our working model is that RalA facilitates trafficking of TJ and ciliary constituents to 

the plasma membrane via the Exocyst, while RalB regulates endocytosis and/or recycling of 

these components. This and future work will significantly increase our understanding of how 

these GTPases function together, and in collaboration with the Exocyst, to regulate epithelial 

polarization. 

2169 

Out of the loop: The role of Bem1 in actin-independent polarization in S. cerevisiae. 

S. E. Smith 1,2 , R. Li 1,2 ; 1 Stowers Institute for Medical Research, Kansas City, MO, 2 Dept of 

Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 

Cell polarization is vital for a range of important processes across the spectrum of life, from cell 

migration in human development to polarized cell division in budding yeast, but the mechanisms 

of polarization are still not well understood. A key player is the small GTPase Cdc42, the 

“master regulator” of polarity. In budding yeast, polarization of Cdc42 requires neither spatial 

cues nor cytoskeletal structures, but occurs via a dynamic, diffusion-based process. One model 

consists of a positive feedback loop in which a small, stochastic accumulation of active Cdc42- 

GTP recruits the adaptor molecule Bem1, which then recruits the activator of Cdc42, Cdc24, 

resulting in an increased local active pool and further recruitment. In this work, we disrupted 

individual steps in the proposed feedback loop by abrogating the ability of Bem1 to interact with 

either Cdc42 or Cdc24. We then investigated the effect of the disruption on diffusion-based 

polarization. As expected, we found that abrogation of the Bem1-Cdc24 interaction resulted in a 

complete loss of polarization. However, we were surprised to find that Cdc42 polarization was 

still robust when the Cdc42-Bem1 interaction was disrupted, even though Bem1 did not localize 

to the polar cap. This finding invalidates the current feedback loop model, and suggests that 

Bem1 may have a location-independent role in activating Cdc24. 

2170 

Does ciliary autotomy in the kidney contribute to PKD? 

C. Yeaman 1 , N. Ma 1 ; 1 Anatomy & Cell Biology, University of Iowa, Iowa City, IA 

Primary cilia are active signaling centers present on most differentiated cell types. Cells may 

lose their cilia either by resorption into the cell, or shedding (autotomy) from the cell surface. 

Autotomy is an ancient process, but its physiological significance in mammals is unclear. We 

have shown that diverse agents trigger ciliary shedding in renal epithelial cells both in vitro and 

in vivo. This occurs by a distinct mechanism than that described for ciliary resorption, as 

inhibitors of resorption fail to block autotomy and intact cilia are recovered in the media of 

cultured cells and in the urine of mice following treatment with autotomy-inducing agents. 

Shedding of primary cilia promotes dramatic changes to epithelial cells, including compositional 

and functional changes to tight junctions, induction of partial epithelial-to-mesenchymal 

transition and dysregulated cell cycle control. A full-length cilium is not required to trigger 

autotomy-induced epithelial remodeling. It occurs in cells that express stunted pro-cilia, such as 

non-differentiated epithelial cells and cells lacking essential ciliogenesis components (e.g. 

IFT88). We hypothesize that autotomy liberates the maternal centriole from its tethered state at 

the apical plasma membrane, thereby facilitating its differentiation into a signal-generating

MONDAY 

centrosome. In renal epithelial cells, ciliary autotomy is dependent on the catalytic activity of 

Nek8 and the Exocyst vesicle tethering complex. jck mice, which develop autosomal-recessive 

polycystic kidney disease and harbor a mutation in the NEK8 gene, exhibit substantially 

elevated levels of ciliary shedding in vivo. Therefore, we propose that ciliary autotomy is a 

physiologic phenomenon in the kidney, and that dysregulated autotomy may contribute to PKD 

by altering trans-epithelial fluid transport, tissue morphogenesis and growth regulation. 

Cancer Cell Biology I 

2171 

Evaluating the effect of South African herbal extracts on breast cancer cells. 

M. Choene 1 , L. Motadi 1 ; 1 Genetics, University of the Witwatersrand, Johannesburg, South Africa 

South African plants have been used for generations by traditional healers due to their antibacterial, 

anti-viral, anti-fungal and anti-amoebic properties. Now with the emergence of natural 

product research in the science fraternity, a lot of these indigenous plants have been showing 

great potential as an alternative cancer treatment to orthodox treatments. For our research, our 

aim was to investigate the anti-breast cancer activities of three South African plant extracts: 

Kedrostis Foetidissima, Euphorbia Mauritanica and Elytropappus Rhinocerotis. We looked at 

the effect of these extracts on breast cancer cells since it is one of the leading cancers in 

females in South Africa. The herbal extracts were screened for their cytotoxicity in two breast 

cancer cell lines MCF-7 and YMB-1 by means of an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl 

tetrazolium bromide) assay. MTT is a simple colorimetric assay used to measure cell 

cytotoxicity, proliferation or viability. The cells were treated with varying concentrations (10, 30, 

50 and 100µg/ml) of the extracts, then the MTT assay was conducted to determine the IC50 

(concentration of extract that causes 50% cell death) of these herbal extracts on the cell lines. 

We found that Elytropappus Rhinocerotis had no significant effect on both cancer cell lines, 

Kedrostis Foetidissima exhibited cytotoxicity on both cell lines with 48% cytotoxicity at 100µg/ml 

(IC50 > 100µg/ml) on MCF-7 cells and 40% cytotoxicity at 100µg/ml (IC50 > 100µg/ml) on YMB- 

1 cells. Euphorbia Mauritanica showed 53% cytotoxicity at 100µg/ml and 37% cytotoxicity at 50 

µg/ml (50µg/ml 100 µg/ml) on MCF-7 but had no significant effect on YMB-1. For those 

extracts which exhibited cytotoxicity on the breast cancer cells, we then aimed to investigate if 

the cells were dying due to apoptosis and not necrosis. We then employed the use of a flow 

cytometer using Annexin V, which can detect cells that are alive, undergoing apoptosis and 

necrosis (Annexin V has a strong calcium dependent affinity to phosphatidylserine residues, 

which are normally hidden in the cytoplasmic plasma membrane but translocate to the cell 

surface during apoptosis, acting as a probe to detect apoptosis). It was then observed that the 

herbal extracts inducing death via apoptosis and not necrosis. We can conclude that the herbal 

extracts K. Foetidissima and E. Mauritanica exhibit some level of cytotoxicity on breast cancer 

cells. It is also promising that they cause cells to undergo apoptosis showing great potential in 

their use in breast cancer natural product research and drug design sector. 

2172 

Traditional herb in cancer. 

L. R. Motadi 1 , A. Thafeni 1 , M. Choene 1 ; 1 School of Molecular Biology, University of the 

Witwatersrand, Johannesburg, South Africa 

Euphorbia mauritanica and Kedrostis hirtella extracts can induce anti-proliferative activities in 

cancer cells.

MONDAY 

In recent times a significant proportion of the drugs used to treat cancer are either natural 

products or compounds discovered based on the study of natural products and their interaction 

with cellular targets. While the use of synthetic combinatorial libraries has enhanced the pace of 

drug discovery, the extraordinary structural diversity intrinsic to natural products assures that 

they will continue to find utility in defining novel cellular targets for therapeutic intervention, and 

also as drug candidates and structural leads for chemical optimization. With our increased 

understanding of the molecular mechanisms underlying cancer progression and cell death, this 

work aims to target E. mauritanica and K. hirtella as new therapeutic targets for treatment in 

cancer in particular Breast and Lung cancer. Accordingly, the study was aimed to investigate 

possible molecular mechanisms that are associated with the potential anti-carcinogenic property 

of this agrofield weed. MCF-7 and A549 cells were exposed to different concentrations (0-100 

mg/ml) of the crude methanolic extract of E. mauritanica and K. hirtella to evaluate their growth 

inhibitory and apoptosis inducing effects. The extract elicited a dose- and time-dependent 

inhibition of cell proliferation, followed by a concomitant decrease in cell viability. The observed 

cytotoxicity was linked to the induction of apoptosis as determined by Flow cytometer Annexin V 

features known to be associated with the advent of apoptosis. Real time quantitative RT-PCR 

analyses of Bax, Bcl-2, RBBP6 and p53 exhibited aberrant expression profiles of these genes 

under various treatment conditions. Taken together, the data suggest that the crude methanolic 

extract of E. mauritanica and K. hirtella contains bioactive compounds that may be beneficial in 

the treatment of malignant growths. However, the mechanisms behind this activity still need to 

be researched. 

2173 

A natural small molecule YCG185 inhibits angiogenesis both in vitro and in vivo. 

Y. Kim 1 , H. Jung 1 , H. Kwon 1 ; 1 Chemical Genomics National Research Laboratory, TRCP, 

Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, 

Seoul, Korea 

Angiogenesis, a formation of new blood vessels from pre-existing ones, plays a critical role in 

normal and pathological phenotypes including solid tumor growth and metastasis. Accordingly, 

the development of new anti-angiogenic agents has been considered as an efficient strategy for 

the treatment of cancer and other human diseases related with angiogenesis. To identify such 

small molecules, 300 crude extracts of natural plants were subjected to cell-based screening 

toward the proliferation of human umbilical vein endothelial cells (HUVECs). As the result, the 

active principle of YCG185 was identified as a new anti-angiogenic agent. YCG185 inhibited the 

proliferation of HUVECs at IC50 of 6.6 μg/mL without showing any toxicity to the cells. YCG185 

significantly suppressed in vitro angiogenesis such as VEGF-induced tube formation and 

chemoinvasion. Moreover, the compound inhibits chorioallantoic membrane in vivo 

angiogenesis at non-toxic doses. In addition, YCG185 decreased the expression levels of 

hypoxia inducible factor-1a and its target gene, VEGF, in a dose-dependent manner. Taken 

together, these results demonstrate that YCG185 could be a novel natural small molecule 

targeting angiogenesis. 

2174 

Genetic Modification of Breast Cancer Gene Targets Using ZFN Technology Reveal 

Differential Responses to Drug Sensitivity. 

G. L. Davis 1 , C. Corman 1 , L. Daley 1 , G. Pegg 1 , H. Zakeri 1 , Z. Zhang 1 , G. Wemhoff 1 ; 1 Sigma 

Aldrich, St. Louis, MO 

Breast cancer tumorigenesis is a complex disease where multiple signaling pathways 

participate in cell proliferation and invasion. The heterogeneity of this disease presents a

MONDAY 

challenge in developing therapuetic treatments because patients respond to therapies with 

varying degrees of sensitvity 1,2. For example, previous studies have demonstrated that 

activation of the MEK/MAPK pathway, loss of cell-cell adhesion, and enahnced epithelial-tomesenchymal 

transition confer resistance to the breast cancer therapeutic, gefitinib 2,3,4. 

With the advent of zinc finger nuclease (ZFN) technology, it is possible to generate cancer 

relevant mutations in one or more endogenous genes. Zinc finger nucleases have been 

optimized to target specific genes where they induce a double-strand break adjacent to their 

binding site. Double strand-breaks are either repaired by non-homologous end joining (NHEJ) or 

homology dependent repair (HDR). As a result, cell lines harbor insertions, deletions, or 

integrations within the targeted gene of interest. 

We have utilized zinc finger nuclease technology to target and disrupt endogenous breast 

cancer relevant genes, SYK, ESR2, BCR, and APC, in the near normal mammary epithelial line, 

MCF10a. These gene targets play a pivotal role in cellular proliferation, migration and cell 

adhesion and may thereby play a contributing role in gefitinib sensitivity 5,6,7,8,9,10. In this 

study, we aim to investigate how loss of SYK, ESR2, BCR, and APC gene function, in ZFN 

engineered cell lines, affect sensitivity towards the tyrosine kinasae inhibitor, gefitinib. 

2175 

Gene Specific Regulation of NFκB-Dependent Anti-Apoptotic Genes in Metastatic 

Prostate Cancer Cells by Bortezomib. 

S. Manna 1 , B. Singha 1 , T-P. Chang 1 , S. Sanacora 1 , I. Vancurova 1 ; 1 Biological Sciences, St. 

John's University, Queens, NY 

Prostate cancer is the third most common cause of death from cancer in men of all ages. One of 

the critical factors in progression to the metastatic prostate cancer is the increased activity of the 

transcription factor NFκB, which induces synthesis of anti-apoptotic genes, such as Bcl-2, cIAP- 

1 and cIAP-2, thus promoting inhibition of apoptosis and resistance to chemotherapy. 

Bortezomib is a potent 26S proteasome inhibitor that has been used for the treatment of 

multiple myeloma and mantle cell lymphoma, and has shown promising results in several other 

types of cancer, including the metastatic prostate cancer. However, the molecular mechanisms 

of bortezomib function in prostate cancer cells are not fully understood. In this study, we 

investigated the mechanism of bortezomib function in the metastatic prostate cancer PC-3 cells. 

Our data demonstrate that in PC-3 cells, bortezomib induces translocation of the NFκB 

inhibitory protein, IκBα, from the cytoplasm to the nucleus, thus inhibiting the constitutive NFκB 

activity in these cells. However, our results indicate that the regulation of NFκB activity by the 

bortezomib-induced nuclear IκBα is gene specific. While the expression of NFκB-dependent 

anti-apoptotic genes cIAP-1 and cIAP-2 is inhibited by bortezomib, expression of Bcl-2 is not 

suppressed. Differences in the transcriptional regulation by the bortezomib-induced nuclear 

IκBα might hold the key for development of more effective therapies for the advanced forms of 

prostate cancer and other cancers characterized by the constitutive activation of NFκB and 

resistance to chemotherapy. 

Funded by: NIH grants GM079581 and AI085497 to I.V.

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2176 

The neuroprotective efficacies and anticancer properties of various botanical extracts, 

Rue chalpanesis and Hibiscus sabdariffa, against neurotoxic-induced Parkinson's 

disease and pancreatic adenocarcinoma in vitro. 

T. Zeleke 1 , H. Tsegaye 1 , A. Bettica 1 ; 1 Biology, Manhattanville College, Purchase, NY 

Polyphenolic antioxidants have demonstrated anticarcinogenic activities by inhibiting or delaying 

the oxidation process caused by free radicals. Many extracts, containing anthocyanins, 

flavonoids, or phenolic compounds, have been shown to be strong antioxidants and possible 

neuroprotective agents. Common neurodegenerative disorders, such as Parkinson's disease 

(PD), may show neuronal cell loss as an accumulation of reactive oxygen species (ROS) due to 

increased oxidative stress. Roselle extract from the tropical plant Hibiscus sabdariffa contains 

primarily anthocyanins and protocatechuic acid, which can exhibit antioxidant, anti-inflammatory 

and antitumorigenic properties. Rue extract, from the tropical plant Ruta chalepensis, may also 

protect cells from oxidative stress-induced free radicals. The planetary structures of acridone, a 

major constituent in the Rue extract, can be inserted between a double stranded DNA and 

interfere with cellular machinery, contributing to its antitumor properties. For plant extractions, 

dried plant samples of R. chalapensis are centrifuged, vortexed, and agitated in the cold for 24 

hours. H. sabdariffa (10g) is boiled in water, followed by filtration. To determine inhibition of 

growth rate in pancreatic adenocarcinoma cell lines, PANC-1 cells are seeded at a 

concentration of 6x104 cells/well in 96-well plates. After seeding and adherence to the well 

plate, all cells are allowed one to two population doubling times (PDT) before and after 

treatments. Single drug 24 hour treatments for dose response curves in ten fold dilutions in 

water determine effective IC50 values for Ruta and Hibiscus extracts. To determine the 

neuroprotective efficacies of these extracts with relationship to an oxidative stress-induced in 

vitro PD model, pretreatments of SK-N-SH neuroblastoma cells with these extracts against the 

neurotoxic insults, MPP+ and 6-OHDA were undertaken. The cells are pretreated in 96-well 

plates to formulate dose-response curves, with each of the serially-diluted extracts added for 24 

hours before a potent dose of MPP+, a mitochondrial complex I inhibitor, or 6-OHDA, a potential 

ROS generator. The cells are exposed to each insult first for 24 hours for posttreatment studies, 

followed by the extracts for 24 hours. Cotreatments with each extract and the neurotoxic insults 

were performed to elucidate possible preconditioning or gene activation versus actual 

mechanisms of action. Cell viability/ proliferation of triplicate data sets are assessed either 

through the MTT mitochondrial (for PANC-1 cells) or Neutral Red lysosomal assay (for 

neuroblastoma cells). All data are statistically analyzed using a one-way ANOVA with Tukey’s 

post hoc tests. Preliminary dose responses for R. chalepensis and H. sabdariffa extracts show 

effective doses of 10-4-10-6 g/ml for SK-N-SH cells. The neuroprotective efficacies of the 

botanical extracts against the neurotoxin-induced PD model may indicate their successful use in 

the prevention or treatment of Parkinson’s disease. Effective IC50 values may lead to possible 

chemopreventive strategies for certain cancer cell types as well. 

2177 

Gefitinib resistance of cancer cells correlated with TM4SF5-mediated 

epithelialmesenchymal transition. 

M. Lee 1 , J. W. Lee 1,2 ; 1 Department of Pharmacy, Reseach Inst of Pharmaceutical Sciences, Cell 

Dynamics Res Ctr, College of P, Seoul National University, Seoul, Korea, 2 Cancer Research 

Institute, College of Medicine, Seoul National University, Seoul, Korea 

Although cancers can be initially treated with the epidermal growth factor receptor (EGFR) 

inhibitor, gefitinib, continued gefitinib therapy does not benefit the survival of patients due to 

acquired resistance through EGFR mutations, c-Met amplification, or epithelial-mesenchymal

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transition (EMT). It is of further interest to determine whether mesenchymal-like, but not 

epithelial-like, cancer cells can become resistant to gefitinib by bypassing EGFR signaling and 

acquiring alternative routes of proliferative and survival signaling. Here we examined whether 

gefitinib resistance of cancer cells can be caused by transmembrane 4 L six family member 5 

(TM4SF5), which has been shown to induce EMT via cytosolic p27Kip1 stabilization. Gefitinibresistant 

cells exhibited higher and/or sustained TM4SF5 expression, cytosolic p27Kip1 

stabilization, and mesenchymal phenotypes, compared with gefitinib-sensitive cells. Conversion 

of gefitinib-sensitive to -resistant cells by introduction of the T790M EGFR mutation caused 

enhanced and sustained expression of TM4SF5, phosphorylation of p27Kip1 Ser10 

(responsible for cytosolic location), loss of E-cadherin from cell-cell contacts,and gefitinibresistant 

EGFR and survival signaling activities. Additionally, TM4SF5 overexpression lessened 

the sensitivity of NSCLC cells to gefitinib. Suppression of TM4SF5 or p27Kip1 in gefitinibresistant 

cells via the T790M EGFR mutation or TM4SF5 expression rendered them gefitinibsensitive, 

displaying more epithelial-like and less mesenchymal-like characteristics. Together, 

these results indicate that TM4SF5-mediated EMT may have an important function in the 

gefitinib resistance of cancer cells. [This work was supported by NRF by senior researchers 

program (Leap research, 2011-0001160) and Global Frontier Project grant (NRF-M1AXA002- 

2010-0029778), and a grant of the Korean Health Technology R&D Project (A100727), 

MHWFA, Korea to JW Lee]. 

Key words: epithelial-mesenchymal transition; drug resistance; EGFR; lung cancer; 

tetraspanin 

2178 

Bioavailability Enhancing Strategy for Oral Administration of Doxorubicin. 

Y. ZHAO 1 , M. L. Forrest 1 ; 1 Pharmaceutical Chemistry, the University of Kansas, Lawrence, KS 

Doxorubicin (DOX) has been used as an anticancer drug to treat a variety of cancers. Due to 

the first pass effect, DOX has been only administrated intravenously. Recent studies have 

shown that isoflavonoid, i.e. quercetin, can improve oral bioavailability of doxorubicin by 

blocking the activity of p-glycoprotein. In addition, quercetin is absorbed primarily by gut lymph. 

Our hypothesis is that oral administrated doxorubicin-quercetin prodrug (DQ) will improve oral 

bioavailability and potentially enhance the absorption of DOX in gut. We have successfully 

synthesized DQ. The IC50 of DOX, quercetin and DQ in 4T1.2 cells were 5.7 µM, 49.2 µM and 

14.04 µM, respectively. Both free DOX and DQ were orally fed to Sprague Dawley female rats. 

Pharmacokinetic study showed that 1h after drug administration, only DQ, but not free DOX, 

gave an active form of DOX in the blood serum. This study showed that the doxorubicinquercetin 

prodrug strategy can be used to improve oral bioavailability of DOX. 

2179 

Induction of Intracellular Superoxide in Chronic Myelogenous Leukemia Cells Leads to 

an Antioxidant Response Downstream of NFE2L2. 

L. F. Gemta 1 , F. Alvarez-Calderon 1,2 , K. Hansen 1 , J. DeGregori 1,3 ; 1 Biochemistry and Molecular 

Genetics, University of Colorado - Anschutz Medical Campus, Aurora, CO, 2 Medical Scientist 

Training Program, University of Colorado - Anschutz Medical Campus, Aurora, CO 3 Molecular 

Biology, University of Colorado - Anschutz Medical Campus, Aurora, CO 

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by the 

BCR-ABL fusion gene. Bcr-Abl tyrosine kinase inhibitors including Imatinib have been used to 

treat CML in patients. These inhibitors fail to eliminate all types of Bcr-Abl+ leukemia especially 

in advance and blast crisis phases. In this study, we identified that inhibiting mitochondrial

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metabolism with oligomycin A, an inhibitor of mitochondria ATP-synthase, sensitizes CML cells 

to Imatinib. The combination of 1 µM Imatinib and 5-10 nM oligomycin A resulted in elimination 

of CML cells without inhibition of mitochondrial respiration. Although Annexin V/Propidium 

Iodine analysis showed that most of the cells died by apoptosis, the exact mechanism by which 

the combination therapy successful eliminated these cancer cells is unknown since the doses of 

OA used are ~100-fold lower than the IC50. The level of HIF-1α and downstream targets of Bcr- 

Abl including p-Akt, p-ERK, and p-STAT5 were not affected by the treatment. The combination 

therapy increased intracellular superoxide levels and induced expression of the antioxidant 

response downstream of NFE2L2 expression. Normal and cancerous cells protect themselves 

from the damage caused by high levels of superoxide by activating their antioxidant responses. 

These responses include the induction of the transcription factor nuclear factor (erythroidderived 

2)-like 2 (NFE2L2), which in turns regulates the expression of its downstream target 

genes including superoxide dismutase-2 (SOD2), gamma-glutamyltransferase 1 (GGT1), 

glutamate-cysteine ligase catalytic subunit (GCLC), and heme oxygenase (decycling) 1 

(HMOX1). The combination therapy inhibits mitochondrial metabolism without inhibiting 

mitochondrial respiration and leads to increases in superoxide levels and compensatory 

increases in antioxidant responses. 

2180 

Genome-Wide RNAi Screen for Tetraploid-Specific Lethality in Cancer Cells. 

T. S. Kuroda 1 , R. K. Dagher 1 , D. Pellman 1 ; 1 Pediatric Oncology, Dana-Farber Cancer Institute, 

Howard Hughes Medical Institute, Boston, MA 

Cancer cells are often genetically unstable, resulting in aneuploid genomes. The consequences 

of aneuploidy are poorly understood, but it has been suggested to play roles in tumor 

aggressiveness, chemotherapy resistance, and metastasis. 

One important route to an aneuploid genome may be through doubling of the genome, leading 

to unstable tetraploid cells. Many cell division errors ultimately manifest as a failure of 

cytokinesis. Especially in cells defective in p53 function, tetraploid cells proliferate, but generate 

both high rates of whole chromosome aneuploidy and defects in the maintenance of 

chromosome structural integrity. Our laboratory has demonstrated that in p53-null primary cells, 

tetraploidy promotes tumor development and the resulting tumors display markedly abnormal 

genomes (Fujiwara T. et al. Nature 2005, 437: 1043). It is now known that several human 

cancer-inducing mutations-- e.g. RB loss or loss of the adenomatous polyposis coli tumor 

suppressor-- also predispose cytokinesis failure and tetraploidy. These findings highlight a need 

to understand in detail the physiological changes that accompany tetraploidy, which might 

provide with a new strategy for cancer therapeutics. 

To gain an understanding of physiological alterations associated with tetraploidy in cancer cells, 

we have established tetraploid cell lines derived from a near diploid human colorectal cancer 

cell line HCT116. Compared to diploid cells, anaphase FISH showed that tetraploid cells have a 

higher rate of chromosome mis-segregation—with levels similar to chromosomally unstable 

cancer cells. 

Using these paired tetraploid and diploid cell lines, we have performed a genome-wide RNAi 

screen to identify genes whose knockdown selectively affect the viability of chromosomally 

unstable tetraploid cells. After secondary validation of the screen, we have identified 61 

tetraploid-selective lethality genes. Consistent with the known p53-dependent barrier to 

proliferation of tetraploid and aneuploid cells, and validating the overall approach, one 

tetraploid-selective lethality gene is MDM2, an E3 ligase for p53.

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2181 

Growth Inhibitory Effects of Death Receptor 5 Agonist, Drozitumab alone and in 

Combination with the Novel Spirocyclic Lignan Ramonanin A. 

K. J. Chavez 1 , B. Stone 1 , S. Lipkowitz 1 ; 1 Laboratory of Cellular and Molecular Biology, National 

Institutes of Health, National Cancer Insititute, Bethesda, MD 

Clinically, breast cancers can be divided into distinct subtypes that express estrogen receptors 

(ER), progesterone receptors (PR), those that have amplification of HER2/Neu, and those that 

lack expression of ER or PR and lack amplification of Her2/Neu (so called triple negative breast 

cancer or TNBC). TNBC has a poor outcome compared to the other subtypes of breast cancers. 

Previously, we have shown that a recombinant GST fusion protein with TNF-related Apoptosis 

Inducing Ligand (GST-TRAIL) selectively kills triple-negative breast cancer cells with 

mesenchymal features by activation of TRAIL receptor 2 (TRAIL-R2). In this study we have 

characterized the growth inhibitory effects of a clinically relevant agonistic antibody to TRAIL- 

R2, referred to as Drozitumab. To determine the selectivity of Drozitumab, we chose a panel of 

15 breast cancer cell lines including 3 ER/PR positive cell lines, 4 HER2/Neu amplified cell 

lines, and 8 triple-negative cell lines. Treatment of these cells with Drozitumab selectively killed 

TNBC cell lines with mesenchymal features. ER positive, HER2/Neu amplified and TNBC cell 

lines with epithelial features were resistant to Drozitumab induced cell death. Drozitumab 

induced caspase activation (measured by activation of the initiator caspase 8, activation of the 

downstream caspases 3/7, and PARP cleavage) and the toxicity of Drozitumab was blocked by 

the pan-caspase inhibitor z-VAD-FMK. This was identical to the pattern seen with GST-TRAIL. 

Cross-linking with an anti-FC antibody enhanced the efficacy of Drozitumab as shown by the 

more rapid appearance of cleaved caspase 8, greater caspase 3/7 activity, and more rapid 

PARP cleavage. Furthermore, we show that a novel family of Spirocyclic lignans from 

Guaiacum sp. (Ramonanins) enhances Drozitumab -induced apoptosis in MBA-MD231and 

HCC38 triple-negative breast cancer cell lines. In conclusion, our data indicates that Drozitumab 

selectively inhibits growth of mesenchymal phenotype triple-negative breast cancer cell lines in 

a caspase dependent fashion and that cell death is enhanced in the presence of Ramonanin A. 

This data provides evidence that TRAIL-R2 targeted therapies may have therapeutic potential in 

the treatment of TNBC with mesenchymal features. 

2182 

The effective inhibition of growth rate and induction of apoptosis in adherent and 

suspension cancer cell lines by the administration of various Panax (Ginseng) species. 

N. Yousaf 1 , A. Luiso 1 , T. Zeleke 1 , A. Bettica 1 ; 1 Biology, Manhattanville College, Purchase, NY 

Ginseng (Panax) is a perennial plant with fleshy roots that contains Rg1, Rg2, Rf and other 

ginsenoside groups. A popular traditional medicine for centuries, ginseng is believed to enhance 

memory, stimulate the immune system, reduce cholesterol levels, and fight depression and 

insomnia. Various Panax species, such as Korean, American, and Chinese Red or White 

Ginseng, have been shown to enhance memory and stimulate the immune system. Ginseng 

extracts have also been considered as adaptogens, natural substances that aid in homeostatic 

balance. The red ginseng, in its various forms, differs from white ginseng in its processing. The 

Korean ginseng, grown in cooler climates, has higher ginsenosides/saponin count than Chinese 

ginseng. Panax red ginseng, either Chinese or Korean, is considered to be more potent than the 

American ginseng, Panax quinquefolium. To determine the anticancer effects of the various 

ginseng species, the inhibition of growth rate and the induction of apoptosis is determined for 

both an adherent pancreatic adenocarcinoma cell line (PANC-1) and a suspension B-cell 

leukemic cell line (SUP-B15). B cell chronic lymphocytic leukemia (B-CLL) has long been 

considered a disease in which B-CLL cells accumulate due to a presumed defect in their

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apoptotic mechanism. Previous morphologic observations suggest that B-CLL cells are resting 

cells with rarely detected cell cycle activity in the blood. For single and combination treatments, 

cells will be seeded at a density of 5x104 cells/well in 96-well plates. For suspension cultures, 

round bottom 96 well plates are used. When seeding round bottom well plates, the cells are 

allowed to sediment, forming a relatively loose monolayer. All plated cells are allowed one to 

two population doubling times (PDT) before treating. Single drug 24 hour treatments for dose 

response curves in ten fold dilutions determine effective IC50 values for each of the ginseng 

species. Results from these preliminary dose response studies determine whether consecutive 

(24 hours of one species followed by 24 hours of second species at effective doses) or additive 

drug trials (1/2 effective dose of each species together for 24 hours), is considered. The 

treatment course is followed by 1-2 PDT and assayed by one of two different methods. All 

treatment plates are repeated in triplicate. To determine inhibition of growth rate and cell 

viability, the MTT cell proliferation assay is performed with adequate washing. The optical 

density is read between 490-550nm and adjusted for background. The Caspase-3 colorimetric 

protease assay quantifies caspases that recognize the amino acid sequence DEVD, and since 

caspase 3 is an effector caspase, the level of apoptosis under the test conditions can be 

determined. All data are statistically analyzed using a one-way ANOVA with Tukey’s post hoc 

tests. Treatments with American Ginseng showed protection in the nanogram ranges. Korean 

Ginseng was more effective individually and, in combination treatments, the Chinese 

Red/Chinese White Ginseng extracts were most effective overall. Considering the growth rate 

inhibition and induction of apoptosis on adherent and suspension cancer cultures, ginseng 

species are potential anticancer agents. 

2183 

Isoform specific function of the formin FMNL2. 

C. Péladeau 1 , A. Heibein 1 , G. L. Lahaie 1 , S. Copeland 1 , J. Copeland 1 ; 1 Cellular and Molecular 

Medicine, University of Ottawa, Ottawa, ON, Canada 

Cancer cell metastasis is induced by actin-dependent cell migration and is consequently 

affected by the regulated function of cytoskeletal remodelling proteins. Formins, a highly 

conserved family of actin remodelling proteins, have been shown to provoke motility during 

metastasis and invasion. Indeed, formin-like 2 (FMNL2) has been shown to increase motility 

and facilitate invasion when overexpressed in colorectal cancer cells (CRC). Furthermore, 

knocking down the expression of FMNL2 inhibits amoeboid-style invasion by melanoma cells in 

vitro. FMNL2 mRNA is subject to alternative splicing and results from our lab, and others, 

suggest that the resulting encoded proteins are likely to differ in their regulation, subcellular 

localization and activity. The main objectives of this study are to identify the FMNL2 splice forms 

expressed in various cancer cell lines and determine if FMNL2 activity is required by these cells 

for invasion. In this study we have used RT-PCR to identify four FMNL2 splice forms (ITM, YHY, 

PMR and TQS) expressed in non-invasive (SW480) and invasive (SW620) colorectal cancer 

cells, as well as in highly invasive A375 melanoma cells. Preliminary data from cell-based 

assays suggest that there are functional differences between our identified FMNL2 isoforms in 

terms of both auto-regulation and their effects on actin dynamics. In addition, our qPCR data 

suggests that an “invasive” isoform of FMNL2 may be preferentially expressed in highly invasive 

cells. The data obtained in this study will lead to further understanding of the role of FMNL2 in 

invasion and metastasis as well as identify specific targets for the development of future antimetastatic 

therapies.

2184 

Profilin-1 downregulation promotes transendothelial migration of breast cancer cells 

through increased VEGF secretion. 

M. Joy 1 , P. Roy 2 ; 1 Bioengineering, University of Pittsburgh, 2 Bioengineering and Pathology, 

University of Pittsburgh, Pittsburgh, PA 

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Transendothelial migration of cancer cells is necessary for both intravasation and extravasation 

steps of tumor metastasis. Clinical correlation and xenograft studies from our laboratory have 

revealed that downregulation in the expression of profilin-1 (Pfn1: an actin-binding protein) is 

associated with increased metastatic potential of breast cancer cells. The aim of the present 

study was to evaluate the effect of loss of Pfn1 expression on transendothelial migration of 

breast cancer cells. We found that stably silencing Pfn1 expression through shRNA increases 

the ability of MDA-MB-231 breast cancer cells to transmigrate through a confluent monolayer of 

human vascular endothelial cells (VEC), and this can be rescued by re-expression of an RNAiresistant 

version of Pfn1. Immunostaining for beta-catenin (a component of adherens junctions) 

showed that Pfn1 knockdown facilitates tumor cell-induced disruption of cell-cell junctions in 

VEC. Biochemical analyses of conditioned media derived from MDA-231 cells revealed that 

secretion of several factors that promote tumor metastasis including VEGF (a potent disruptor of 

endothelial cell-cell junctions) is significantly increased upon loss of Pfn1 expression. Silencing 

VEGF expression abrogates the stimulatory effect of Pfn1 KD on transendothelial migration thus 

suggesting that Pfn1 downregulation promotes transendothelial migration of breast cancer cells 

through increased VEGF secretion. These findings set the stage for future in vivo studies 

examining Pfn1’s role in regulating tumor cell dissemination into the vasculature. 

2185 

Microarray analysis of differentially expressed genes in MDA-MB-231 breast cancer cells 

treated with the laminin-derived peptide C16. 

E. S. Santos 1 , V. M. Freitas 1 , M. S. Menezes 1 , G. M. Machado-Santelli 1 , R. G. Jaeger 1 ; 

1 

Departament of Cell and Developmental Biology, Institute of Biomedical Sciences of The 

University of Sao Paulo, Sao Paulo, Brazil 

Human breast cancer constitutes a worldwide health care problem. During breast cancer 

development and progression, tumor cells are engaged in a complex interplay with the 

surrounding microenvironment. In this scenario, extracellular matrix molecules play important 

roles influencing malignant behavior. An increasing number of evidences have shown that 

peptides derived from laminin cleavage are involved in tumor progression. Among them, C16 

(KAFDITYVRLKF), derived from laminin-111 gamma-1 chain, is a cell-adhesive peptide that 

increases cell migration and invasion, enhances metastasis, and promotes angiogenesis. This 

prompted us to analyze whether C16 would regulate gene expression in cells derived from 

human breast cancer (MDA-MB-231). Cells were treated with C16 or scrambled peptide control 

(FKLRVYTIDFAK) for 24 hours. After treatment, gene expression was analyzed by microarray. 

Total RNA was extracted, biotin-labeled cDNA was generated and hybridized to Human Gene 

1.0 ST Array (Affymetrix). GeneChips were stained and scanned. Expression Console software 

converted gene fluorescence intensities into numerical values. We found that C16 regulates 

expression of 80 genes in MDA-MB-231, including genes directly involved in processes 

associated with cancer. Among those genes, FGFR3, GPNMB and SPOCK1 expression was 

increased by C16 treatment, as confirmed by quantitative Real-Time PCR. FGFR3 encodes a 

tyrosine kinase receptor, which regulates various biological processes, including cell migration 

and angiogenesis. GPNMB encodes a transmembrane glycoprotein involved in angiogenesis 

and promotion of migration, invasion and bone metastasis of breast cancer cells. SPOCK1, on 

the other hand, encodes a proteoglycan associated with cell adhesion and increased migration.

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To correlate gene expression with function, we analyzed whether C16 would regulate invasion, 

proliferation and apoptosis in MDA-MB-231 cells. Functional results also indicated that C16 

significantly increases MDA-MB-231 cell invasion, as shown by assays in Boyden chambers 

coated with Matrigel. However, no effect on cell proliferation and apoptosis was observed. We 

concluded that laminin-derived peptide C16 regulates gene expression and enhances invasion 

of metastatic MDA-MB-231 breast cancer cells. 

2186 

Laminin-derived peptide C16 induces invasion and invadopodia activity in human oral 

squamous cell carcinoma and fibrosarcoma cells. 

A. S. Siqueira 1 , R. Rafael 1 , M. P. Pinto 1 , V. M. Freitas 1 , R. G. Jaeger 1 ; 1 Cell and Developmental 

Biology, Institute of Biomedical Sciences - University of Sao Paulo, Sao Paulo, Brazil 

Our Laboratory has been studying the effects of laminin-derived peptides in tumor biology. 

Here, we addressed whether peptide C16 (KAFDITYVRLKF, gamma 1 chain) would stimulate 

invasion activity in cell lines derived from oral squamous cell carcinoma (OSCC) and 

fibrosarcoma (HT1080), two malignant tumors with different origins and poor prognosis. Using 

Boyden chambers coated by Matrigel, we observed that C16 increased invasion rate in both cell 

lines compared to scrambled control peptide (C16SX). Tumor cells that actively invade 

surrounding tissues depend on invadopodia to degrade extracellular matrix barriers. 

Invadopodia are actin-rich membrane protrusions associated with proteolytic activity. Therefore, 

we analyzed the role of C16 on invadopodia activity of OSCC and HT1080 cells, through a 

fluorescent substrate degradation assay. Measurement of digestion spots (black areas in 

fluorescent background) showed that C16 significantly increased invadopodia activity of both 

neoplastic cell lines compared to C16SX. Invadopodia dynamics in C16-treated cells was futher 

explored using time-lapse 4D fluorescence microscopy. Time-lapse videos of living cells 

transfected with GFP-cortactin and cultured on fluorescent gelatin were acquired at 5 min 

interval (total 1-2 hours). Invadopodia extensions through the fluorescent substrate were 

evaluated with acquisition of at least ten Z sections per time point, using a piezoelectric device 

coupled to the microscope objective. Measurements of degraded areas showed that C16 

increased invadopodia activity over time compared to C16SX. We also searched for signaling 

pathways related to C16 effects in OSCC and HT1080 cells. This peptide increased p-ERK 

expression in both cell lines compared to control.Furthermore, inhibition of ERK signaling 

pathway with MEK inhibitor U0126 decreased C16-related invasion and invadopodia activity. 

These results demonstrated that C16 effects in these cell lines may be related to ERK 1/2 

pathway. We conclude that C16 increases invasion and invadopodia activity in cells derived 

from oral squamous cell carcinoma and fibrosarcoma, probably through ERK signaling pathway. 

Support: FAPESP (08/57103-8; 09/17336-6) and CNPq (304986/2009-7). 

2187 

ADAMTS-1 regulating migration, invasion, and invadopodia formation in breast cancer 

cells. 

V. M. Freitas 1 , J. B. Amaral 1 , E. S. Santos 1 , R. G. Jaeger 1 , F. R. Mangone 2 , M. A. Nagai 2 , G. M. 

Machado-Santelli 1 ; 1 Department of Cell and Developmental Biology, Institute of Biomedical 

Sciences of The University of Sao Paulo, Sao Paulo, Brazil, 2 Department of Radiology, School 

of Medicine of The University of Sao Paulo, Sao Paulo, Brazil 

ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the 

ADAMTS family of metalloproteases. This enzyme is related to pathological processes such as 

inflammation and cancer. In spite of ADAMTS-1 biological relevance, the mechanisms 

underlying its involvement in tumor biology remain elusive. Here we sought to investigate the

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role played by ADAMTS-1 regulating migration and invasion of malignant mammary cell lines 

(MDA-MB-231 and MCF7). We observed variable levels of ADAMTS1 mRNA expression in a 

series of primary breast tumors. MDA-MB-231 cells with reduced expression of ADAMTS-1 

exhibited increase in migration and invasion. Cancer cells depend on invadopodia to degrade 

and invade surrounding extracellular matrix. Invadopodia are membrane protrusions with 

enzymes required for extracellular matrix components (ECM) degradation. Silencing of 

ADAMTS-1 by siRNA increased invadopodia activity in MDA-MB-231 cells, as shown by 

fluorescent degradation substrate assays. Cortactin and MT1-MMP are important invadopodia 

proteins. Immunoblot of cells transfected with ADAMTS-1 siRNA showed augmentation of 

cortactin and MT1-MMP expression compared to controls. Regulatory mechanisms underlying 

ADAMTS-1 effects may be related to VEGF, a growth factor involved in migration and invasion. 

MDA-MB-231 cells with silenced ADAMTS-1 showed increase of VEGF concentration in the 

conditioned medium compared to control. This conditioned medium also induced HUVEC 

tubulogenesis. Furthermore, MBA-MB-231 showed high expression of VEGF receptor 

(VEGFR2) compared to MCF7 cells. We may assume that ADAMTS-1 effects in tumor 

invasiveness may be related to VEGF and VEGFR expression. We suggest that decrease of 

ADAMTS-1 stimulates migration, invasion and invadopodia formation in breast cancer cells. 

These effects are probably regulated by VEGF and VEGFR. 

2188 

Analysis of endothelium mimicry by the CSC-like cells derived from iPS cells. 

S-I. Matsuda 1 , A. Mizutani 1 , T. Kasai 1 , A. Satoh 1 , T. Kudoh 1 , L. Chen 1,2 , M. Seno 1 ; 1 Graduate 

School of Natural Science and Technology, Okayama University, Okayama, Japan, 2 Tianjin 

Central Hospital of Bynecology Obstetrics, China 

Cancer stem cells (CSCs) are capable of self-renewal and differentiation, which has been 

considered to be responsible for tumor initiation and to contribute to cancer resistance. In 

addition, CSC-like cells in glioblastoma have also been demonstarted to mimic or differentiate 

into vascular endothelium. Eradication of these CSCs is considered critical part of successful 

anti-cancer treatment, but detailed analysis of CSCs is still slow because of their rarity and 

difficulty of maintenance in culture. 

Recently, we have established stable CSC-like cells from mouse induced pluripotent stem 

(miPS) cells (Mizutani et al., 2011 ASCB meeting), which were cultured in the conditioned 

medium of several kinds of mouse cancer cell lines P19, Lewis lung carcinoma (LLC), B16 and 

MC.E12. In particular, miPS cells converted to CSC-like cells in the conditioned medium of 

Lewis lung carcinoma (miPS-LLCcm) cells exhibited extensive angiogenesis in vivo. In this 

study, we evaluated these miPS-LLCcm cells for the potency to mimic vascular endothelium. 

First we inquire about their stemness by assessing the expression of stemness marker genes, 

Oct3/4, Sox2, Klf4 and Nanog. Compared with miPS, these markers except Oct3/4 tend to be 

up-regulated in the obtained CSC-like cells implying some difference from normal stem cells. 

Simultaneously, the expression of vascular endothelial markers, VE-cadherin and VEGFR2, 

were also found upregulated in miPS-LLCcm and miPS-B16cm cells. However, only miPS- 

LLCcm cells exhibited tube formation on MatrigelTM, which is consistent with the results of 

tumorigenicity in vivo. Since this in vitro tube formation was observed in the absence of VEGF, 

VEGFR2 might not directly be involved in the angiogenesis. When miPS-LLCcm cells were 

cultured in the presence of puromycin, the tube formation was not observed. This impotency 

was recovered when puromycin was removed from the culture media. Taking these into 

consideration miPS-LLCcm cells should have potential of differentiation, which is responsible for 

vasculogenic mimicry in vivo, maintaining the heterogeneous population in the culture. This 

speculation was supported by the flow cytometoric analysis for GFP expression in miPS-LLCcm 

cells showing approximately 30 % was GFP positive. Thus, miPS-LLCcm cells in this study

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have successfully been shown to have CSC-like characters, such as cancer stemness, 

malignant behavior and capacity to mimic vascular endothelium. Our CSC-like cells might be a 

good model to explain the mechanism of tumor angiogensis in detail not limiting in glioblastoma. 

2189 

The HT29 and DLD1 colorectal cancer cell lines contain cancer stem cells. 

C. B. Penny 1 , B. L. Milner 1 , V. E. Gibbon 1 , P. Ruff 1 ; 1 Oncology, University of Witwatersrand Med 

Sch, Johannesburg, South Africa 

Cancer stem cells (CSCs) similar to embryonic stem cells are defined by their abilities to selfrenew 

and additionally retain the ability to form tumours ectopically. They have been identified in 

several human malignancies, recently being associated with functional properties of drug 

resistance, invasion and migration. This resistance to conventional chemotherapies may lead to 

tumour relapse and the arisal of new tumours. Using confocal microscopy, we have identified 

putative colon cancer stem cells (CSC) in the HT29 and DLD-1 colon cancer derived cell lines. 

These lines representative of stage 3 colorectal adenocarcinoma, co-express the cell surface 

markers, CD133 (prominin 1) and epithelial cell adhesion marker (EpCam). Upon FACS 

analysis of these cell lines, using CD133 (PE-Miltenyi Biotech) and EpCam antibodies (FITC-BD 

Biosciences), two distinct cell populations were gated; within the HT29 cell line, 64.63% of gated 

events were positive for EpCam and 35.30% expressed both EpCam and CD133; and in the 

DLD1 cell line, 46.77% of gated events were positive for EpCam expression and 52.83% for 

both markers. Subsequently, magnetic cell separation was used to isolate pure populations of 

these cells from each cell line, with a CD133 antibody labeled with superparamagnetic particles 

(Miltenyi Biotech). The purified CD133+ cells were cultured in StemPro human embryonic stem 

cell medium (serum free) (GIBCO), to minimize the possibility of differentiation. The cells were 

grown both as adherent cultures and as three dimensional spheroids, these being more 

representative of in vivo tumours. To further investigate the “stemness” of these cells, the 

expression of the pluripotency markers Nanog, Sox 2 and Oct3/4 were assessed by confocal 

microscopy. All of these transcription factors essential to stem cell identity are present in the 

CD133+ cells. In summary, the CSCs isolated here, represent a novel population of cells for 

evaluating cancer therapies directed against pluripotent cells. 

2190 

Floating spherical cell colonies from human adenocarcinoma of the lung are enriched in 

stem-like phenotypic markers. 

A. Tesei 1 , C. Arienti 1 , G. Paganelli 1 , A. Pasini 2 , G. Brigliadori 1 , D. Calistri 1 , E. Giordano 2 , W. 

Zoli 1 ; 1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori 

(I.R.S.T.), Meldola (FC), Italy, 2 Laboratory of Cellular and Molecular Engineering, University of 

Bologna - Campus of Cesena, Cesena (FC), Italy 

It is presently widely accepted that tumor growth rely on a niche of so-called cancer stem cells 

(CSCc), or tumor-initiating cells (TICs), endowed with self-renewal properties. We recently 

showed that a subset of normal lung epithelial-specific stem cells might reside within the 

bronchoalveolar duct junction (Tesei A., et al. Cell Proliferation 2009; 42: 298-398). The aim of 

the present work was to isolate and characterize CSCc/TICs from a human adenocarcinoma 

cell line (RAL) that we originally established at the Istituto Scientifico Romagnolo per lo Studio e 

la Cura dei Tumori (I.R.S.T.) at Meldola (Gasperi-Campani A., et al., Cancer Genetics and 

Cytogenetics 1998; 107:11–20) and from surgical human tissue samples. Single-cell 

suspensions derived from the RAL cell line were cultured in ultralow attachment plates in a 

serum-free medium. Neoplastic lung tissue samples, derived from patients affected by 

adenocarcinoma at different stages, were mechanically and enzymatically dissociated before

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expansion in culture. Expression of stemness related genes in cell clusters was evaluated with 

RT-PCR and Real Time RT-PCR. Single-cell suspensions gave rise to floating spherical 

colonies within about ten days. Lung-spheres obtained by our originally established lung 

adenocarcinoma cell line RAL, resulted enriched in CD133 and OCT-3/4 transcripts. RT-PCR 

analysis of tumor spheres obtained from surgical tissue samples showed a higher expression of 

BCRP-1, CD133, BMI-1, OCT-3/4, Lef-1, CD44 and Slug, when compared to original tissue. A 

gradual loss of CD133 expression was evident as the tumors become more undifferentiated. 

Evaluation of the level of CpG methylation in OCT-3/4 and CD133 gene promoters was also 

performed by means of bisulfite sequencing in cells growing in floating spherical RAL cell 

colonies and in the original adherent cell culture. They both showed a constant hypermethylated 

status suggesting that this level of transcriptional control is not involved in modulating the 

expression of these stem-like phenotypic markers. 

2191 

Generation of Cancer Stem Cell Model from Mouse iPS Cells. 

A. Mizutani 1 , S-I. Matsuda 1 , T. Kasai 1 , T. Kudoh 1 , L. Chen 2 , M. Seno 1 ; 1 Okayama University, 

Okayama, Japan, 2 Tianjin Central Hospital of Bynecology Obstetrics, China 

Objective; Heterotopic transplantation of embryonic stem cells and induced pluripotent stem 

(iPS) cells have been shown to form teratoma, a benign tumor exhibiting heterogeneous but 

normal phenotype of differentiated cells, but not teratocarcinoma. Since the stem cells niche has 

been thought to play a significant role for the maintenance and the differentiation of stem cells, 

we hypothesized that cancer niche could drive stem cells into malignant cells, particularly into 

cancer stem cells (CSCs). The presence of CSCs in tumor has been considered as a reason for 

a poor prognosis. Until now, no model of cancer stem cell line is established as an effective tool 

to study CSC. To investigate CSC in detail, we tried to generate cancer cells from mouse iPS 

(miPS) cells, in which green fluorescent protein (GFP) has been inserted into the 5â€ 

untranslated region of the Nanog gene. 

Methods and Results;A ccording to our hypothesis, miPS cells were cultured in the conditioned 

medium of various cancer derived cells (P19, LLC, B16, and MC.E12) for 4 weeks. The survived 

population of the cells was evaluated for tumorigenicity in nude mice. The miPS cells cultured in 

the conditioned medium from Lewis lung carcinoma cells (miPS-LLCcm) developed tumor with 

rapid growth and exhibited remarkable angiogenesis. Histology of the tumor derived from miPS- 

LLCcm showed the malignancy, while that of teratoma derived from miPS cells showed no 

malignancy. Morphologically stem-like cells expressing GFP was observed in the primary 

culture of the malignant tumor derived from miPS-LLCcm cells. These cells were found to grow 

as spheroid with GFP expression in suspension culture. The spheroid cells were also found to 

be highly tumorigenic in nude mice. The expression of the stem cell marker genes, such as 

Nanog, Cripto, Oct3/4, Sox2, Klf4, were detectable in miPS-LLCcm cells as much as in miPS 

cells. 

Conclusion; In this study, we cultured miPS cells in the conditioned mediun of cancer derived 

cells, which was regarded as cancer niche converting the normal stem cell into CSC cells. The 

resultant cells formed spheroids in suspension culture and developed malignant tumor in vivo 

exhibiting extensive angiogenesis. In addition, these cells showed a capacity of self-renewal 

and expression of the markers associated with stem cell properties of undifferentiated state. 

Thus we concluded these cells as a model of CSC. This model should critically be helpful for the 

CSC study and for the development of effective cancer therapies.

Cell Biology of Microbes and Parasites 

2192 

Molecular mechanism underlying MinE-induced membrane deformation. 

Y-L. Shih 1,2 ; 1 Institute of Biological Chemistry, Academa Sinica, Taipei, Taiwan, 2 Institute of 

Biochemical Sciences, National Taiwan University, Taiwan 

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The Min system of E. coli mediates placement of the cell division septum at midcell. We recently 

reported that MinE can directly associate with the cell membrane through its N-terminal domain. 

An interesting observation is the ability of MinE to induce membrane tubule formation in vitro, a 

property similar to several eukaryotic proteins involved in membrane trafficking, such as 

dynamin and Bar-domain proteins. To investigate the mechanism underlying the MinE-induced 

membrane deformation, we used time-lapse fluorescence microscopy to characterize the 

membrane deformation in vitro. We recorded massive tubulation from giant liposomes and 

initiation of membrane tubule formation and lipid clustering on the supported lipid bilayers. 

Furthermore, we found that residues 2-9 are capable of forming an amphipathic helix of 1-2 

helical turns when interact with the membrane.We used the tryptophan blue shift assays to 

determine the helical face that interacts with the membranes. CD spectral analyses suggested 

that conformational changes in MinE 1-31 occurred in the presence of liposomes. We conclude 

that the alpha helix of MinE 2-9 appears to work in concert with the charge residues R10/K11/K12 

to mediate the membrane association of MinE. It is known that insertion of an amphipathic helix 

into the membranes can induce local curvature changes that are able to destabilize the cell 

membranes and initiate membrane tubule formation. In conclusion, MinE not only shares 

common structural motifs to proteins involved in membrane trafficking, but is also active in 

inducing membrane deformation. We therefore uncovered the mechanism of how MinE may 

sculpt the membrane curvature. 

2193 

Biochemical and functional characterization of GlpQ homolog of Xenorhabdus 

nematophila. 

P. Singh 1,2 , R. Bhatnagar 1 , N. Banerjee 2 ; 1 School of Biotechnology, Jawaharlal Nehru 

University, Delhi, India, 2 Insect Resistance Group, International Centre for Genetic Engineering 

and Biotechnology, New Delhi, India 

Xenorhabdus nematophila is a gram negative bacteria living in symbiotic association with a soil 

nematode of the genus Steinernema and as a pathogen stage in insect hosts. The bacteria 

produces different exoenzymes which help in degradation of macromolecules to provide 

nutrients necessary for development of both the bacteria and the nematode. In the present 

study, we have characterized an extracellular enzyme glycerophosphodiester 

phosphodiesterase (GDPD) encoded by glpQ gene of X. nematophila. The DNA sequence 

encoding the enzyme was cloned, expressed and purified. We used the recombinant protein for 

biochemical characterization. The enzyme was able to hydrolyse glycerophosphodiesters like 

glycerophosphorylcholine, glycerophosphoethanolamine and to a lesser extent serine and 

inositol esters of phospholipids. The enzyme is thermostable at 50°C for upto 4 hours, retaining 

85% of initial activity. It has an optimum pH of 9.5. The enzyme activity was stimulated by 

Ca 2+ ions. Activity profile of GDPD in different growth phases of X. nematophila showed 

maximum activity in mid log phase. The enzyme was largely localised in periplasm and 

extracellular medium while negligible activity was detected in cytosol. It is also displayed on the 

cell surface of the bacteria, as treatment with pronase abolished all the enzyme activity from the 

outer membrane preparation. Based on the crystal structure of GlpQ of E. coli a homology 

model of Xenorhabdus GlpQ was created. The residues Glu-59, Asp-61 and His-74 located in

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the active site of the enzyme were changed to alanine using site directed mutagenesis. Enzyme 

activity was abolished in all the three protein variants validating the crucial role of these residues 

in the active site of the enzyme. Since the bacteria is exposed to different unfavourable 

environments in the hosts so we determined glpQ profile in nutritional stress conditions. 

Transcriptional analysis of glpQ specific mRNA has shown upregulation of the gene in response 

to phosphate starvation. Similarly enzyme activity was increased by 1.6 folds when the cells 

were starved for phosphate upto 7 hours. To further analyse it with the growth phase of bacteria 

it was shown that upregulation was mainly associated with the early log phase of the bacteria. 

Stationary phase cells did not respond to phosphate starvation. Bioinformatic analysis of the 

upstream region of glpQ showed presence of two promoters. This was confirmed by primer 

extension analysis which clearly indicated promoters P1 and P2 about ~80 and ~565 

nucleotides upstream of the start codon of the gene. To explore the function of both the 

promoters in phosphate regulation, lacZ fusion constructs were prepared and promoter activity 

was determined by β-galactosidase assay which indicated that promoter located 80 bp 

upstream of the start codon has a role in phosphate regulation of glpQ gene. 

2194 

The role of a bacterial SMC in chromosome segregation. 

M. Schwartz 1 , L. Shapiro 1 ; 1 Developmental Biology, Stanford University, Stanford, CA 

The bacterial genome must be completely replicated and segregated prior to the completion of 

cell division in order to ensure that each daughter cell receives one complement of the genome. 

The Structural Maintenance of Chromosomes (SMC) protein is conserved from bacteria to 

humans and is thought to play important roles in chromosome organization and segregation. 

We have identified a point mutation (SMC-E1076Q) in the ATPase domain of Caulobacter SMC 

that disrupts chromosome segregation and cell division. This mutation caused a dominantnegative 

phenotype in which DNA replication was able to proceed, but segregation was 

inhibited, resulting in an accumulation of parS centromeres at one pole. Surprisingly, DNA was 

still deployed throughout the cell and the cellular positions of other chromosomal loci were in the 

wild-type order relative to the parS centromere. Loss of ATP hydrolysis, either by this point 

mutation or by the utilization of non-hydrolyzable ATP caused abnormally stable binding of SMC 

to DNA in vitro. We propose that SMC spuriously links the duplicated chromosomes 

immediately after passage of the replication fork when the two chromosomes are in very close 

proximity. In wild type cells, ATP hydrolysis opens the SMC dimer, freeing one chromosome to 

segregate to the opposite pole. SMC is more likely to trap DNA from a single chromosome once 

the replicated chromosomes are physically separated, thereby condensing the chromosome. In 

this model, the loss of ATP hydrolysis causes the SMC-E1076Q dimer to remain bound to both 

chromosomes, inhibiting segregation. The linking of the two chromosomes can be reinforced by 

additional mutant dimers binding elsewhere on the neighboring chromosomes. 

2195 

Binding of recombinant soluble Influenza A hemagglutinins to cell surface sialic acids of 

cultured cells and tissue sections. 

A-K. Sauer 1 , M. Bohm 1 , C. Schwegmann-Weßels 1 , G. Herrler 1 ; 1 Institute of Virology, University 

of Veterinary Medicine Hannover, Hannover, Germany 

The major viral envelope glycoprotein of influenza A viruses is the hemagglutinin (HA) which is 

responsible for host cell recognition and virus entry. It is synthesized as an uncleaved precursor 

in the host cell secretory pathway where it is posttranslationally modified and forms trimers. The 

receptor determinants for influenza A viruses are sialic acids of glycolipds and/or glycoproteins. 

Avian influenza strains preferentially bind to sialic acids linked to galactose in α2,3-conformation

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whereas human strains prefer α2,6-linked sialic acids. To distinguish between both forms, the 

plant lectins MAA and SNA, respectively, are used in many studies. Due to the huge diversity of 

oligosaccharide structures and the different hemagglutinin subtypes, those two plant lectins are 

not sufficient to characterize the binding properties of influenza hemagglutinins. 

To get a closer insight into the cellular interaction partners of influenza A viruses we generated 

soluble hemagglutinins that can be used as lectins for detection of those sialoglycoconjugates 

that are recognized by influenza viruses. Connection of the ectodomain to the Fc-component of 

a human IgG and to a modified GCN4 leucin zipper motif as trimerization domain resulted in 

chimeric proteins that are secreted into cell culture supernatant after transfection and were 

purified by FPLC. The binding of the soluble HAs can be demonstrated by immunofluorescencebased 

assays.. 

These recombinant soluble HAs of the avian subtypes H7 and H9 were found to bind to the 

surface of different cell lines, trachea sections of chicken and turkey and sections of the porcine 

lung. On all permanent mammalian cell lines tested, the H9 shows stronger binding compared 

to the H7 protein. On trachea sections of chicken and turkey a 2fold higher amount of H9 is 

needed to establish an equal binding pattern compared to that of H7 despite the presence of 

both sialic acid linkage types as indicated by the lectin stain. Porcine lung sections show more 

SNA staining on the surface whereas MAAII stains cells in basal layers of the epithelium. Only 

H9 binds to porcine lung sections but not as widely distributed over the surface as the SNA 

stain. 

These experiments show that a lectin staining alone cannot sufficiently depict the distribution of 

the cellular interaction partners of influenza A viruses. Soluble hemagglutinins promise to be a 

valuable tool to visualize potential influenza binding sites on cells and tissues. 

2196 

Adaptation of avian influenza viruses of the subtype H9N2 to avian and human 

respiratory epithelial cells. 

M. Erdt 1 , M. Bohm 1 , H. Petersen 2 , C. Winter 1,2 , S. Rautenschlein 2 , C. Schwegmann-Weßels 1 , G. 

Herrler 1 ; 1 Institute of Virology, University of Veterinary Medicine Hannover, Foundation, 

Hannover, Germany, 2 Clinic for Poultry, University of Veterinary Medicine Hannover, Foundation 

Influenza viruses initiate infection by binding of the haemagglutinin (HA) to sialic acid residues 

presented by cell surface components. Viruses isolated from different host species may differ in 

their preference for the type of sialic acid, e.g. N-acetylneuraminic acid (Neu5Ac), or for a 

linkage type connecting the sialic acid molecule to the neighboring sugar. Influenza viruses from 

mammalian hosts prefer α-2,6-linked sialic acids whereas most avian influenza viruses 

prefentially recognize Neu5Ac attached via an α-2,3-linkage to galactose. Avian viruses of the 

H9 subtype are an exception, because several strains of H9 influenza viruses have been shown 

to favor α-2,6-linked sialic acids. This finding was surprising because in some avian species α- 

2,6-linked sialic acids are expressed in very low amounts, at least in the respiratory epithelium, 

a primary target for influenza virus infection 

We analyzed the adaptation of an egg-grown avian influenza virus of the subtype H9N2 (strain 

A/chicken/SaudiArabia/CP7/1998) to the respiratory epithelium of chicken and turkeys. Tracheal 

organ cultures (TOC) served as a culture system which preserves the setting of epithelial cells 

as present in the avian trachea. 

The parental virus was passaged four times in either chicken or turkey TOCs. The progress of 

infection was monitored by determining the viral titer induced by this virus at various time points 

post infection. In the course of the passages, the virus underwent an adaptation process 

indicated by an increase in the viral titer. Infection of chicken TOCs by the chicken TOCadapted 

virus resulted in a 3.5-fold increase in the viral titer compared to the egg-grown virus.

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By contrast, infection of turkey TOCs with the turkey TOC-adapted virus was characterized by a 

nearly 10-fold increase in viral titer. 

Furthermore, Calu-3 cells, a permanent human cell line with features of the bronchiolar 

epithelium when grown under air-liquid interface conditions (ALI), was infected with these avian 

viruses. The influence of several passages in these cells was monitored and analysis of cell 

culture supernatants showed that avian H9N2 viruses in Calu-3 cells reached higher titers than 

in the TOC system. In the future these experiments should be extended to primary human 

respiratory epithelial cells. 

2197 

Peritoneal Macrophages Employ Predominantly αxβ2 But Not αMβ2 For Candida albicans 

Recognition. 

S. Jawhara 1 , D. Soloviev 1 ; 1 Molecular Cardiology, Cleveland Clinic Lerner Research Institute, 

Cleveland, OH 

Candida albicans is an opportunistic yeast pathogen that represents the fourth most frequent 

cause of nosocomial bloodstream infections in the United States. Recently, we demonstrated 

that the integrin αMβ2 is the principal leukocyte receptor for the filamentous form of C. albicans. 

In the present study, we explored the role of another β2 integrin, αxβ2 (CD11c/CD18, p150,95), 

in the model of antifungal defenses. Although αxβ2 is highly homologous to the αMβ2, its role 

remains unclear in the immune response to fungal infections. Employing αx-knock out (KO) 

mice in the murine model of systemic candidiasis, we first demonstrated that mice deficient in 

αxβ2 had significantly increased susceptibility to the invasive fungal infection. We observed that 

C. albicans induced mortality in αx-KO mice more rapidly (2- to 3-fold faster) than when the 

same inoculum was used in wild-type mice. Notably, the fungal burden was significantly higher 

in both the brain and kidneys of αx-KO than wild-type mice. Subsequently, the histopathology 

examination of both kidneys and brain from C. albicans-infected αx-KO mice showed a higher 

presence of filamentous growth and tissue damage than in wild-type mice. Elimination of αxβ2 

significantly impedes peritoneal macrophages recruitment to the place of infection and their 

capacity for anti-fungal activity, while having virtually no effect on the neutrophil`s function. In 

contrast, αM-elimination mostly affected the antifungal activity of neutrophils but not that of 

peritoneal macrophages. Additionally, αxβ2 elimination increased the expression of interleukin- 

6, but not the tumor necrosis factor alpha (TNFα). Taken together, these data indicate that αxβ2 

is essential for C. albicans elimination by macrophages and plays a role in innate antifungal 

immunity. 

2198 

The role of TrpM7 in Salmonella pathogenesis. 

A. M. Young 1 , J. E. McCombs 1 , S. Van Engelenburg 1 , W. W. Blakeslee 1 , A. E. Palmer 1 ; 

1 Chemistry and Biochemistry, University of Colorado Boulder, Boulder, CO 

Salmonella subspecies are enteropathogenic gram-negative bacteria responsible for food-borne 

illness in humans ranging from gastroenteritis to typhoid fever. The infection of a range of host 

cells by Salmonella is driven by the injection of bacterial effector proteins through a needle-like 

type III secretion system (T3SS). Effector proteins hijack host cell machinery and intracellular 

signaling pathways. These interactions promote invasion of the cell through cytoskeleton 

rearrangement and the engulfment of the bacteria into a Salmonella containing vacuole (SCV). 

Salmonella invasion is known to induce an elevation in host cell Ca 2+ , however the molecular 

mechanism of this event has remained undefined. Fluctuations in Ca 2+ activate a diverse array 

of downstream signaling processes including gene transcription, actin polymerization, and 

endocytosis. Given these roles, it is likely that Salmonella manipulates and utilizes host cell Ca 2+

MONDAY 

signaling during invasion. In this work, we investigate the origin and nature of the Ca2 + elevation 

by examining Salmonella invasion of non-phagocytic cells at the single cell level. Localized Ca 2+ 

elevation was observed to result from influx through a transient receptor potential cation channel 

(TRPM7) at sites of invasion. This study identifies a novel mechanism by which TRPM7 is 

activated by a Salmonella effector, SopB, through its ability to modulate phosphoinositides, 

indicating that TRPM7 can be gated by rare signaling phospholipids. 

2199 

Defining the roles of inclusion membrane microdomain proteins in chlamydial 

pathogenesis. 

J. Mital 1 , E. Lutter 1 , T. Hackstadt 1 ; 1 Rocky Mountain Labs/NIH, Hamilton, MT 

Chlamydia trachomatis is the leading cause of sexually transmitted disease in the Western 

world and of infectious blindness in the world. Chlamydiae are obligate intracellular bacteria that 

reside within a membrane bound vacuole termed the inclusion, which does not fuse with the 

endosomal pathway but selectively interacts with vesicular pathways to acquire lipids and 

nutrients from the host cell. The inclusion is extensively modified by insertion of chlamydial 

proteins (Incs), which are ideally positioned to mediate host-pathogen interactions. There is little 

functional information regarding Inc proteins. Once internalized, the nascent inclusion 

repositions itself at the centrosome in a process that is dependent upon chlamydial protein 

synthesis, microtubules and dynein. Inclusion repositioning is independent of the cargo linker 

p50 dynamitin, indicating that a bacterial effector may directly link the inclusion to dynein. We 

previously described a subset of Incs (B, 101, 222, 850) that colocalize with host Src-family 

kinases in microdomains in the inclusion membrane. These microdomains are closely apposed 

to host centrosomes, indicating a probable role in microtubule interaction. Knockout and 

inhibitor studies show that Src family kinases are involved in the microtubule dependent 

processes of inclusion repositioning, sphingomyelin trafficking and are essential for normal 

chlamydial development. Here we report studies of the functional roles of the microdomain Incs 

in chlamydial pathogenesis. When ectopically expressed in HeLa cells, Inc850 consistently 

colocalizes with host centrosomes. Inc850 contains a dynein light chain binding domain and has 

been shown by yeast two-hybrid assays to interact with dynein light chain subunits. This 

suggests that inclusion microdomains comprised of chlamydial and host proteins are directly 

involved in microtubule dependent trafficking of the nascent chlamydial inclusion. The full role of 

these microdomains in pathogenic processes remains to be elucidated. 

2200 

The PDZ1 domain of MAGI-1 rescues CAR Ex8 from MAGI-1 mediated degradation and 

augments adenoviral infection. 

A. O. Kolawole 1 , R. Yan 1 , P. Sharma 1 , K. J. Excoffon 1 ; 1 Biological Sciences, Wright State 

University, Dayton, OH 

A major factor in virus entry into cells is localization and abundance of the primary receptor. The 

Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for group B 

coxsackievirus and many serotypes of adenovirus. In most epithelia, a seven exon isoform of 

CAR (CAR Ex7 ) is exclusively localized at the basolateral surface where it behaves as a 

homophilic adhesion protein and is inaccessible for viral infection. However, in welldifferentiated 

human airway epithelia, we recently discovered an alternatively spliced, low 

abundance eight exon isoform of CAR (CAR Ex8 ) that is localized apically, where it may initiate 

apical viral infection. The two isoforms differ only in the last 26 (CAR Ex7 ) or 13 (CAR Ex8 ) amino 

acids of the cytoplasmic domain, which suggests that some intracellular interactions may differ. 

One such differential interaction involves MAGI-1, an essential PDZ domain containing protein

MONDAY 

with 6 distinct PDZ domains (PDZ0-5), known to be involved in cell polarization and cancer. The 

molecular basis of the CAR-MAGI-1 interaction was investigated by adenovirus infection, 

immunocytochemistry, MAGI-1 siRNA knockdown, yeast two hybrid assay, coimmunoprecipitation, 

in vitro translation, and fluorescence resonance energy transfer. In nonpolarized 

cells, MAGI-1 caused a loss of CAR Ex8 at the protein level and reduced adenovirus 

infection. In contrast, MAGI-1 siRNA knockdown increased adenovirus infection, suggesting that 

endogenous MAGI-1 regulates CAR Ex8 levels. The key PDZ-based interactions between CAR 

and MAGI-1 were then identified and confirmed. Both CAR isoforms strongly interacted with 

PDZ3. CAR Ex8 also interacted with PDZ1. Co-expression of CAR Ex8 with isolated MAGI-1 PDZ3 

significantly reduced CAR Ex8 cell surface expression and adenovirus infection but did not reduce 

total CAR Ex8 protein levels. These data suggest that MAGI-1 PDZ3 is responsible for retaining 

CAR Ex8 within the cell but degradation of CAR Ex8 likely requires MAGI-1 interacting 

proteins/domains.Co-expression of the isolated PDZ1 domain of MAGI-1 with CAR Ex8 did not 

affect adenovirus infection. However, PDZ1 was able to rescue CAR Ex8 from MAGI-1-mediated 

degradation, thereby supporting adenovirus infection via CAR Ex8 in the presence of MAGI-1. 

These data suggest that molecules directed at facilitating or inhibiting the interaction between 

PDZ1 and CAR Ex8 may be able to regulate the levels of CAR Ex8 and hence increase or decrease 

epithelial susceptibility to adenoviral infection. 

2201 

The VP1 Subunit of JC Polyomavirus Drives Trafficking to the ER for Productive Viral 

Infection. 

A. Derdowski 1 , C. Nelson 1 , M. Maginnis 1 , B. O'Hara 1 , W. Atwood 1 ; 1 Molecular Biology, Cell 

Biology and Biochemistry, Brown University, Providence, RI 

There are at least nine different human polyomaviruses. Two of these, JC polyomavirus (JCV) 

and BK polyomavirus (BKV), are long-established as human pathogens in immunocompromised 

hosts causing progressive multifocal leukoencephalopathy (PML) and nephropathy, 

respectively. The remaining human polyomaviruses have all been discovered within the last 

decade, establishing this class of viruses as emerging opportunistic pathogens. Polyomaviruses 

are coated with 72 pentamers of the viral attachment protein VP1. VP1 is responsible for 

attachment to the host cell surface and implicated in trafficking to the ER and nucleus for 

productive infection. The VP1 subunit of JCV was bacterially expressed and purified by FPLC. 

VP1 pentamers were then directly labeled and viral entry and trafficking were analyzed by 

confocal microscopy in real-time. We demonstrate for the first time that the JCV VP1 pentameric 

subunit drives viral trafficking through Rab 5 and 7-positive vesicles to the ER for productive 

infection, and interference of this trafficking with pharmacological inhibitors reduces infection. 

This work not only highlights the importance the viral VP1 pentamer in polyomavirus trafficking, 

but also establishes a viral trafficking model for emerging polyomaviruses that lack cell culture 

systems. 

2202 

Inhibition of Rab5 activation by Exotoxin S of Pseudomonas aeruginosa. 

S. Mustafi 1 , N. Rivero 1 , M. Barbieri 1 ; 1 Florida International University, Miami, FL 

Pseudomonas aeruginosa is a human pathogen that frequently causes severe hospital-acquired 

infections. Exotoxins secreted from Type III secretion system of Pseudomonas aeruginosa are 

critical molecules to disrupt mammalian epithelial cell membrane trafficking and signaling. One 

of them also known as Exotoxin S, is a bi functional protein which shows ADP ribosyltransferase 

activity and targets several intracellular proteins, including Ras and Rab small GTPases. Here, 

we demonstrate that Rab5, but not Rab4 and Rab7, play a critical role during early steps of the

MONDAY 

invasion of Pseudomonas aeruginosa. We show that invasion of live Pseudomonas aeruginosa, 

but not heat inactivated Pseudomonas aeruginosa, down-regulates of Rab5 activity in 

macrophages. In addition, invasion of live Pseudomonas aeruginosa lacking Exotoxin S was 

unable to inactivate Rab5. Furthermore, we also found that overexpression of Rin1 partially 

reverses the inactivation of Rab5 during invasion of live Pseudomonas aeruginosa. These 

observations are consistent with a model, in which the presence of Exotoxin S may interfere 

with the Rab5 cycle activity in macrophage 

2203 

The ATPase activity of EccC is required for ESX-1 secretion in Mycobacterium 

tuberculosis. 

D. Dovala 1 , O. Rosenberg 1 , J. Cox 1 ; 1 University of California, San Francisco, San Francisco, CA 

The human pathogen Mycobacterium tuberculosis (Mtb) utilizes a specialized type VII secretion 

system, ESX-1, to circumvent host defenses during macrophage infection. ESX-1 mutants are 

severely attenuated, highlighting the importance of this secretion system. While many 

substrates and components of ESX-1 have been identified, the mechanistic details of how these 

substrates are pumped into the host cell remain elusive. The Mtb proteins EccCa and EccCb 

contain several putative FtsK-like ATPase domains and form a complex which is required for 

ESX-1 secretion. FtsK domains in other proteins are known to exert force on substrates, and 

thus may serve as the pump in the ESX-1 translocon. We show that the EccC complex forms an 

ATPase with three functional ATPase domains. We show that all ATPase domains are required 

for successful secretion of ESX-1 substrates from Mtb, and genetic approaches have identified 

the roles of each domain in complex formation and substrate binding. 

2204 

Host Innate Receptor Regulation is Mediated by Influenza Virus Proteins. 

D. J. Vigerust 1,2 , V. L. Shepherd 1,2 ; 1 VanderbIlt University SOM, Nashville, TN, 2 Department of 

Veteran Affairs, Nashville, TN 

Influenza virus A (IVA) infection is a significant cause of morbidity and mortality each year. 

Interactions between the influenza virus and cells of the innate immune system – specifically 

macrophages – may exacerbate acute symptomology and immune evasion and disease 

outcome, but the interaction between influenza and the macrophage remains poorly understood. 

Host-pathogen dynamics may prevent the proper function of the macrophage in the clearance of 

harmful pathogens. Our current hypothesis is that host innate defense molecules such as the 

cell surface mannose receptor (MR) may be utilized as a receptor of entry for viruses such as 

influenza, and that molecules such as MR and MHC-I are targeted for down-regulation by 

influenza A viruses leading to dysregulation of macrophage function. In previous work from our 

laboratory the HIV-1 Nef protein was shown to interact with the MR tail to down-regulate the 

surface receptor expression. The interaction between the MR and Nef was localized to two 

specific regions of Nef. Sequence comparison of the known interacting domains of the HIV-1 

Nef protein and the sequence of influenza A NS1 and PB1 revealed similarity in several motifs 

that indicate a potential for MR regulation by influenza. Our initial data suggest that infection of 

MR-expressing cells results in a significant decrease in surface expression of MR by 48 hours 

post-infection. Further, in vitro transient transfection of macrophages with influenza proteins 

leads to removal of MR from the surface at levels approaching 30% for NS1 and PB1. Removal 

of MHC-I from the cell surface was also seen as early as 5 hours post-transfection. These data 

support the hypothesis that influenza proteins regulate two important host innate defense 

molecules in a fashion that is similar to that seen with HIV-1 Nef.

New Technologies and Frontiers 

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2205 

High-throughput cellular assays using a well-less plate format. 

G. Quinones 1 , K. Nicholes 1 , M. Lye 2 , N. Kim 2 , J-P. Stephan 1 ; 1 Genentech, South San Francisco, 

CA, 2 Curiox Biosystems, Singapore 

Cellular assays represent a great opportunity for researchers to test various molecules in a 

more biologically relevant context than biochemical assays. Many of these cell-based assays 

could be adapted for high-throughput and high content screening assays, providing 

investigators the opportunity to interrogate large number of samples and conditions. Despite 

significant technical advances made during the last few years, high-throughput/high content 

cellular assays still suffer from key limitations in working with a large number and variety of cell 

lines. A key limitation is the use of suspension cell lines, especially when multi-step staining 

procedures are required. Here, we demonstrate that the use of a well-less plate system which 

significantly improves the flexibility of our high content screening platform. The well-less format 

utilizes surface tension to maintain the cell population on the glass surface of plate in 2.5mm 

diameter drops, while a gentle buffer exchange allows the cells to remain on the plate surface 

throughout a variety of experimental procedures. We performed cell viability experiments in the 

presence of the antimitotic agent monomethyl auristatin E (MMAE) on the suspension cell lines 

U-937 and K-562 in immunofluorescence and chemiluminescence viability assays, showing that 

in both assays we achieve an EC50 of 4.4uM and 9.1uM respectively. We also performed 

immunofluorescence-based, protein-protein interaction assays using a large library of single 

transmembrane, multi-transmembrane and secreted proteins expressed in COS7 cells. The 

expressed library was incubated with tagged bait proteins to interrogate novel protein-protein 

interactions at the cell surface. This resulted in the successful expression and binding of several 

known ligands to their respective receptors, i.e. PD-1 binding to PD-L1, NGF to NGFR, PVRL2 

to PVRIG, and HVEM binding to BTLA. We also examined changes in cell morphology using 

both established and primary human cell lines infected with fluorescent organelle-specific 

baculovirus constructs. Live imaging of the infected cells revealed alterations in the trafficking 

patterns of EEA1-labeled early endosomes in the presence of the dynamin inhibitor Dynasore. 

We also observed changes in the actin and tubulin cytoskeletal structure in the presence of 

Cytochalasin D and Blebbistatin. The added flexibility in terms of cell lines and readouts enables 

the screening platform to truly perform high-throughput and high content assays to examine a 

wide variety of cellular processes. 

2206 

Solutions for the preparation of samples for integrated correlative microscopy. 

M. A. Karreman 1,2 , A. V. Agronskaia 1 , E. G. Van Donselaar 2 , K. E. Vocking 2 , H. C. Gerritsen 1 ; 

1 Molecular Biophysics, Utrecht University, Utrecht, Netherlands, 2 Biomolecular Imaging, Utrecht 

University, Utrecht, Netherlands 

The Integrated Laser and Electron Microscope is a novel tool for correlative microscopy, 

combining a fluorescence microscope (FM) and a Transmission Electron Microscope (TEM) 

within one set-up. Here, a specially designed laser scanning fluorescence microscope is 

mounted into one of the side ports of a conventional Tecnai 12 TEM. First, the region of interest 

is identified based on fluorescence labeling of the sample. This region can then effortlessly be 

relocated in the TEM and subsequently investigated at high resolution. Due to the integrated 

nature of this set-up, the sample (eg. sections of cells on a grid) needs to be suitable both for 

FM and TEM. Unfortunately, the heavy metal stain employed to contrast the biological material 

for TEM has a quenching affect on the fluorophore required for FM imaging. Furthermore, most

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fluorophores are optimized for an aqueous environment and lose a large part of their brightness 

in a dry environment, which is needed for the TEM. 

Here, we demonstrate an optimized sample preparation protocol for iLEM. By creating a 

distance between the metal stain and the fluorophore, the quenching effect can be prevented. 

This is achieved by performing a block staining, so the biological material in the section is 

already contrasted. The fluorescent label on the section is then physically separated from the 

stain in the section. We show that the intensity and the lifetime of the fluorophore are not 

affected by the heavy metal stain bound to the biological material in the section. Furthermore, 

the choice of fluorophores for iLEM imaging is critical. Although in wet conditions Alexa 488 

fluoresces brighter than the TRITC dye, in a dry environment the opposite is the case. We have 

found that the lifetime and intensity of Alexa 488 is severely reduced in a dry environment 

whereas the TRITC dye is unaffected. 

Therefore, we can conclude that the sample preparation can be optimized by physically 

separating the heavy metal stain from the fluorophore. Also, the choice of fluorophores is of high 

importance due to their different properties in a wet or dry environment. 

2207 

Imaging promoter activity with Intracellular MultiAptamer Genetic tag (IMAGEtag). 

J. Ray 1 , I. Shin 1 , M. Zhao 2 , M. Ilgu 1 , V. Gupta 3 , J. Beasley 3 , L. Peng 2 , G. Kraus 3 , M. Nilsen- 

Hamilton 1 ; 1 Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, 

2 Optical Sciences, The University of Arizona, Tucson, AZ, 3 Chemistry, Iowa State University, 

Ames, IA 

A detailed understanding of the processes involved in gene transcription is necessary to answer 

many fundamental biological questions. Imaging techniques have been developed in the last 

decade with the capability of exploring the elements of gene expression and monitoring the 

fates of selected mRNAs. In this study we describe an aptamer-based system to monitor 

promoter activity in living cells and in real time, which we have named as IMAGEtag 

(Intracellular MultiAptamer Genetic tag). We have cloned and expressed a string of multiaptamers 

downstream of the GAL1 promoter in Saccharomyces cerevisiae and have incubated 

these cells with fluorescent-labeled aptamer target molecules followed by induction of the 

promoter. This technology uses FRET (Förster resonance energy transfer) to tell us that when 

the promoter is active and provides a dynamic transcriptional profile of the cells. In this work we 

have used three different types of multiaptamers (viz. PDC aptamer, tobramycin aptamer and 

neomycin aptamer) with their respective ligands, which have been individually labeled with cy3 

and cy5 dyes. With this noninvasive technology we are able to measure gene expression in real 

time of individual living cells. This allows an evaluation of the intercellular variation in gene 

expression. We have also used this method to measure the rate of transcriptional elongation. 

The IMAGEtag system has the potential of being used to track the real-time levels of gene 

expression at the cellular level in a broad range of cell types.

MONDAY 

2208 

In Situ Microscopic Visualization and Relative Quantification of Inorganic Polyphosphate 

Stores by 4',6-Diamidino-2-Phenylindole (DAPI)-stainning. 

F. M. Gomes 1,2 , I. Ramos 1,2 , W. Girard-Dias 2 , E. Machado 1,3 , K. Miranda 2,3 ; 1 Laboratorio de 

Entomologia Medica, Instituto de Biofísica Carlos Chagas Filhos, UFRJ, Rio de Janeiro, Brazil, 

2 Laboratorio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofisica Carlos Chagas Filho, 

UFRJ, Rio de Janeiro, Brazil, 3 Laboratorio de Biotechnologia, Diretoria de Programas, 

INMETRO, Rio de Janeiro, Brazil 

Inorganic polyphosphate (polyP) is a widely distributed biological polymer composed of 

phosphate residues linked by phosphoanhydride bonds. They play key biological roles such as 

phosphate and energy reservoir, metal homeostasis, regulation of transcription factors and 

transcription fidelity, as well as regulation of several enzyme activities [1]. Nevertheless, polyP 

has remained poorly studied mainly due to the limited methods for polyP quantification and in 

situ visualization. 4',6-diamidino-2-phenylindole (DAPI) is a commonly used nuclei stain that 

binds to double stranded DNA, exhibiting a fluorescence emission maximum around 450 nm. 

Interestingly, it has been shown that polyP is able to shift DAPI emission maximum to a higher 

wavelength around 525-550 nm displaying a greenish-yellowish color distinct from the blue 

nuclei-signals [2]. This DAPI-polyP interaction has been shown to be specific and to generate 

proper quantum yield for microscopic observation. Nevertheless, although a few studies have 

focused on the spectroscopic properties of analytical grade sodium polyphosphate-DAPI 

fluorescence, there are no consistent reports concerning methods of polyP visualization using 

DAPI. In addition, the lack of a general protocol for polyP staining hinders its wider utilization as 

a polyP-visualization tool. In the present study, we report a systematic evaluation of different 

protocols of DAPI-staining for the detection of polyP stores in different biological models. 

Suspensions of subcellular fractions of insect eggs and parasites containing polyP granules 

showed a clean DAPI-polyP fluorescence after brief incubation with DAPI, without previous 

treatments. Staining of Poly P stores in intact cells usually required aldehyde fixation and/or 

detergent permeabilization, as exemplified in DAPI-polyP fluorescence obtained from Eimeria 

parasites. DAPI-polyP signals were also detected from cryosections of mild-fixed, OCTembedded 

midgut of insects. In addition, a DAPI-polyP fluorimetric method was applied to 

quantify polyP mobilization during early egg development of the insect Rhodnius prolixus. The 

method was validated by the parallel measurement using a more widely accepted polyP 

quantification protocol that uses a recombinant yeast exopolyphosphatase. Taken together, our 

results strongly support the broader use of DAPI as a valuable tool for both polyP visualization 

and quantification in different biological models. Nevertheless, one should take into account that 

sample preparation may vary according to the model used and that care should be taken when 

localizing and quantifying polyP from different biological models. 

[1] Rao, N.N., M.R. Gómez-García, and A. Kornberg, Inorganic polyphosphate: essential for 

growth and survival. Annual review of biochemistry, 78, 605-647 (2009) . 

[2] Aschar-Sobbi, R., et al., High Sensitivity, Quantitative Measurements of Polyphosphate 

Using a New DAPI-Based Approach. Journal of Fluorescence, 18 (5), 859-866 (2008).

MONDAY 

2209 

Teachable tool for standardization of human induced pluripotent stem cell colony 

selection from live cell microscopy image sequences. 

S-J. J. Lee 1 , H. Lai 1 , W. T. Hendriks 2 , Z. A. Kenyon 1 , S. V. Alworth 1 , C. Nakada 3 , L. Daheron 4 , 

Y. Kiyota 3 , L. L. Rubin 2 , C. A. Cowan 2 ; 1 DRVision Technologies LLC, Bellevue, WA, 

2 Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Cambridge, 

MA, 3 Instruments Company, Nikon, Yokohama-city, Kanagawa,Japan 4 Harvard University, 

Harvard Stem Cell Institute, Cambridge, MA 

In a 2006 breakthrough study, Yamanaka and Takahashi proved that somatic cells could be 

reprogrammed to an embryonic-like state using four transcription factors. The induced 

pluripotent stem cell (iPSC) field has since progressed at a remarkable pace impacting basic 

research, drug discovery, bioproduction, drug screening, personalized medicine and cell 

therapy. One of the key challenges facing the industry is that iPSC, or iPSC-like colony 

selection remains technically challenging, and thus state-of-the-art protocols in a broad range of 

iPSC applications are often of low yield and un-predictable quality. Tools and techniques for the 

standardization of colony selection in real time systems, such as microscopy incubators are 

particularly valuable, to reduce the variability of outcomes and lower costs. In particular, tools 

that can predict future outcomes are useful because it allows productive colonies to be selected. 

We developed a teachable image recognition tool that can be taught to perform the 

quantitative analysis and classification of phase contrast and fluorescence images 

automatically. To enable the standardization of iPSC colony selection we created an image 

analytic by teaching. The analytic performs fully automated colony detection, colony feature 

measurement and iPSC colony classification in human iPSCs. Importantly, the classification 

step in the analytic is updateable by teaching. This allows the tool to be iteratively improved 

over time as the assay protocol is improved and colony true outcomes are determined by 

monitoring the complete re-programming process. 

The objective of this initial study was to validate our tool for the prediction of human 

iPSC colony outcomes. Registered images covering the whole well were acquired on the Nikon 

BioStation CT and stitched in our tool to create a large (~20,000 x 20,000 pixels) composite 

image sequence of the whole well include phase constrast and a viral-GFP construct that is 

silenced upon full reprogramming. We validated the tool by predicting week four outcomes using 

image frames up to week three. Ground truth is established by a loss of the GFP reporter. Our 

hypothesis is that the prediction using images up to week three can accurately predict each 

colonies' week four outcome as quantified by a loss in GFP fluorescence. We test the 

hypothesis by assessing the sensitivity and specificity of the week three prediction accuracy. 

We conclude that the hypothesis is supported with high prediction accuracy. Future studies will 

look towards real time colony selection and future outcome (e.g. differentiation) prediction. 

2210 

The Use of Discrete Polyethylene Glycol Linkers to Reduce Non-Specific Staining in 

Immunofluorescence. 

R. S. Brody 1 , M. Vermillion 1 , A. A. Rampersaud 1 , P. D. Davis 2 , D. Moothard 3 ; 1 QuantaBiodesign, 

Columbus, OH, 2 QuantaBiodesign, Powell, OH, 3 American Qualex, San Clemente, CA 

The objective of this study was to develop fluorescein conjugated antibodies for tissue and cell 

imaging that are brighter and show less non-specific staining than standard fluorescein 

isothiocyanate (FITC) conjugates. This objective was achieved by conjugating fluorescein to an 

antibody through a discrete polyethylene glycol (dPEGÂ®) linker. This study will be extended to

MONDAY 

the use of dPEGÂ® linkers to conjugate proteins with other fluorescent dyes with different 

excitation and emission wavelengths. 

METHODS.Fluorescent antibody conjugates were prepared by attaching fluorescein (FL) to one 

end of a dPEGÂ® linker that contains 12 ethylene glycol units and a terminal Nhydroxysuccinimide 

ester (FL-dPEGÂ®12-NHS). FL-dPEGÂ®12-NHS was conjugated to goat 

anti-rabbit IgG (GAR) and goat anti-mouse IgG (GAM) using standard NHS ester reaction 

conditions followed by purification over size exclusion columns. The same antibodies were also 

labeled with FITC for comparison. Cell based images were made using paraformaldehyde-fixed 

MCF-7 cells that were treated with 0.3% Triton X-100 and then incubated with a primary mouse 

monoclonal antibody against a golgi protein (GM130) followed by incubation with either FLdPEGÂ®12-GAM 

or FITC-GAM. Cell nuclei were labeled with DRAQ5 and imaging was done 

with a Zeiss 510 scanning laser confocal microscope. Fixed tissue sections were investigated 

by incubating them with a rabbit monoclonal antibody against a cancer bio-marker protein 

followed by incubation with either FL-dPEGÂ®12-GAR or FITC-GAR. Fluorescence was 

detected using a Nikon TE2000-E inverted epi-fluorescent microscope. For both the cell and 

tissue analyses, the intensity of the specific labeling and the fluorescence background for each 

conjugate was evaluated. 

RESULTS. At high incorporation levels, fluorescein that is attached to an antibody through a 

dPEG12Â® linker shows much less self-quenching than fluorescein attached to an antibody via 

a FITC label. The fluorescence of FL-dPEGÂ®12 labeled antibodies in solution increases 

almost linearly with fluorescein incorporation until approximately 20 fluorescein molecules are 

attached to each antibody. FL-dPEGÂ®12-GAM was found to bind specifically to the target 

mouse monoclonal in the MCF-7 cells, giving images with moderate intensities and low 

backgrounds. FITC labeled GAM gave images with similar intensities but much higher 

backgrounds. 

Cancerous cells could be clearly imaged in tissue sections using FL-dPEGÂ®12-GAR. In 

contrast, FITC-GAR gave very high backgrounds with similar sections that made detection of 

cancerous cells problematic. 

2211 

Computational Quantitative Image Analysis of Tissue Elongation in the Drosophila 

Embryo. 

M. Westacott 1 , A. Zommer 1 , T. Blankenship 1 , D. Loerke 1 ; 1 University of Denver (DU), Denver, 

CO 

Elongation of the Drosophila embryonic epithelium is driven by cell intercalation, which causes 

the tissue to narrow in one dimension and lengthen in the other. The remodeling of the epithelial 

sheet during this process is a powerful system to illuminate the role of cell-cell adhesion and 

membrane traffic in generating the necessary forces for re-organization of the cellular 

architecture, and 4-D in vivo imaging of the Drosophila embryo allows us to measure cell 

interface remodeling dynamics on the second timescale. We have developed an automated 

computational assay to quantitatively measure the dynamics of epithelial sheet remodeling. This 

assay is based on the segmentation of cells/interfaces to yield a 'skeletonized' representation of 

the sheet, the tracking of features across planes and in time, and the in silico reconstitution of 

the 3D spatial structure and network topology of the epithelial sheet and its temporal 

remodeling. This assay allows quantitative measurement of interface dynamics and cellular 

reorganization, as well as an unbiased measurement of protein localization asymmetries within 

the epithelial sheet in multi-color imaging experiments. We have measured the temporal 

dynamics of interface contraction/elongation, and observe the propagation of remodeling

MONDAY 

dynamics both laterally within the sheet and in the vertical direction (from apical to basolateral 

plane). 

2212 

A simple, versatile method for GFP-based single molecule localization microscopy. 

J. Ries 1 , C. Kaplan 1 , E. Platonova 1 , H. Eghlidi 1 , H. Ewers 1 ; 1 Institute of Biochemistry, ETH 

Zurich, Zurich, Switzerland 

Single molecule localization-based superresolution microscopy methods such as PALM or 

STORM, have been breakthrough techniques of the last years. Until now however, they require 

special fluorescent proteins to be cloned or high-affinity antibodies to be generated for specific 

labeling. On the other hand, many laboratories will have most of their constructs in GFP form 

and entire genomes are available as functional GFP-fusion proteins. 

Here, we report a method that makes all these constructs available for superresolution 

microscopy by targeting GFP with tiny, high-affinity antibodies coupled to blinking dyes. It thus 

combines the molecular specificity of genetic tagging with the high photon yield of organic dyes 

and minimal linkage error. 

Direct STORM on microtubules labeled with our novel antibodies showed that indeed the 

linkage error was minimal, whereas the large size of standard antibodies resulted in an 

additional error of >10 nm in immunolabeling. 

The brightness of our labels enabled us to perform rapid time-lapse dSTORM and sptPALM on 

living neurons expressing the outer membrane protein GPI-GFP. Three-dimensional dSTORM 

on microtubules using the bi-plane approach allowed us to distinguish overlapping microtubules 

with an axial separation of ~100 nm. 

Using a budding yeast GFP-tag genomic library we could readily image several GFP-tagged 

proteins targeted to specific intracellular locations. 

In summary, targeting of GFP-labeled constructs with tiny antibodies provides fast and simple 

access to superresolution microscopy of virtually any known protein in cells. Since for several 

organisms the entire genome is available as GFP-tagged constructs, all these proteins are 

immediately accessible without the requirement for cloning or the generation of antibodies. 

Finally, due to a simple one-step labeling protocol, our technique opens the door to highthroughput 

localization analysis of entire genomes at the nanoscopic level in cells. 

2213 

Next Generation Sequencing Analysis of the Evolutionary Impact of Ploidy. 

A. Selmecki 1 , P. A. Richmond 2 , R. Dowell 2,3 , D. Pellman 1 ; 1 Department of Pediatric Oncology, 

Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 2 Molecular, Cellular, 

Developmental Biology, University of Colorado, Boulder, CO, 3 Biofrontiers Institute, University of 

Colorado 

The role of polyploidy—increased copy number of chromosomes relative to an organism’s 

accepted euploidy—on cells under environmental stress is poorly understood. Ploidy is common 

in cancer so it is important to understand how ploidy impacts evolution. We performed an 

evolution experiment on strains of Saccharomyces cerevisiae with different ploidy (haploid, 

diploid, and tetraploid) under selection for growth on a low-glucose, high raffinose media. After 

240 generations, the parental strain and seven of the surviving strains (2 diploid and 5 

tetraploids) were sequenced via next generation sequencing technology. We analyzed the 

sequenced strains to identify mutations causal for survival and compared the mutations 

between the strains of different ploidy. Preliminary results reveal more mutations in the 

tetraploid strains relative to their diploid counterparts. In addition, we have benchmarked

MONDAY 

variation-calling software with both “in silico” generated synthetic datasets, as well as our 

evolved strains for calling SNPs and indels at higher ploidy (N > 2). 

2214 

An automated method for tracking punctate objects in live cell imaging data. 

A. Ma 1 , J. Metz 2 ; 1 Albert Einstein Coll Med, Bronx, NY, 2 Albert Einstein College of Medicine, 

Bronx, NY 

Continuing advances in fluorescence optical microscopy are yielding an unprecedented surge in 

live cell imaging data, leaving manual analysis intractable. We have developed a general and 

model-free algorithm for automatic tracking of single-particle objects in crowded and noisy 

environments in live cell time-lapse imaging data. In this algorithm, we divided objects in the 

imaging data into two categories: those with unique image patterns and those without. We first 

use multi-scale bidirectional template matching procedure to identify objects with unique image 

patterns. From object linkings made for these objects, we can extract valuable information of the 

dynamics of the system. Such information will be used as inputs to the dynamic model based 

procedure that we use to track the objects without uniquely recognizable image patterns. The 

versatility and robustness of the method were demonstrated by both synthetic and experimental 

time lapsed imaging data. 

2215/L124 

Enabling Biology with the Structural Biology Knowledgebase. 

M. Gabanyi 1 , J. Westbrook 1 , D. Micallef 1 , Y-P. Tao 1 , R. Shah 1 , W. McLaughlin 2 , T. Schwede 3 , P. 

Adams 4 , W. Minor 5 , H. Berman 1 ; 1 Chemistry and Chemical Biology, Rutgers University, 

Piscataway, NJ, 2 The Commonwealth Medical College, Scranton, PA, 3 Swiss Institute of 

Bioinformatics, University of Basel, Switzerland, 4 Physical Biosciences, Lawrence Berkeley 

National Labs, 5 University of Virginia Medical School, Charlottesville, VA 

The PSI Structural Biology Knowledgebase (SBKB, http://sbkb.org) is a scientific search portal 

that returns comprehensive biological, structural, and methodological information about 

proteins. Information related to 3D protein structures and the sequences targeted by worldwide 

structural genomics efforts are combined with links to open biological resources to give an 

integrated view of a protein. Using the information found on the SBKB, researchers can make 

informed decisions about their projects and what to do next. It is created by the Protein 

Structure Initiative in collaboration with the Nature Publishing Group, and features the latest 

research and technology advances each month to enable researchers in a broad range of 

biomedical fields. 

2216 

Identification of Ca2+/CaM as a target protein of a new hypoxia-inducible factor 1-alpha 

inhibiting small molecule using phage display biopanning. 

B. Kim 1 , K. Lee 2 , H. Jung 1 , H. Kwon 1 ; 1 Chemical Genomics National Research Laboratory, 

TRCP, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei 

University, Seoul, Korea, 2 Dongguk University, Seoul, Korea 

Hypoxia inducible factor-1 alpha (HIF-1α) is an essential transcription factor subunit that 

regulates expression of proteins involving in angiogenesis and metastasis such as VEGF. 

Accordingly, HIF-1α represents an emerging therapeutic target for development of novel 

anticancer drugs. We recently developed a new synthetic small molecule inhibiting HIF-1α 

(YCG-6), which results in decrease of VEGF mRNA expression. Here, the anti-proliferative and 

the anti-angiogenic activity of the compound on human umbilical vascular endothelial cells

MONDAY 

(HUVECs) were validated through MTT assay and in vitro tube formation and chemoinvasion 

assays. Furthermore, identification of the target protein of the compound was conducted by 

using phage display biopanning. As the result, calmodulin (CaM) was isolated as a plausible 

target protein and Ca 2+ is required for binding of the compound to CaM. Moreover, direct 

interaction of the CaM and compound was confirmed by Surface Plasmon Resonance analysis. 

Virtual molecular docking study suggested that the compound acts as a CaM antagonist by 

binding to hydrophobic pocket of the protein. Moreover, the cell proliferation was arrested by the 

compound as like other CaM antagonizers did. The effect of YCG-6 on Ras/ERK signaling and 

prostate specific antigen expression in LNCaP androgen-sensitive cells also clearly reveal that 

YCG-6 acts as HIF-1α inhibitor by targeting CaM. 

2217 

Exploring Atomic Force Microscopy for Single Cell Manipulations. 

R. Afrin 1 , U. S. Zohora 2 , H. Uehara 2 , S-I. Machida 1 , T. Watanabe-Nakayama 2 , M. Saito 1 , A. 

Ikai 1 ; 1 Innovation Laboratory, Tokyo Institute of Technology, Yokohama, Japan, 2 Life Science, 

Tokyo Institute of Technology, Yokohama, Japan 

The atomic force microscope (AFM) is a versatile tool for imaging, force measurement and 

manipulation of proteins, DNA and living cells basically at the single molecular level. In the 

cellular level manipulation, extraction and identification of mRNA’s from defined loci of a cell, 

insertion of plasmid DNA and pulling of membrane proteins, for example, have been reported 

from our group [1]. We used AFM as a novel tool to create holes at defined loci on the cell 

membrane for visualization of intracellular structure through the hole and for targeted gene 

delivery into living cells as the ultimate purpose of the application of nano-medicine. Targeted 

gene delivery was successfully performed by inserting an AFM probe that was coated with the 

Monster Green Fluorescent Protein phMGFP Vector for transfection of the cell. Following 

targeted transfection, the gene expression of GFP was observed and confirmed by the 

fluorescence microscope with high accuracy. Using this AFM probe technology as a fishing 

nano-device we successfully harvested intra-cellular mRNA’s and study mRNA expression in 

single living cells without damage to the cells and analyzed by PCR method. For this, an AFM 

tip was inserted into a living cell to extract mRNAs, which were then analyzed for the number of 

adhered mRNA through multiplication by RT-PCR and quantitative PCR [2]. We performed 

quantitative measurement of mRNA at different loci within individual living cells before and after 

stimulation with fetal bovine serum. Before activation, mRNAs were found mostly near the 

nucleus, whereas after activation, they were also found in the frontal regions of locomotive cells. 

The results agreed with previous experiments of FISH [3]. Asymmetric localizations of cellular 

proteins and mRNAs are important for cell functions such as division, differentiation and 

development. The localization of speci?c mRNA generates cell polarity by controlling the 

translation sites of speci?c proteins and thereby restricting their locations to appropriate cellular 

regions. This method can also be applied to study such important aspects of mRNA distribution 

within living cells. 

References: 

1. Afrin R. et al., (2009) J. Mol. Recognit. 22:363-372. 

2. Uehara H. et al., (2009) Methods Mol Biol. 544:599-608. 

3. Kislauskis EH et al., (1997) J. Cell Biol. 136:1263-1270 .

MONDAY 

2218 

High Throughput Passive Rheology Assay for Cancer Cell Mechanics. 

L. D. Osborne 1 , J. Cribb 1 , V. Swaminathan 2 , R. Spero 1 , E. O'Brien 1 , R. Taylor 3 , R. Superfine 1 ; 

1 Dept. of Physics & Astronomy, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 

2 Laboratory of Cell and Tissue Morphodynamics, National Heart, Lung, and Blood Institute, 

3 Dept. of Computer Science, The University of North Carolina at Chapel Hill 

While the dynamic nature of cellular structure enables a cell to sense and respond to 

environmental forces, this complexity introduces a significant degree of variability in the 

mechanical properties of individual cells within a population. Understanding the composition of 

cell populations is critical as cells become cancerous and their mechanics change due to 

alterations in cytoskeletal structure. To address this need, we are developing a high throughput 

system that utilizes passive microbead rheology to characterize cancer cell mechanics. Here, 

we report on the progress towards a parallel array of 12 independently functioning imaging 

systems capable of gathering mechanical measurements for a 96-well specimen plate in under 

10 minutes. Using our single imaging system prototype, we show that the thermal diffusion of 

1um beads connected to integrin surface receptors via fibronectin can distinguish ovarian 

cancers with varying metastatic potentials. With a sampling of over 500 beads, we report moduli 

differences between cancer cells and within cancer cell populations. Additionally, we show 

differences in cell moduli caused by substrate stiffness. Our results support previously 

documented work describing the inverse relationship between mechanical stiffness and invasion 

behavior. This demonstrates the value of our high throughput passive rheology assay as a 

screening tool for studying specific signaling pathways involved in mechanotransduction. 

2219 WITHDRAWN 

2220 

Toxic metal detection in foodstuff. Synthetic biology approach used to create 

biosensors. 

A. Bartos 1,2 , B. Wang 1 , M. Buck 1 , J. Schumacher 1 ; 1 Imperial College London, London, United 

Kingdom, 2 Technical University of Lodz, Poland 

The objective of the presented research was to explore possibilities of using redesigned E.coli 

genetic regulatory systems to quantify foodborne metals for analytical purposes. Trace amounts 

of exogenous metals are known food contaminants. Metallic ions such as mercury, nickel, led, 

copper, cadmium, mercury etc. are amongst most toxic agents found in commercially available 

food products. Adsorbed and accumulated over years they lead to chronic health disorders. 

Ordinarily used instrumental chemistry methods (graphite furnace atomic absorption 

spectrometry, inductively coupled plasma atomic emission spectroscopy, inductively coupled 

plasma mass spectrometry) can go down to ng/dm3 in their limits of detection, but have a set of 

drawbacks. We designed a microbiological transcription-responsive biosensor to identify trace 

amounts of toxic metals. Such recombinant senses a number of metals, resulting in transcription 

of genes regulated by inducible protein factors: ZraR in the case of Zn2+, Pb2+; CusR for Cu2+, 

NikR for Ni2+ etc. We have combined the promoter sequences of GlnK, ZraP, NorV, PspA, 

PrpB, CusC to the coding sequence of GFP to obtain metal responsive fluorescent outputs. 

Conducted research provides scope and prospects of recombinant E.coli as biosensors, with 

emphasis of detection limits confined by cells responsibility to molecular signalling and viability 

to grow in toxic environment. NCM3722, MC1061 and Top10 strains were induced at 

OD600=0.05 and, separately, at OD600=0.4 with 10 metal solutions known to activate the 

corresponding signalling pathways. Results were obtained with the use of plate reader in the 

form of fluorescence and OD600 measurements over time past induction. They indicate

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successful use of bacterial cultures to sense metals with detection limits below the milimolar 

range. 

2221 

Integrated Laser and Electron Microscopy: in search of the causes of FSHD Muscle 

Dystrophy. 

M. Karreman 1,2 , E. van Donselaar 2 , S. Agronskaia 1 , W. Voorhout 3 , T. Verrips 2 , H. Gerritsen 1 ; 

1 Science Faculty, Molecular Biophysics, Utrecht, Netherlands, 2 Dept. of Biology, Biomolecular 

Imaging, Utrecht, Netherlands, 3 Life Sciences Division, FEI Company, Eindhoven, Netherlands 

Facio Scapulo Humeral Dystrophy (FSHD) is the third most common type of muscle dystrophy, 

and affects 1:20,000 people per year worldwide. Currently, much is known about the genetic 

background of this disease, and how FSHD affects the patients. However, the cellular 

processes involved in FSHD are hitherto not identified. 

We set out to study the cellular causes and effects of FSHD with a novel tool for correlative 

microscopy. The Integrated Laser and Electron Microscope (iLEM) combines a fluorescence 

microscope (FM) and a transmission electron microscope (TEM) within one set-up. This allows 

for the localization of regions of interest over a large field of view with the FM, and subsequent 

ultrastructural analysis of these areas with the TEM. Since both microscopes are integrated 

within one set-up, the correlation between the FM and the TEM is extremely fast and very 

accurate. 

FSHD is known to be a heterogeneous disease; its expression can vary from muscle to muscle 

and from cell to cell. Ultrastructural studies of affected cells will help us to gain more insight in 

the processes involved in this disease. The iLEM is employed to navigate to the FSHD affected 

regions, so that these areas can subsequently be studied at high resolution. We aimed to 

localize FSHD affected cells with the iLEM in muscle biopsies from patients. At a certain stage 

of muscular dystrophy, creatine kinase forms crystalline structures in the mitochondria. This 

protein was used as a marker to navigate to the FSHD affected cells in the biopsy; next the 

crystalline structures and their cellular context was studied at high resolution. 

In conclusion; due to the heterogeneity of the expression of FSHD, the iLEM is a unique tool to 

rapidly navigate to the cells of interest and study the effects of this disease with high resolution. 

2222 

A novel single chain Rac biosensor for the study of tumor cell migration. 

Y. Moshfegh 1 ; 1 Albert Einstein College of Medicine, Bronx, NY 

The p21 Rho family of small GTPases are heavily involved in cell motility and migration. 

Specifically, Rac is critical for cell protrusion at the leading edge, and has been shown to be 

overexpressed in several types of tumors. However, the exact role of Rac in relation to the other 

Rho GTPases still remains unclear, because previous approaches for the study of these 

molecules are limited due to their lack of sub-cellular spatiotemporal resolution. By developing 

genetically encoded, single-chain fluorescent biosensors, we are able to bypass this limitation 

and visualize protein activation as well as localization in real time, making this innovation a 

necessary and powerful tool for the study of small G-proteins and cell migration. 

To study the function of the Rac during cell protrusion, and also its relationship to the other Rho 

GTPases, we developed a novel, genetically-encoded fluorescent Rac1 biosensor, based on an 

intra-molecular design. This new biosensor is a massive improvement over the previousgeneration 

Rac biosensor because there is an equimolar distribution of FRET donor and

MONDAY 

acceptor and this produces a more accurate readout. Our design maintains the correct Cterminal 

lipid modification of full-length Rac1, enabling proper interaction with upstream 

regulators, including GEFs, GAPs, and GDIs. We constructed this new single-chain biosensor 

by modifying the placement of the binding domain in relation to the first fluorescent protein, to 

achieve the proper orientation for optimal interaction between the GTPase and its binding 

domain. In addition, we incorporated an internal autoinhibitory mechanism into our design, to 

modulate the binding domain affinity. 

This new biosensor will allow the real-time visualization of the spatiotemporal dynamics of Rac 

in live carcinoma cells. In particular, we will be able to explore the molecular basis for the 

mechanism of Rac, Rho, and Cdc42 coordination at the leading edge. 

2223 

An inducible, reversible system for the rapid and complete destruction of proteins in 

mammalian cells. 

D. Fachinetti 1 , A. J. Holland 2 , D. W. Cleveland 3 ; 1 Department of Cellular and Molecular 

Medicine, Ludwig Institute for Cancer Research, La Jolla, CA, 2 Department of Cellular and 

Molecular Medicine, University of California, Ludwig Institute for Cancer Research, La Jolla, CA, 

3 Department of Cellular and Molecular Medicine, University of California, Ludwig Institute for 

Cancer Research 

Inducible degradation in living cells is a powerful approach for identifying the function of a 

specific protein or protein complex. Here we demonstrate that an auxin-inducible degron (AID) 

system is capable of controlling the stability of AID-tagged proteins that are in either nuclear or 

cytoplasmic compartments and even when incorporated into protein complexes. Induced 

degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as 9 

minutes and proceeding to completion with first order kinetics. Auxin-inducible, degron-mediated 

instability is demonstrated to be rapidly reversible. Induced degradation is shown to initiate and 

continue in all cell cycle phases, including mitosis, making it especially useful for identifying 

function(s) of proteins of interest during specific points in the mammalian cell cycle. 

2224 

An Application Specific Microfluidic Format for Dynamic Live Cell Studies. 

M. Allen 1 , P. Hung 1 , T. Gaige 1 , P. Lee 1 ; 1 CellASIC Corp., Hayward, CA 

Studying living mammalian cells in vitro is critical to understanding basic biology, signaling 

pathways, drug effects, and disease models. While the tools and techniques for cellular analysis 

have advanced rapidly over the past decades, the cell culture platform has remained largely 

undeveloped since the Petri dish. In light of growing evidence that the cellular culture 

environment is critical for sustaining physiologically relevant phenotype and behavior, we have 

developed a microfluidic cell culture platform to control various cellular microenvironment 

parameters including reagent perfusion, temperature, and gas composition, all through a 

universal control system used in tandem with different application specific microfluidic plates. 

Each plate fits a standard microplate format containing four independent micro-fabricated PDMS 

culture chambers which can be addressed by up to 6 different reagents. The microfluidic plate 

houses all necessary solutions in simple layout of wells that are readily accessed during 

experimental setup using a pipette. A 170-micron thick glass coverslip bottom enables high 

magnification imaging on an inverted microscope. Dynamic environment changes within plates 

are controlled through software that integrates the universal control system with real-time image 

analysis to form a closed feedback loop. The utility of the platform was demonstrated with a 

number of applications. First, automated immunostaining was demonstrated using HT-1080 

fibrosarcoma cells. Cells were cultured in the microfluidic plate to the desired density, then

MONDAY 

automatically washed, fixed, permeablized, blocked, and fluorescently stained with antibodies 

using a series of solutions and programmed exposure times, significantly simplifying the 

traditional protocol. Second, promyelocytic HL-60 cells differentiated into granulocytes were 

tracked to show a chemotactic response to a dynamic chemical gradient for live cell migration 

studies. Third, real-time transfection optimization was performed using HeLa cervical carcinoma 

cells revealing expression of GFP-tagged tubulin via live cell fluorescence microscopy. Fourth, 

3D cell culture was demonstrated with MCF10A mammary epithelial cells cultured in Matrigel 

with continuous perfusion to track acinus formation. This application specific design philosophy 

promises to bring dynamic cell microenvironment control within reach for a large range of usergenerated 

cell biology applications to complement current advanced cellular analysis 

techniques. 

2225 

Use of Short Tandem Repeats for Rapid Authentication of Human Cell Lines. 

T. Reid 1 , K. Wilson 2 , C. Kraemer 3 , M. Baird 4 ; 1 DNA Diagnostics Center, Cincinnati, OH, 

2 University of Cincinnati, OH 3 DNA Diagnostics Center, 4 DNA Diagnostics Ctr, Fairfield, OH 

Human cell lines are used worldwide in biological and biomedical research, and accurate data 

interpretation depends on unambiguous identity of a cell line relative to its original source. 

Contamination and misidentification of human cell cultures has been identified as a longstanding 

issue within the scientific community, and steps are now being taken to address the 

problem. 

Short-tandem-repeat (STR) DNA profiling provides an accurate, reliable, and standardized 

method for authentication of human identity. Currently, the ATCC is considering proposed 

standards for cell line authentication utilizing human STR typing, and many journals have 

adopted the NIH-recommended policy and mandate cell line authentication as a requirement for 

publication. 

Here we demonstrate the validation and use of two different STR systems to authenticate 

human cell lines as well as detect contamination by mouse feeder cells and other human cell 

lines. Threshold levels for detection of complete profiles as well as contamination are compared 

between the systems, and typical typing issues specific to cell lines are discussed. 

2226 

Monitoring Changes in NF-kB Pathway Regulation Using Highly Sensitive Multipex 

Bioluminescent Reporter Assays. 

D. Hughes 1 , M. dobbs 1 , J. Narahari 1 , J. Choi 1 , A. Deshpande 1 , B. Webb 1 ; 1 Thermofisher 

Scientific, Rockford, IL 

The study of complex cellular signaling pathways requires powerful and specific tools to monitor 

changes in gene activation or repression. In order to accurately monitor these processes, 

reporter gene assays are commonly used. We have developed a series of next generation 

multiplexed luciferase reporters for studying gene regulation. These reporters were developed 

to improve the sensitivity and convenience of conventional luciferase reporter systems. First, we 

have used two naturally secreted luciferase genes, Gaussia luciferase from the Marine copepod 

Gaussia princeps and Cypridina luciferase from the Marine ostracod Cypridina noctiluca to 

develop a novel dual secreted reporter system. This Gaussia/Cypridina dual system enables 

monitoring transcriptional regulation of two promoters within tissue culture media without the 

need for cell lysis using sequential addition of luciferase substrates. Importantly, both Gaussia 

luciferase and Cypridina luciferase are considerably brighter than traditional Firefly luciferase 

reporters. Second, we have utilized a mutant form of the Italian Firefly Luciferase from Luciola

MONDAY 

cruciata that has a red-shifted emission spectrum to develop a dual luciferase assay with 

Gaussia luciferase in which the light output of the two luciferases are spectrally resolvable. This 

Gaussia/Red Firefly dual spectral assay allows simultaneous monitoring of two promoters in a 

single read assay through addition of both substrates and then spectral interrogation of the 

resulting light output. In the present study, we utilized both techniques, multiplexing by spectral 

separation using Gaussia/Red Firefly, and multiplex assays relying on sequential addition of 

substrates two secretory luciferases, Gaussia/Cypridina to monitor changes in NFkB promoter 

activity in response to small molecule agonists. Our results demonstrate the utility of dual 

secreted luciferase assays for sensitive real time monitoring of NFkB reporter activity in the 

media and simultaneous detection of spectrally resolvable luciferases using filter based 

detection. 

2227 

Optimization of Protein Purification Using Small-Scale Separation Columns. 

R. Tobias 1 , L. Hoang 1 , C. Suh 1 , D. Gjerde 1 ; 1 PHYNEXUS, INC., San Jose, CA 

Biophysical and functional characterization of therapeutic candidates requires that proteins are 

well purified and enriched post-expression. Currently the process for adequate preparation 

requires that sufficient quantities of material be scaled up and processed in a time consuming 

manner using expensive chromatography equipment. As improvements in functional and 

analytical assays increase throughput and reduce the quantity of protein required for analysis, 

the availability of efficient small-volume protein purification methods would be of high value to 

researchers in earlier stages of drug discovery and development. Recent advances in the area 

of miniaturized, high-throughput tools for purification, enrichment and desalting of proteins 

eliminate bottlenecks associated with traditional protein purification processes. By performing 

high-performance functional protein separations on small samples in parallel, it is now possible 

to obtain more relevant data in a completely automated format. Data from protein separations in 

micro-scale chromatography columns is presented here along with optimized conditions 

enabling functional and analytical characterization of therapeutic proteins purified by this unique 

format. 

2228 

Alternative Digital Microfluidic Devices for Cell Assays. 

N. Thorne 1 , B. Demchak 1 , L. Pontiggia 1 , S. Freire 1 ; 1 University of the Sciences, Philadelphia, PA 

Digital microfluidics (DMF) is a technique for transport of droplets by an electric potential applied 

to an array of electrodes, and has appeared as an alternative to the conventional fluid transport 

in microchannels. DMF enables automation and control of droplets unparalleled by any other 

technique of fluid transport, without the need for pumps, tubes, or valves (1). In particular, we 

are interested in developing DMF platforms to study ciliary motion. 

Usually, the fabrication of DMF devices requires access to well-equipped facilities and the 

training of students in a variety of techniques (e.g, metal deposition, photolithography, etc.). This 

is often expensive and time consuming, limiting the access to this technique, particularly for 

researchers at undergraduate institutions. 

We developed an alternative method for fabrication of DMF devices (2). One key aspect is that 

miniaturized interelectrode gaps, thought as essential for optimal performance, are not a 

requirement for droplet actuation; altogether, the strategy enables fast fabrication of robust and 

reliable devices at low costs.

MONDAY 

However, this technique requires relatively high voltages for DMF operation (typically 500 

VRMS), which contrasts with the low voltages (~120 VRMS) used in conventional, clean-room 

based devices. This has been a point of criticism, with the claim that high voltages might be 

detrimental to biological specimens. 

Tetrahymena thermophila (SB 255) cells, well suited for research purposes due to the large 

number of cilia, were used for tests. Droplets (10 µL) containing cells were actuated on DMF 

devices. We will present results showing that no changes were observed in the vitality and 

proliferation of these cells after transported in the alternative DMF devices. This corroborates 

previous results showing that the voltage across the droplet in a DMF device is an insignificant 

fraction of the total applied voltage, indicating very little, if any, effects on cells (3). 

We are currently evaluating non-specific adsorption of analytes to surfaces, which effects device 

performance. We have chosen a simple and well-established assay, cell deciliation with 

dibucaine. We will show that the minimum concentration of dibucaine for a complete and almost 

instantaneous loss of cilia is close to the used in conventional, off-chip deciliation (1.3 mM)(4). 

This indicates little non-specifc adsorption of dibucaine to surfaces in this assay. 

Since adsorption to surfaces might be analyte dependent, we are also looking for collaborations 

to develop other assays of interest, to further characterize the devices. However, these 

preliminary results pave the way to on-chip deciliation. Future studies will focus on 

characterizing motion of extracted cilia, directly on a microfluidic device. This work has been 

done by undergraduate students (Biological Sciences and Physics). 

References: 

1 Fair, R. B. "Digital microfluidics: is a true lab-on-a-chip possible?" Microfluidics and 

Nanofluidics 2007, 3, 245-281. 

2 Thorne, N.; Lamberto, M.; Mazza, A.; Freire, S. L. S. "An Alternative Strategy for 

Fabrication of Robust and Flexible Digital Microfluidic Devices" Pacific Northwest Journal of 

Undergraduate Research and Creative Activities 2011, 2 

3 Barbulovic-Nad, I.; Yang, H.; Park, P. S.; Wheeler, A. R. "Digital microfluidics for cellbased 

assays" Lab Chip 2008, 8, 519-526. 

4 Thompson, G. A., Jr.; Baugh, L. C.; Walker, L. F. "Nonlethal deciliation of Tetrahymena 

by a local anesthetic and its utility as a tool for studying cilia regeneration" J Cell Biol 1974, 61, 

253-257. 

2229 

Deciphering the Intermediate Filament Structure(s) using cryo Electron Microscopy and 

Novel Metal Labels. 

R. Kirmse 1 , C. Bouchet-Marquis 1 , M. Pagratis 1 , A. Hoenger 1 ; 1 MCDB, University of Colorado at 

Boulder, Boulder, CO 

Intermediate filaments (IFs) are critical building blocks of the cell´s cytoskeleton. Together with 

actin filaments and microtubules they are responsible for various cell functions such as 

mechanical stress resistance, signal transduction, and material transport. In contrast to actin 

and tubulin a detailed 3D structure of IF proteins and grown filaments remains elusive. 

Crystallization of IF proteins for example is extremely difficult caused by their elongated shape. 

Regardless, cryo electron microscopy and tomography provides the opportunity to observe 

proteins in 3D. By rapid freezing we are able to keep the IFs hydrated and as close to their 

native state as possible. Subsequently cryo tomography allows reconstructing the 3D structure

MONDAY 

of the observed sample in great detail. In addition we are developing new labeling methods 

utilizing cloneable high-density markers to further label the IF substructure. These markers will 

enable us to draw conclusion about the basic underlying arrangements of the IF proteins in the 

mature filaments.

TUESDAY, DECEMBER 6- Late Abstracts 3 

Actin Cytoskeleton 

TUESDAY 

2230 

Depletion of Arp2/3 complex reveals critical role of lamellipodia in haptotaxis, but not 

chemotaxis. 

C. Wu 1 , J. E. Bear 2,3 ; 1 Cell and Developmental Biology, UNC-Chapel Hill, Chapel Hill, NC, 

2 UNC-Chapel Hill, Chapel Hill. CA 3 HHMI 

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain 

Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be 

critical for cell motility, this notion has not been rigorously tested. Using fibroblasts derived from 

Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks 

lamellipodia. This line shows defective random cell motility and relies on a filopodia-based 

protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of 

Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, these cells respond 

normally to shallow gradients of PDGF indicating that lamellipodia are not required for 

chemotaxis. Conversely, these cells cannot respond to a gradient of extracellular matrix 

(haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal 

adhesions suggesting that one principle function of lamellipodia is to organize cell-matrix 

adhesions in a spatial coherent manner. 

2231 

A Peptide Mimetic of Heat Shock Protein Beta 6 Alters 3t3 Migration through a Cofilin- 

Dependent Mechanism. 

C. Smoke 1 , D. N. Derkach 1 , D. Eng 1 , J. Uhlenkamp 1 , K. B. Perkins 1 , M. R. Sheller 1 ; 1 Capstone 

Therapeutics, Tempe, AZ 

Prior research has shown that AZX100, a 24-amino acid peptide mimetic of Heat Shock Protein 

β6 (HSPβ6), reduces filamentous actin and focal adhesions in myofibroblasts, cells instrumental 

in the process of dermal scar healing. It has also been shown that HSPβ6 binds to 14-3-3, 

leading to dephosphorylation of cofilin. Dephosphorylated cofilin severs filamentous actin and 

increases the pool of globular actin. Investigating the manner in which AZX100 affects cofilin 

dephosphorylation, alters actin dynamics and influences migratory behavior in myofibroblasts 

could elucidate the role of AZX100 in scar healing. 

The present study used 3T3 fibroblasts treated with transforming growth factor β-1 (TGFβ-1) 

alone or in the presence of varying concentrations of AZX100 for one hour. Fibroblasts were 

treated with TGFβ-1 to induce differentiation to myofibroblast-like cells. Cell lysates were 

resolved by 2-dimensional gel electrophoresis to determine the cofilin-to-phospho-cofilin 

expression levels. AZX100 treatment of the 3T3 cells significantly increased the percent of 

dephosphorylated cofilin compared to cells treated with TGFß-1 alone (73% vs. 40% 

respectively; p =0.006). Additionally, live cell imaging was performed to study the effect of 

various doses of AZX100 on the actin cytoskeleton. Treatment with 1 and 10 μM AZX100 

produced the greatest change in the actin cytoskeleton as measured by overall reduction in cell 

area. Doses of AZX100 greater than 10 μM and lower than 1 μM did not significantly alter the 

actin cytoskeleton compared to control cells. Finally, myofibroblast migration was analyzed 

through the Electric Cell-substrate Impedence Sensing (ECIS) system, which measured 

changes in resistance over time. AZX100 treatment resulted in a dose-dependent reduction of 

migration over one hour compared to TGFβ-1 alone, with a maximal reduction of 53%. In

TUESDAY 

conclusion, AZX100 increases cofilin dephosphorylation inmigratory cells. The associated 

migration pattern was also altered, which may be due to morphological changes resulting from 

the liberation of dephosphorylated cofilin from 14-3-3. 

2232 

Biophysical Linkage between Local Mechanical Properties and Global Cellular 

Responses in Living Fibroblasts: Actin Cytoskeleton Perspectives. 

H-C. Harn 1 , Y-K. Wang 2 , H-H. Lin 3 , C-M. Cheng 4 , M-J. Tang 1,3 ; 1 Institute of Basic Medical 

Sciences, National Cheng Kung University, Tainan, Taiwan, 2 Dept. of Medicine, Skeleton-Joint 

Research Center, National Cheng Kung University, 3 Department of Physiology, College of 

Medicine, National Cheng Kung University, Tainan, Taiwan, 4 Institute of Nanoengineering and 

Microsystems, National Tsing Hua University, Hsinchu, Taiwan 

Each organ in our body has different mechanical properties—ranging from 200 Pa (brain) to 310 

MPa (Achilles tendon)—serving as the structural-based scaffolding and the inherent force 

source of mechanical stimulation for single cells. The cellular response under mechanical 

stimulation is highly tuned to influence a cell’s structure provided by the cytoskeleton. The 

cytoskeleton governs a diversity of cellular behaviors such as cell division, motility, and 

morphology. Solon et al. have observed that fibroblasts adjusted their spreading areas and actin 

cytoskeleton organization in order to match the change of an external environment. We 

therefore hypothesize that actin cytoskeleton could be a key determinant to the changes in cell’s 

mechanical properties in response to different mechanical stimuli. In this study, we attempted to 

explore the mechanical properties of an individual actin filament in living fibroblasts (NRK49F), 

and to determine its contributions to the elasticity of the cell as a whole with external physical 

stimuli. We used a biological atomic force microscope to (i) scan a living fibroblast in a liquid 

environment, (ii) indent around the periphery in a living fibroblast, and (iii) indent on top of the 

nucleus in a living fibroblast to determine Young’s moduli of specific locations, respectively. We 

also constructed cell’s 3D images via confocal fluorescent microscopy, in order to probe the 

morphological responses of actin cytoskeleton with mechanical stimuli. The results showed that 

a fibroblast changed its local and global elasticities under substratum stiffness. The organization 

of actin filaments—the number of actin filaments at a specific location that we probed in a living 

fibroblast—were strongly affected by the change with substrate stiffness. Furthermore, even for 

a single fibroblast seeded on a stiffer substrate showed a distinct difference (20 and 200 kPa) in 

the elasticity of actin filaments; this could correspond to the variations in the thickness of actin 

filaments and the distribution of actin filaments around the cell periphery. This study connects 

the local mechanical properties of actin filaments to the overall elasticity of a living cell in 

response to mechanical stimulation. It highlights the important contribution of actin filaments 

could play in regulating the behavior and function of a cell. 

2233 

Stereocilia F-actin polymerization is coupled to the mechanotransduction machinery in 

the auditory hair bundles. 

V. Michel 1,2 , E. Caberlotto 1 , D. Weil 1 , C. Petit 1,3 ; 1 Pasteur Institut-INSERM, Paris, France, 

2 Université Pierre et Marie Curie, Paris, France, 3 Collège de France, Paris, France 

In the hair bundles of the auditory hair cells, the mechanotransducer channels are gated by tiplinks, 

extracellular filaments that interconnect the stereocilia and stretch from the tips of 

stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. Tip-links 

consist of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes 

USH1D and USH1F, respectively. In this study, we found that upon the post-natal 

disappearance of the tip-links, caused by the deletion of one of its components, cadherin-23, or

TUESDAY 

of one of its anchoring proteins, sans, stereocilia from the short and medium but not of the high 

stereocilia row undergo a dramatic reduction in length. This indicates that the 

mechanotransduction machinery has a positive effect on F-actin polymerization. The loss of the 

tip-links and the resulting loss of functional MET channels is what stops the renewal of the short 

and middle rows of stereocilia in these mutant mice. We can conclude that the tip-link plays an 

unsuspected, direct or indirect role on the undergoing actin polymerization processes in the 

different stereocilia rows, which ensures the development and the dynamic maintenance of the 

hair bundle’s staircase pattern. 

2234 

Mutual regulations of actin cytoskelton remodeling and peroxisome proliferator-activated 

receptor-γ on early adipocyte differentiation. 

H. Nobusue 1 , N. Onishi 2 , Y. Oki 1 , T. Shimizu 2 , T. Chiyoda 2 , H. Saya 2 , K. Kano 1 ; 1 Laboratory of 

Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, Fujisawa, Japan, 

2 Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School 

of Medicine, Shinjuku-ku, Japan 

Adipocyte differentiation at an early stage is directly regulated by gene expression of a master 

regulator such as peroxisome proliferator-activated receptor-γ (PPARγ). During adipogenesis 

actin cytoskelton remodeling defines important events of the differentiation process; this is 

characterized by the conversion of filamentous actin from stress fibers to cortical actin 

structures. Here, we examined the regulatory relationship between actin cytoskelton remodeling 

and PPARγ in early adipocyte differentiation. Depending on adipogenic induction, actin stress 

fibers were immediately disrupted prior to the expression of PPARγ. And treatment with 

cytoskeletal fixation agent phalloidin maintained actin fiber structures even after adipogenic 

induction, and caused a down-regulation of PPARγ. In addition, ectopic expression of activated 

RhoA, which is known to promote the formation of actin stress fibers, inhibited the disruption of 

actin stress fibers and PPARγ expression after adipogenic induction, and these effects were 

recovered by treatment with the RhoA kinase inhibitor Y-27632. Moreover, treatment of the actin 

polymerization inhibitor cytochalasin D in active RhoA-expressing cells caused the disruption of 

actin stress fibers, and as a result, PPARγ expression was rescued. On the other hand, 

introduction of the PPARγ-specific shRNA resulted in maintained the high-level expression of 

fibronectin (FN) matrix and inhibited the reorganization into adipocyte-specific cortical actin 

structures during adipocyte differentiation. And transfection of FN-specific siRNA in PPARγknockdown 

cells after adipogenic induction caused the formation of cortical actin structures. In 

conclusion, our findings indicate that actin stress fiber disruption directly induces PPARγ 

expression, and that PPARγ regulates the reorganization into adipocyte-specific cortical actin 

structures through the down-regulation of FN matrix, and provide a novel insight into the mutual 

regulatory mechanisms between actin cytoskelton remodeling and adipogenic transcription 

factors in early adipocyte differentiation. 

2235 

The Carboxy Termini of Human Myopodin Isoforms Influences Non-Muscle Myosin II- 

Dependent Myopodin Subcellular Localization. 

F. Kai 1 , R. Duncan 1,2 ; 1 Department of Microbiology, Dalhousie University, Halifax, NS, Canada, 

2 Department of Biochemistry & Molecular Biology and Department of Pediatrics, Dalhousie 

University, Halifax, NS, Canada 

More than 80% of patients with invasive prostate cancer have deletions within their myopodin 

gene, suggesting that myopodin is a reliable predictive marker of prostate cancer metastasis. 

However, the functions of myopodin in cancer metastasis remain unknown. Biochemical studies

TUESDAY 

have revealed that myopodin is an actin-binding protein. Since actin rearrangement is an 

essential process involved in cell migration, it has been postulated that myopodin might 

suppress cancer metastasis by modulating the actin cytoskeleton. Presently, five human 

myopodin (hMYO) splicing variants, each with a unique amino and/or carboxy-termini, have 

been identified. However, it is unclear whether the unique sequence of each isoform 

differentially regulates myopodin function. To examine if these splicing variants function 

distinctly, we ectopically expressed myopodin isoforms in invasive prostate cancer cells (PC3) 

and benign prostatic hyperplasia cells (BPH-1). Immunofluorescent staining revealed that the 

different myopodin isoforms induce distinct actin structures within the cell body. Each isoform 

interacts distinctly with these actin structures, but none of the isoforms colocalize with actin near 

the cell periphery. Truncation analysis indicated the unique carboxy-terminal sequences 

regulate the distinct localization of myopodin isoforms with the actin cytoskeleton. Furthermore, 

the restrictive subcellular localization of some myopodin isoforms was abrogated by inhibitors 

that affect downstream effectors in the activated RhoA pathway (i.e. the ROCK inhibitor Y- 

27632 and the non-muscle myosin II inhibitor blebbistatin). Taken together, these results 

indicate the carboxy-termini of the human myopodin isoforms regulate their ability to 

differentially alter and co-localize with actin cytoskeletal structures in a non-muscle myosin IIdependent 

manner. 

FuiBoon Kai is supported by a trainee award from The Beatrice Hunter Cancer Research 

Institute with funds provided by The Terry Fox Foundation Strategic Health Research Training 

Program in Cancer Research at the Canadian Institutes of Health Research. 

2236 

Surface-bound VASP controls Arp2/3 complex-dependent actin gel growth. 

P. Noguera 1 , A. Lamora 1 , J. Plastino 1 ; 1 Physico-chimie Curie (PCC) - UMR 168, Institut Curie, 

Paris, France 

The Drosophila Enabled/Vasodilator Stimulated Phosphoprotein (Ena/VASP) protein family is 

involved in numerous actin based processes such as filopodia formation and lamellipodia 

protrusion, but the precise function and mode of action of Ena/VASP proteins remain 

controversial. To address this question, we use an in vitro assay composed of beads coated 

with actin polymerization activators, incubated in a protein mix that supports the growth of an 

actin gel on the bead surface. Over time, the symmetrical gel breaks open to form a comet tail 

that propels the bead forward, mimicking in simplified conditions the dynamics of the actin 

cytoskeleton at the plasma membrane of moving cells. Here we use beads that activate the 

actin-related protein (Arp) 2/3 complex, an actin polymerization nucleator, and additionally 

recruit wild-type and mutant forms of murine VASP to the bead surface. We show that VASP 

increases the rate of actin comet growth. We further show that this activity requires the F-actin 

binding capacity of VASP, its tetramerization domain and its profilin recruiting site, but that the 

G-actin binding site is dispensable. When VASP is not targeted to the bead surface, it has no 

effect on bead speed, despite the fact that it is present throughout the actin comet. In another 

series of experiments, we provide evidence that VASP focuses polymerization near the bead 

surface in the absence of capping protein via its F-actin binding capacity. Overall these results 

shed light on the molecular mechanism of Ena/VASP proteins, and on how these proteins enter 

into the actin dynamics equation in vivo.

TUESDAY 

2237 

Rho-GTPase mediated regulation and membrane targeting of human leukocyte formins 

FMNL1 and FMNL2. 

S. Kuehn 1 , J. Block 2 , K. Rottner 2 , M. Geyer 1 ; 1 Department of Physical Biochemistry, Max Planck 

Institute of Molecular Physiology, Dortmund, Germany, 2 Institute of Genetics, University of 

Bonn, Bonn, Germany 

Dynamic actin filament structures required for cell motility and migration are regulated by 

nucleation and elongation factors, such as members of the formin family. The large, 

multidomain proteins FMNL1 and FMNL2 belong to the subgroup of Diaphanous-related formins 

(DRFs). The FH2 domain-mediated actin assembly activity of DRFs is inhibited by an 

intramolecular interaction between the N-terminal regulatory region and the C-terminal DAD 

domain. This inhibition is released via binding of Rho-GTPases to the GBD and FH3 domain. 

Additional cofactors might be required for full activation, localization or membrane targeting. 

We here show that human FMNL2N interacts specifically with GppNHp-bound Cdc42 in the 

micromolar affinity range. Cdc42 binds to FMNL2N via switch I, switch II as well as the insert 

helix that is unique for Rho-GTPases. In FMNL2, binding to Cdc42 involves the GBD and all 

armadillo repeats of the FH3 domain. The structure thus provides for the first time an effectorspecific 

binding of the Rho-specific insert helix. We could determine crucial residues, which can, 

if mutated, switch the specificity of FMNL2 for Cdc42 to Rac1. We identified a basic insert that 

disrupts the canonical FH3 repeat fold. This insert binds acidic phospholipids and negatively 

charged liposomes in vitro, suggesting plasma membrane association through electrostatic 

interactions. Additionally, FMNL2 is N-terminally myristoylated, a fatty acid modification that 

drives membrane binding. Together with the C-terminal prenylation of the GTPase, a large 

membrane binding surface of the dimeric formin complex assembles that sustains membrane 

association during migration. In vivo experiments reveal that both, the basic insert and the 

myristoyl moiety, are responsible for plasma membrane targeting of the actin polymerization 

factor. 

2238 

Watching the motions of the cell wall synthesis machinery and underlying cytoskeleton 

in B. subtilis with high precision particle tracking. 

E. C. Garner 1 , X. Zhuang 2 , W. Wang 3 , R. Bernard 4 , D. Rudner 4 , T. MItchison 5 ; 1 Harvard 

University, Cambridge, MA, 2 Chemistry and Chemical biology, 3 Physics, Harvard University, 

Cambridge, MA, 4 Microbiology, Harvard University, Cambridge, MA, 5 Systems Biology, Harvard 

University, Cambridge, MA 

Rod-shaped bacteria elongate by the action of cell-wall synthesis complexes linked to 

underlying dynamic MreB filaments, but how these proteins function to allow continued 

elongation as a rod remains unknown. To understand how the movement of these filaments 

relates to cell wall synthesis, we characterized the dynamics of MreB and the cell wall 

elongation machinery using high-resolution particle tracking in Bacillus subtilis. We found that 

both MreB and the elongation machinery move in linear paths across the cell, moving at similar 

rates (~20nm / second) and angles to the cell body, suggesting they function as single 

complexes. These proteins move circumferentially around the cell, principally perpendicular to 

its length. We find that the motions of these complexes are independent, as they can pause and 

reverse, and also as nearby complexes move independently in both directions across one 

surface of the cell. Inhibition of cell wall synthesis with antibiotics or depletions in the cell wall 

synthesis machinery blocked MreB movement, suggesting that the cell wall synthetic machinery 

is the motor in this system. We propose that bacteria elongate by the uncoordinated,

TUESDAY 

circumferential movements of synthetic complexes that span the plasma membrane and insert 

radial hoops of new peptidoglycan during their transit. 

2239 WITHDRAWN 

2240 

Computational models of embryonic wound healing. 

M. A. Wyczalkowski 1 , V. D. Varner 1 , L. A. Taber 1 ; 1 Biomedical Engineering, Washington 

University in St. Louis, St. Louis, MO 

The objective of this work is to characterize experimentally the healing of circular and slash 

wounds in epithelia of early stage chick embryos and to develop a finite element model of 

wound healing dynamics. 

Early stage chick embryos were harvested and cultured ex ovo. Circular and slash wounds 

(~100um in size) were made in the blastoderm and allowed to heal. The healing process was 

captured with time lapse microscopy, and wound area and shape were characterized as 

functions of time. The contractile response of the tissue to wounding was implemented in a finite 

element model, and the results were critically compared to experimental results for both wound 

types. 

The embryonic wounds displayed a two-phase behavior: rapid wound healing lasting about a 

minute, followed by a more gradual healing to full closure within tens of minutes. This behavior, 

as well as more complicated wound geometry trends (e.g., aspect ratio), were reproduced by 

the model in which 1) wounding induces a rapid isotropic contraction of the tissue surrounding 

the wound followed by 2) a slower formation and contraction of a relatively thin fiber ring 

bordering the wound edge. 

The computational model is able to capture the non-trivial healing dynamics of wounds in 

embryonic epithelia, lending support to a hypothesis where wound healing is governed by two 

distinct cellular mechanisms. 

2241 

Nuclear protein MARVELD1 regulates activity of Rho GTPases and inhibits cell adhesion 

and cell motility. 

S. Wang 1 , J. Hu 1 , F. Han 1 , M. Shi 1 , J. He 1 , S. Liu 1 , L. Yue 1 , Y. Li 1 ; 1 Harbin Institute of 

Technology, Harbin, China 

A number of cytoplasmic factors regulate cell adhesion on an extracellular matrix (ECM), which 

plays a crucial role in cell motility, invasion, cell signal transduction and gene expression. 

Activity of Rho GTPase regulates F-actin dynamics. The focal adhesions and associated F-actin 

structures reorganization led to the alteration of focal adhesion anchored actin stress fibers. 

However, its regulation via nuclear proteins remains obscure. In this study, we identified a 

nuclear protein, MARVELD1 that could suppress cell spreading and movement. Overexpression 

of MARVELD1 in A549 cells decreased the expression level of integrin β1 and 

vinculin, and further led to dephosphorylation of FAK at Tyr 397. Also, knockdown of 

MARVELD1 in HeLa cells and H520 cells could upregulate integin β1 and vinculin, and promote 

FAK Tyr397 phosphoylation. Moreover, we found that activity of Cdc42/Rac1/RhoA was 

regulated. And MARVELD1 partially colocalized with SC35 and interacted with nuclear cap 

binding protein subunit 2, CBP20. In addition, we explored that pre-mRNA processing of several 

focal adhesion proteins, including vinculin and actinin, was affected by MARVELD1.

TUESDAY 

Our studies demonstrate that the nuclear protein MARVELD1 plays a role in pre-mRNA 

processing of some important FAC genes and regulates activity of Rho GTPases, and thereby 

inhibits cell adhesion and cell motility. These studies provide a novel regulatory mechanism of 

cell-ECM adhesion by nuclear protein in tumor cells. 

Molecular Motors 

2242 

The Tail region inhibits the motor activity of human myosin VIIA at molecular and cellular 

level. 

T. Sakai 1 , R. Ikebe 1 , M. Ikebe 1 ; 1 Microbiology and Physiological Systems, University of 

Massachusetts Medical school, Worcester, MA 

Myosin VIIA is a responsible gene for human Usher Syndrome type 1B, which causes hearing 

and visual loss, and plays a role in human auditory function. We previously reported that 

Drosophila myosin VIIA is a monomer and the tail domain inhibits the actin activated ATPase 

activity under low Ca2+ condition. However, it is unknown whether the tail-inhibition mechanism 

is operating in cells. Another critical issue is whether or not mammalian myosin VIIA is regulated 

by similar mechanism since it has been known that regulation mechanism of myosin is often 

quite different between vertebrate myosin and its non-vertebrate counterpart. To address these 

questions, we studied the regulation mechanism of human myosin VIIA (HuM7A) at both 

molecular and cellular level. 

We studied the inhibitory function of the tail by examining the actin-activated ATPase activity of 

HuM7A. The C-terminal deletion (-40 amino acids) of the full-length HuM7A significantly 

increased the ATPase activity. This result suggested that the C-terminal end of tail region 

inhibits the motor activity. The result is similar to the study of Drosophila myosin VIIA, 

suggesting that tail-inhibition mechanism is also operating in HuM7A. 

Our earlier study showed that the dimer formation of GFP-HuM7A HMM or GFP-HuM7A fulllength 

induces the filopodia tip localization of human myosin VIIA in cells, suggesting that 

HuM7A moves along actin bundles in filopodia to the tips. Using this system, we examined the 

inhibitory activity of HuM7tail in cells. We co-expressed the tail with the forced-dimer of HuM7A 

HMM, and studied the effect of the tail on HuM7AHMM translocation to the filopodial tips. 

Expression of the entire tail significantly inhibited the translocation of HuM7A HMM to the 

filopodial tips. We also found that translocation of the forced-dimer of full-length HuM7A is 

significantly attenated and this was facilitated by deletion of the C-terminal 40 amino acids. 

These results are consistent with the tail-inhibition of the ATPase activity in vitro. Present results 

suggested that the tail-induced regulatory mechanism is common between vertebrate and nonvertebrate 

myosin VIIA and is operating in vivo. 

2243 

Myosin-Va is implicated in cell adhesion and spreading. 

A. Ramao 1 , G. P. Nader 1,2 , E. M. Espreafico 1 ; 1 Cellular and Molecular Biology, FMRP-USP, 

Ribeirão Preto, Brazil, 2 Pathology and Cell Biology, Columbia University, New York, NY 

The main objective of this work was to assess the role of myosin-Va in cell adhesion and 

spreading. Here we show that MYO5A mutant melanoma cells exhibit deficient adhesion and 

spreading abilities, and lack the usual organization of the actin cytoskeleton. Myosin-Va 

expression in these cells restored actin bundles, induced lamelipodial/filopodial growth and led 

to redistribution of focal adhesions towards the cell cortex. It is well known that depolymerization 

of microtubules in quiescent fibroblasts leads to an intense formation of focal adhesions

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followed by dissociation of these structures after microtubule repolymerization. To investigate a 

possible role for myosin-Va in the formation step or dissociation of focal adhesions, human 

fibroblasts from a healthy individual (control) or from a patient with deleterious mutation in the 

MYO5A gene were left 48 hours in quiescence and treated with nocodazole for 4 hours. 

Confocal microscopy analysis of treated cells stained for the focal adhesion marker, talin, 

showed no differences between mutant and wild-type cells in the ability of focal adhesion 

formation nor in the disassemble of focal adhesions observed concomitantly to microtubule 

regrowth, 60 minutes after washing out the nocodazole. The same assay was performed with 

myosin-Va knocked-down WM793 human melanoma cells. Knock-down was mediated by a 

lentiviral vector carrying a short-hairpin RNA targeted to MYO5A mRNA (shMYO5A) or, as 

control, an irrelevant shRNA against bacterial LacZ. We observed that, after treatment with 

nocodazole, lamelipodia from WM793-shLacZ cells had numerous peripheral focal adhesions, 

as expected, whereas most of myosin-Va knocked-down cells rather showed ruffled edges and 

lacked focal adhesions. These data suggest that myosin-Va is required for formation of focal 

adhesions in melanocytic cells, but not in fibroblasts. Our laboratory has previously showed that 

the MYO5A-null fibroblasts used here overexpress myosin-Vb and Vc. Therefore, future studies 

should be conducted to answer whether in fibroblasts the formation of focal adhesions is 

compensated by one of the other two myosin-V paralogs, and also to uncover the mechanisms 

by which myosin-V functions in focal adhesion formation in melanocytic cells. 

Financial support: FAPESP, CNPq, CAPES, FAEPA 

2244 

Characterization of the kinesin KIF9 in mammalian cell cycle progression. 

J. E. Hoke 1 , M. E. Rivera 2 , A. M. Billow 1 , L. Alsina 1 , N. J. Quintyne 1 ; 1 Harriet L. Wilkes Honors 

College, Florida Atlantic University, Jupiter, FL, 2 Florida Atlantic University-Honors Coll, Jupiter, 

FL 

The kinesin family of microtubule motors is divided into subfamilies based upon structure and 

function. KIF9 is the founding member of the Kinesin-9 family, a largely uncharacterized group 

of kinesins. It was originally identified by sequence homology to other kinesins and shown to 

interact with the Ras-like GTPase Gem (Piddini et al., 2001). Subsequent studies have indicated 

that KIF9 is vital for flagellar movement in Trypanasoma brucei, likely via interaction with dynein 

(Demonchy et al., 2009) as well as podosome regulation (Cornfine et al., 2011). Additionally, 

Kinesin-9 family members have also been proposed to function in proper microtubule organizing 

center (MTOC) positioning and timely mitotic entry. We have examined KIF9 function in 

mammalian cells by using siRNA-mediated knockdown and overexpression. Our analysis has 

focused on normal progression through S phase and subsequent transition to, and progression 

through, mitosis. We have seen several effects on cell cycle progression. First, a transfected, 

synchronized population of cells shows a notable delay in mitotic entry. Second, there is a delay 

in normal S phase progression. Third, we see that there is a change in mitotic index and 

decrease in the number of anaphase and telophase cells. Finally, we see an increase in the rate 

of multinuclearity, a hallmark of failure of cytokinesis. Taken together, we propose that KIF9 is 

required for normal entry and completion of mitosis, possibly via regulation of the contractile 

ring.

2245 

Kinesin-5 promotes disassembly of long kinetochore microtubules. 

E. S. Tubman 1 , A. Claas 1 , D. J. Odde 1 ; 1 Biomedical Engineering, University of Minnesota, 


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Kinesin-5 is a plus end directed, homotetrameric motor protein that is well known for its role in 

mitosis. It is widely accepted that the motor protein crosslinks antiparallel microtubules, where it 

walks towards the plus end of each, sliding them apart and creating a bipolar spindle. 

Additionally, kinesin-5 in budding yeast plays a role in maintaining a spatial gradient of 

kinetochores (protein complexes that link chromosomes to dynamically assembling microtubule 

plus ends) along the metaphase spindle. Kinesin-5 establishes this gradient by promoting 

disassembly of long kinetochore microtubules. We are interested in determining if kinesin-5 also 

controls the spatial gradient of kinetochore microtubule assembly in other organisms. To test 

this hypothesis we used the infectious yeast Candida Albicans imaged by confocal fluorescence 

microscopy to measure the fluorescence distribution of the tubulin in control cells and mutant 

cells, where one of the two copies of the KIP1 gene (kinesin-5 in Candida) was knocked out. In 

control cells, a bilobed fluorescence distribution indicated that the ends of the kinetochore 

microtubules are drawn to an attractor zone located between the spindle pole and the spindle 

equator, consistent with the distribution in budding yeast. In mutant cells, we found a 

homogeneous fluorescence distribution, indicating a loss of the attractor zone. Our initial results 

here suggest that kinesin-5 acts as a kinetochore microtubule length-dependent disassembly 

promoter in C. Alibcans to mediate the proper alignment of chromosomes across the spindle 

equator during metaphase. 

2246 

Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1. 

J. Xu 1 , B. Reddy 2 , P. Anand 2 , Z. Shu 2 , S. Cermelli 2 , M. Mattson 2 , S. Tripathy 2 , M. Hoss 2 , N. 

James 2 , S. King 3 , L. Huang 2 , L. Bardwell 2 , S. Gross 2 ; 1 University of California, Merced, Merced, 

CA, 2 University of California, Irvine, Irvine, CA, 3 University of Central Florida, Orlando, FL 

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are 

linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a direct head-tail 

interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation 

of kinesin, reversible by the disease-relevant signaling protein, casein kinase 2 (CK2). The 

majority of initially active kinesin (native or tail-less) loses its ability to bind/interact with 

microtubules in vitro, and CK2 reverses this inactivation (~ 4-fold) without altering kinesin’s 

single motor properties. This activation pathway does not require motor phosphorylation, and is 

independent of head-tail autoinhibition. In cultured mammalian cells, reducing CK2 expression, 

but not kinase activity, decreases the force required to stall lipid droplet transport, consistent 

with a reduction in the number of active motors. These results provide the first direct evidence of 

a protein kinase up-regulating kinesin-based transport, and suggest a novel pathway for 

regulating the activity of cargo-bound kinesin. 

2247 

Protein-Protein Interactions within Dynactin’s Shoulder/Sidearm. 

S. A. Ketcham 1 , T. A. Schroer 1 ; 1 Biology, Johns Hopkins University, Baltimore, MD 

Dynactin is a multi-subunit complex that acts as a cargo adaptor and processivity enhancer for 

the microtubule-based motors dynein and kinesin-2. Dynactin interacts with these motors 

through the p150Glued subunit, which along with dynamitin and p24 comprise the 

shoulder/sidearm. Disruption of dynactin with the chaotropic salt KI yields two subcomplexes,

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the shoulder, which contains p24 and dynamitin, and the shoulder/sidearm, which contains p24, 

dynamitin and p150Glued. Although the shoulder/sidearm is released from dynactin as a stable 

subcomplex, the protein-protein interactions that determine its structure remain undefined. Our 

current work suggests that p24 serves as a bridge between dynamitin and p150Glued. 

Recombinant p24 is insoluble, but solubility can be restored by stepwise dialysis out of 

denaturant in the presence of either dynamitin or a fragment of p150Glued. Without either 

binding partner, p24 remains insoluble when subjected to this procedure. We have taken 

advantage of the necessity of a binding partner for p24 solubility to explore p24 structure. 

Renaturation of a series of p24 fragments with dynamitin or p150Glued, coupled with gel 

filtration and velocity sedimentation to analyze the complexes that form, has allowed us to map 

the domains of p24 necessary for its interactions with its binding partners. These results are 

beginning to yield a better understanding of the structure of the shoulder/sidearm and will be 

exploited to gain insight into how the shoulder/sidearm anchors to the Arp1 minifilament. 

Cell Division 

2248 

Cellular Size Checkpoints During G1/S- and G2/M-Transition Mediate Contact Inhibition 

Of Proliferation. 

S. J. Streichan 1 , C. R. Hoerner 1 , D. Holzer 1 , T. Schneidt 1 , L. Hufnagel 1 ; 1 Cell Biology & 

Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany 

Tissue and organ development is characterized by tightly controlled patterns of coordinated cell 

growth, cell division, and cell rearrangements. Non-cancerous epithelial cells in culture show a 

similar behavior in terms of regulated proliferation: initially, they proliferate at high rate and 

compensate for their reduced size after cell division by cell growth. Then, after reaching 

confluence cells undergo size-reductive divisions and the rate of cell division drops markedly, a 

phenomenon called contact inhibition of proliferation (CI). However, it remains elusive how cells 

coordinate growth and division and how during CI the cell cycle of individual cells is regulated 

according to the cell density in the cell collective. 

Here we use fluorescent time-lapse live cell imaging, quantitative image analysis, and 

biophysical modeling to study the link between cell growth and cell proliferation in Madin Darby 

canine kidney (MDCK) cells. Based on the predictions of our physical description we perform 

biophysical tests including active and passive manipulation of cell size. First, we find that cells 

have a critical size when entering S phase. Second, reduced cell size after reentry into the cell 

cycle results in prolonged G2 phase. Third, direct manipulation of cell size by stretching of a 

contact-inhibited cell collective is sufficient to reverse CI as cells reenter the cell cycle and 

progress through mitosis. 

We suggest that mechanical constraints in tissues may delay cell cycle reentry and progression 

by activating size checkpoints acting during the G1/S- and G2/M-transition. 

2249 

Concentration-dependent Correlation of Cell Cycle and Apoptosis Impacts of Anti-Cancer 

Compounds. 

K. Tran 1 , K. Gillis 1 , A. Khan 1 , J. Clor 1 , K. Tyagarajan 1 ; 1 EMD Millipore, Millipore Corporation, 

Hayward, CA 

Recent research has demonstrated the delicate relationship between the opposing processes of 

cell cycle progression and apoptosis. Several anti-cancer compounds have been demonstrated 

to cause apoptosis and subsequently have been shown to cause arrests in specific phases of

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the cell cycle. The study of cell cycle impacts in addition to apoptosis can thus bring meaningful 

understanding of the diverse effects of cellular modulators and compound treatment and provide 

deeper understanding of the inter-relationship between cell cycle and apoptosis. In this study, 

we evaluated a series of anti-cancer compounds with known apoptotic activity to determine the 

sequence of impact on cell cycle versus apoptosis induction for multiple cell types. Jurkat and 

HeLa cells were treated with multiple concentrations of compounds and the impact on cell cycle 

and apoptosis (based on Annexin V binding) on different dosages of treatment were evaluated. 

Our studies demonstrate that multiple compounds could modulate different cell cycle phases 

depending on the concentration and cell type used. Further the sequence of impacts on cell 

cycle and apoptosis could be very different. Our results here demonstrate that treatment with 

Etoposide, a DNA topoisomerase II inhibitor at low concentrations results in the arrest of cells in 

the G2M stage under conditions where little apoptosis is observed. With increased 

concentration of etoposide, increase in the % of cells in the S phase of cell cycle is observed 

along with increased apoptosis. Thus for etoposide, cell cycle impacts were observed at 

significantly lower concentration than impacts on apoptosis. Staurosporine demonstrated both 

an arrest in G2/M phase with concurrent impacts on apoptosis at low concentrations, higher 

concentrations showed increase in the percentage of cells in the G0/G1 phase. Gambogic acid, 

a potential anti-cancer agent resulted in significant apoptosis and death but no impacts on cell 

cycle were observed. Nocodazole demonstrated impacts on cell cycle and apoptosis 

concurrently. Parallel analysis of apoptosis data along with cell cycle effects will be critical for 

understanding the linkage between these processes and provide for a more complete 

understanding of impacts of modulators of cell cycle and cell death and the design of effective 

cellular study models. 

2250 

Phospho-regulation of the anillin-related scaffolding protein Mid1 in fission yeast 

cytokinesis. 

A. K. DeWitt 1 , E. Kranz 1 , J. Phelan 1 , B. Nader 1 , K. L. Gould 2 , D. M. Clifford Hart 1 ; 1 Cell and 

Molecular Biology, Grand Valley State University, Allendale, MI, 2 Department of Cell and 

Developmental Biology, Howard Hughes Medical Institute & Vanderbilt University School of 

Medicine, Nashville, TN 

During cytokinesis, physical separation of one cell into two identical cells occurs through 

constriction of a protein-rich ring structure, called the contractile ring. The contractile ring is 

composed of F-actin, type II myosin and more than 100 cytokinetic ring proteins. In fission 

yeast, the anillin-related protein Mid1 plays a critical role in organizing the early steps of 

contractile ring formation and functions as a scaffold to bridge the cell cortex with the contractile 

ring. Cells lacking mid1 form off-centered, highly disorganized ring structures and exhibit severe 

cytokinesis defects. Coincident with its cortical accumulation, Mid1 becomes hyperphosphorylated. 

Our previous research demonstrates that cyclin-dependent kinase, Cdc2, and 

the polo-like kinase, Plo1, directly phosphorylate Mid1. In addition to consensus phosphorylation 

motifs for Plo1 and Cdc2, Mid1 contains several RXXS motifs, which fit the phosphorylation 

consensus sequence for Sid2 kinase. Sid2 is the most downstream kinase in the Septation 

Initiation Network signaling cascade, which signals from the spindle pole body to trigger 

constriction of the contractile ring. To identify specific sites of phosphorylation, a combination of 

site-directed mutagenesis (serine to non-phosphorylatable alanine) with mass spectroscopy and 

two-dimensional phosphopeptide mapping were used. Both methods identified multiple Sid2 

phosphorylation sites within Mid1. To analyze the significance of Sid2 phosphorylation in vivo, 

phospho-site mutants were generated at the endogenous mid1 locus and examined for cell 

division defects. Interestingly, phospho-site mutants displayed cell division defects, including 

sensitivity to low dose latrunculin A and disorganized actin localization. While phospho-site

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mutants maintained the mitotic spindle checkpoint, the majority of cells exhibited severe polarity 

phenotypes and Mid1 protein levels increased when compared to checkpoint activated cells 

expressing wild-type Mid1. Given that Mid1 departure from the contractile ring coincides with 

Sid2 relocalization to the division site, Sid2 may temporally regulate the interaction of Mid1 with 

the membrane or other contractile ring components; our initial experiments favor Sid2 regulating 

Mid1 dissociation from the cell cortex. 

2251 

Regulation of Myosin-II Dynamics and its Implication in the Assembly of the Cytokinesis 

Machine in S. cerevisiae. 

C. Wloka 1,2 , E. Bi 1 ; 1 Cell and Developmental Biology, University of Pennsylvania, Philadelphia, 

PA, 2 Institut für Chemie und Biochemie, Freie Universität Berlin, Berlin, Germany 

Cytokinesis in animal and fungal cells utilizes a contractile actomyosin ring (AMR) to power the 

ingression of the cleavage furrow. The AMR consists of myosin-II and actin filaments. However, 

how the AMR is assembled and disassembled during the cell cycle is not clearly understood in 

any organism. We have analyzed the dynamics of 13 cytokinesis proteins during the cell cycle, 

including the sole myosin-II, Myo1, in the budding yeast Saccharomyces cerevisiae by 

fluorescence recovery after photo-bleaching. We found that Myo1 is dynamic until the onset of 

anaphase when it becomes progressively immobile. Further analysis indicates that the 

immobility of Myo1 during cytokinesis does not require its motor domain and actin filaments but 

requires the putative assembly domain near its C-terminus, which regulates Myo1 localization 

and cytokinesis. Thus, the putative higher-order assembly is important for Myo1 localization, 

dynamics, and its role in cytokinesis. These data also indicate that the AMR is progressively 

disassembled during its constriction without high turnover. 

We also found that proteins involved in AMR assembly and septum formation, such as Iqg1 

(IQGAP), Hof1 (a F-BAR protein), and Inn1 are immobile whereas those involved in actin ring 

assembly (formin and tropomyosin) are highly dynamic during cytokinesis. Strikingly, the 

immobility of all examined proteins depends on the presence of Myo1. This and the observation 

that Myo1 is required for actin ring assembly suggest that Myo1 plays a scaffolding role in the 

assembly of the cytokinesis machine. 

2252 

Orbit/CLASP plays a role as linker connecting cortical microtubules and actomyosin ring 

in Drosophila male meiosis. 

D. Kitazawa 1 , D. Hayashi 1 , C. Miyauchi 1 , Y. Inoue 1 ; 1 Kyoto Institute of Technology, Insect 

Biomedical Research Center, Kyoto, Japan 

Microtubules (MTs) near the cell cortex are essential for continuous constriction of the 

contractile ring in cytokinesis. However, the way in which MTs interact with the contractile 

apparatus has not been completely unveiled. In previous studies, we have shown that Orbit is 

required for stabilization of the MT populations essential for cytokinesis. Therefore, it is possible 

to speculate that Orbit plays an important role in cleavage furrow (CF) initiation in Drosophila. A 

body of evidence indicating that Orbit possesses a binding activity to both MTs and F-Actin in 

Drosophila and Mammalian cells implies another role in initiation of cytokinesis. In this study, we 

carefully examined localization of GFP-tagged Orbit in Drosophila male meiosis. We showed 

that Orbit changes its localization from around the spindle matrix towards prospective CF 

regions at late anaphase. It continuously stayed on the ring in the middle of the cell. Although 

formation of the Orbit ring depends on both MTs and F-Actin, Orbit maintains its localization on 

the CF even in the absence of Myosin II. We showed that Orbit directly binds to F-Actin and the 

heavy chain of Myosin II on the CF. Surprisingly, ectopic localization of Orbit on the cortex

TUESDAY 

induces contractile ring formation in situ and it was followed by CF ingression. Accumulation of 

Orbit on the CF is dependent on MTs and F-Actin. In order to look for transport proteins 

conveying the Orbit toward the CF, we then performed knockdown experiments to examine 

whether all known MT-based motors and proteins displaying a genetic interaction with Orbit are 

required for cytokinesis. Among several genes that we have shown are critical for cytokinesis, 

we especially focused on CLIP190 and its relationship with Orbit. We found that CLIP190 is 

required for transport of Orbit to the prospective CF site independent of the MT binding domain 

of Orbit. Our current data suggests that Orbit plays a central role in contractile ring formation as 

a linker connecting MTs with Actomyosin ring. 

2253 

Fission yeast cyclin-dependent kinase, Cdc2, regulates the Mid1 scaffolding protein 

during cytokinesis. 

J. B. Hackett 1 , J. Phelan 1 , K. L. Gould 2 , D. M. Clifford Hart 1 ; 1 Cell and Molecular Biology, Grand 

Valley State University, Allendale, MI, 2 Howard Hughes Medical Institute, Department of Cell 

and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 

Cytokinesis is the final stage of cell division and utilizes a highly conserved actin-myosin 

contractile ring that forms medially around the cell to pinch the cell membranes together 

resulting in physical separation of one cell into two. The conserved anillin homolog, Mid1, is a 

foundational part of the fission yeast contractile ring, functioning as the initial organizer and anchoring 

the structure to the cell membrane. Mid1 mutants show severe defects in contractile ring assembly and 

medial placement of the division site. Coincident with contractile ring formation, Mid1 is hyperphosphorylated 

and our previous research identified at least two mitotic kinases that directly 

phosphorylate Mid1. Mid1 phosphorylation by the cyclin-dependent kinase Cdc2 at Threonine 517 

facilitates association between the polo-like kinase, Plo1, and the C-terminus of Mid1. To identify 

additional Cdc2 phosphorylation sites within Mid1, we implemented a combination of mutagenesis and 

two-dimensional phosphopeptide mapping. Mutation of Serine 28 to a non-phosphorylatable alanine 

residue reduced phosphorylation of Mid1 N-terminus and this site fits a consensus motif for Plo1 

binding. Fission yeast cells expressing Mid1 phospho-site mutations show sensitivity to low dose 

latrunculin A treatment and bypass the mitotic spindle checkpoint. Surprisingly, phosphosite mutants 

with an activated mitotic spindle checkpoint continue through mitosis but accumulate as septated cells. 

This research highlights a regulatory role for Plo1 and Mid1 in early and late stages of cytokinesis. 

2254 

Centrosomal Protein 55 Stability Is Negatively Regulated BY P53 Through POLO-Like 

Kinase 1. 

Y-C. Chang 1,2 , P. Ouyang 3,4 ; 1 Graduate Institute of Clinical Medicine Sciences, Chang Gung 

University, Guei-San, Taiwan, 2 Nuclear Medicine and Molecular Imaging Center, Chang Gung 

Memorial Hospital, Guei-San, Taiwan, 3 Anatomy, Chang Gung University, Guei-Shan, Taiwan, 

4 Molecular Medicine Research Center, Chang Gung University, Guei-San, Taiwan 

Centrosomal protein 55 (Cep55), which is localized to the centrosome in interphase cells and 

recruited to the midbody during cytokinesis, is a regulator required for the completion of cell 

abscission. Upregulation of Cep55 and inactivation of p53 occur in the majority of human 

cancers, raising the possibility of a link between these two genes. In this study, we evaluated 

the role of p53 in Cep55 regulation. We demonstrated that Cep55 expression levels are well 

correlated with cancer cell growth rate, and that p53 is able to negatively regulate Cep55 protein 

and promoter activity. Downregulation of expression of Cep55 was accompanied by repression 

of polo-like kinase 1 (Plk1) levels due to p53 induction. Overexpression of Plk1 and knockdown

TUESDAY 

of p53 expression both enhanced the post-translational protein stability of Cep55. BI 2356, a 

selective Plk1 inhibitor, however, prevented Cep55 accumulation in p53 knockdown cells, while 

persistently keeping Plk1 levels elevated. Our results, therefore, indicate the existence of a p53- 

Plk1-Cep55 axis in which p53 negatively regulates expression of Cep55 through Plk1 which, in 

turn, is a positive regulator of Cep55 protein stability. 

2255 

Cleavage furrow organization requires PIP2 mediated recruitment of anillin. 

J. Liu 1 , D. Ceccareli 2 , G. Fairn 3 , F. Sicheri 1,2 , A. Wilde 1 ; 1 Molecular Genetics, University of 

Toronto, Toronto, ON, Canada, 2 SLRI, Toronto, Canada, 3 Hospital for Sick Children, Toronto, 

ON, Canada 

Anillin is a key component of the cytokinetic machinery that is essential for the successful 

completion of cytokinesis. Anillin is a multi-domain protein that has the potential to link the 

actomyosin and septin cytoskeletons. It also has a PH domain involved in targeting anillin to the 

cleavage furrow. We sought to examine the mechanism by which the PH domain targets anillin 

to the cleavage furrow. We find that the PH domain of anillin interacts with phosphatidylinositol 

phosphate lipids (PIPs), including PI(4,5)P2, which is enriched in the furrow. Reduction of 

cellular PI(4,5)P2 or mutations in the PH domain of anillin that specifically disrupt the interaction 

with PI(4,5)P2, interfere with the localization of anillin to the furrow. Reduced expression of 

anillin disrupts symmetric furrow ingression in the middle of the cell. However, symmetrical 

furrow ingression and division plane stability can be rescued in anillin depleted cells by targeting 

ectopically expressed anillin to the furrow using an alternate PI(4,5)P2 binding module. These 

data demonstrate an anillin-PI(4,5)P2 interaction targets anillin to the cleavage furrow and is 

required for stable, symmetrical ingression of the furrow during cytokinesis. 

2256 

Sauron is the Drosophila orthologue of the oncogene GOLPH3. 

S. Sechi 1 , V. Mattei 2 , G. D. Raffa 3 , G. Belloni 3 , G. Colotti 1 , M. G. Giansanti 1 ; 1 Istituto di Biologia 

e Patologia molecolari c/o Università Sapienza, CNR, Roma, Italy, 2 Consorzio Universitario 

Sabina Universitas Rieti, Rieti, Italy, 3 Dipartimento di Biologia e Biotecnologie, Università 

Sapienza, Roma, Italy 

Drosophila male meiosis provides an excellent cell system for the molecular dissection of 

cytokinesis. Drosophila spermatocytes are considerably larger than most somatic cells and the 

spindle assembly checkpoint is not stringent in spermatocytes, allowing the characterization of 

genes whose products are required for multiple stages of cell division in addition to cytokinesis. 

In the course of a large screen for mutants defective in spermatocyte cytokinesis, we have 

isolated sauron (sau), a male sterile mutant with defects in both central spindle formation and 

contractile ring assembly. A P element lethal insertion (l(2)s5379) in the CG7085 gene failed to 

complement the sau 1 mutation. DNA sequencing of the sau 1 allele revealed a single point 

mutation in CG7085, resulting in a Glu-Lys amino acid substitution at the C-terminus of the 

predicted protein. These results together indicate that sau encodes a polypeptide of 294 amino 

acids which is 70% identical to human GOLPH3 (alias Gpp34, Gmx33, MIDAS). GOLPH3 is a 

Golgi protein which has recently been recognized as a potent oncogene amplified in many 

human cancers and implicated in cellular transformation via changes in the activity of mTOR. 

We have generated transgenic flies that express GFP-Sau under the control of tubulin 

promoter. The GFP-Sau protein rescues the defects of sau 1 , confirming that the cytokinesis 

phenotype is the consequence of alterations in the Drosophila GOLPH3 ortholog. We have also 

raised polyclonal antibodies against Sauron and started immunofluorescence analysis in fixed 

spermatocytes. Immunostaining of primary spermatocytes revealed that the endogenous protein

TUESDAY 

is enriched in the Golgi of prophase spermatocytes and accumulates at the cleavage site during 

early telophase suggesting an early role in cytokinesis. 

2257 

Microtubule-free cytokinesis in vertebrate somatic cells: the roles of Plk1 and Aurora B 

kinase. 

E. H. Hinchcliffe 1 ; 1 Hormel Institute, University of Minnesota, Austin, MN 

Cytokinetic furrow formation requires the initiation of cortical contractility, and its restriction to 

the cell’s equator. Following anaphase onset spindle microtubules (MTs) and the mitotic kinases 

Aurora B (Aur B) and Polo-like kinase 1 (Plk1) are thought to drive furrow formation, but how 

these activities are coordinated remains poorly understood. Here we use live-cell imaging and a 

temperature-based spindle MT depolymerization/re-growth assay to examine cytokinesis in 

vertebrate somatic cells. Spindle MTs disassembled in either early anaphase (

2260 

Delivery of the cytokinetic signal to the plasma membrane – analysis of the RhoGEF 

Ect2. 

K-C. Su 1 , T. Takaki 1 , M. Petronczki 1 ; 1 Cell Division and Aneuploidy, Cancer Research UK - 

London Research Institute, Clare Hall, Potters Bar, England 

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Cytokinesis is the final stage of cell division and leads to the birth of two individual daughter 

cells. During the process of cytokinesis, the ingression of cleavage furrow divides the cytoplasm 

of the mother cell. 

In animal cells, cytokinesis is controlled by activation of the small GTPase RhoA, which initiates 

the formation of the contractile ring at the equatorial cell cortex during anaphase. It is well 

established that the mitotic spindle determines the position and activity of the contractile 

machinery at the plasma membrane. However, the precise mechanism by which the 

microtubule-associated signalling complexes control the cytokinetic machinery at the membrane 

is poorly understood. 

The conserved RhoGEF protein epithelial cell transforming sequence 2 (Ect2) is essential for 

cleavage furrow formation and RhoA activation at the equatorial cortex. Ect2, through its Nterminal 

tandem BRCT domains, interacts with the centralspindlin subunit MgcRacGAP leading 

to Ect2 recruitment to the spindle midzone. In its C-terminal region, Ect2 contains a guanine 

nucleotide exchange factor domain and a pleckstrin homology domain, the detailed function of 

either remain to be understood. We are using genetic, cell biological, and biochemical assays to 

address how Ect2 initiates cytokinesis at the plasma membrane in a spatially and temporally 

controlled manner. 

Our analysis will provide new insights into the molecular mechanism that delivers the cytokinetic 

signal to the equatorial cortex during anaphase in animal cells. 

2261 

Control of Mitotic Exit Network (MEN) signaling in S. cerevisiae meiosis. 

M. Attner 1 , A. Amon 1,2 ; 1 David H. Koch Institute for Integrative Cancer Research, Dept of 

Biology, Massachusetts Inst Technology, Cambridge, MA, 2 Howard Hughes Medical Institute 

When cells exit from mitosis, cyclin dependent kinases (CDKs) are inactivated. In budding 

yeast, Cdc14, a protein phosphatase that is released from its inhibitor in the nucleolus during 

anaphase, triggers CDK inactivation. Cdc14 release is controlled by the mitotic exit network 

(MEN), an essential GTPase signaling cascade. How CDK activity is regulated in meiosis is less 

clear. During meiosis, a diploid cell undergoes two rounds of chromosome segregation following 

one round of DNA replication, resulting in the formation of four haploid gametes. At the meiosis 

I-meiosis II transition, CDK activity is thought to be low enough to allow the meiosis I spindle to 

dissemble but high enough to prevent DNA re-replication. To understand how the cell achieves 

this balance at the meiosis I-meiosis II transition, we have characterized the mitotic exit network 

(MEN) in meiosis. We have found that the MEN is not required and not active at the meiosis Imeiosis 

II transition. However, the MEN becomes active during anaphase II and is required for 

timely exit from meiosis II. We further show that MEN signaling in meiosis is regulated differently 

from MEN signaling in mitosis. Whereas localization to the spindle pole body (SPB) is critically 

important for MEN signaling in mitosis, meiotic MEN signaling relies on the regulated binding of 

downstream MEN components and does not depend on SPB components. Taken together, our 

data suggest that exit from meiosis I and II are regulated by different mechanisms than exit from 

mitosis, which may help establish permissive conditions for the meiotic divisions.

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2262 

A midzone-based ruler adjusts chromosome compaction to anaphase spindle length. 

G. E. Neurohr 1,2 , A. Naegeli 2 , I. Titos 1 , D. Theler 2 , B. Greber 2 , Y. Barral 2 , M. Mendoza 1 ; 1 Center 

of Genomic Regulation (CRG), Barcelona, Spain, 2 Institute of Biochemistry, ETH Zurich, Zurich, 

Switzerland 

For successful partitioning of the genetic material during cell division, mitotic chromosomes 

must condense enough to be segregated by the fully elongated spindle, while spindles must 

elongate enough to segregate the bulkiest chromosome. Although both maximal spindle length 

and chromosome size can vary even within a given species, we know little about how cells cope 

with these changes to ensure faithful segregation of the genetic material under such conditions. 

We have managed to fuse the two longest chromosomes of the budding yeast S. cerevisiae to 

generate a yeast strain containing an oversized chromosome. The long arm of this chromosome 

exceeds the longest wild type arm by 50%. Remarkably, these cells were able to cope with the 

presence of the long chromosome without delaying anaphase or increasing spindle length, and 

without slowing down their growth rate. Instead, cells adapted through increasing the 

condensation of the compound chromosome specifically. Consistently, cells carrying the fused 

chromosomes became more sensitive to loss of condensin activity. In addition we observed that 

length-dependent stimulation of condensation took place during anaphase and depended on the 

spindle midzone, aurora/Ipl1 activity, and phosphorylation of histone H3 on Ser10. We propose 

that the spindle midzone functions as a ruler that adapts the condensation of long chromosome 

arms to spindle length to promote their faithful segregation during anaphase, regardless of 

variations in chromosome or spindle length. 

2263 

Identifying Novel Kinases Involved in Mitotic Progression Using Kinome-wide Screens. 

H. Chen 1 , H. Ma 1 , Y. Tsang 1 , C. Cheung 1 , S-S. Ng 1 , J. Zhang 1 , R. Poon 1 ; 1 Division of Life 

Science, Hong Kong University of Science and Technology, Hong Kong, China 

The main purpose of mitosis is to segregate sister chromatids into two nascent cells, such that 

each daughter cell inherits one complete set of chromosomes. Protein phosphorylation is one of 

the main post-translational regulation mechanisms of mitosis. A number of protein kinases such 

as cyclin-dependent kinases, Aurora kinases, Polo-like kinases (PLKs), NIMA-related kinases, 

and spindle assembly checkpoints kinases BUB1, BUBR1 and MPS1, are known to be involved 

in the control of various mitotic processes. Dysfunction of phosphorylation events regulated by 

these protein kinases would cause abnormal chromosome segregation as well as genome 

instability. However, the whole repertoire of mitotic kinases probably remains to be deciphered. 

In this study, we sought to identify novel mitotic kinases with RNAi approaches. Mouse 

fibroblasts NIH3T3 modified with histone H2B-GFP expression was transfected with a mouse 

kinome siRNA library to deplete individual kinases. Novel kinase candidates essential for 

mitosis progression were identified according to an increased mitotic index. Subsequently, the 

candidate mitotic kinases were compared with a previous screen in Drosophila. Several 

matched candidates from the mouse and Drosophila screens were chosen for further analysis in 

human cancer cells. Approaches including flow cytometry, live cell imaging, and immunostaining 

were applied to confirm the functional impact of these candidates in mitosis. These results 

indicate that in addition to the set of well-studied kinases, several novel protein kinases may 

also play critical role in mitosis.

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2264 

A stochastic model of kinetochore–microtubule attachment accurately describes 

chromosome segregation. 

G. Gay 1 , T. Courtheoux 1 , C. Reyes, 1 , S. Tournier 1 , Y. P. Gachet 1 ; 1 LBCMCP, CNRS, Toulouse, 

France 

Erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. 

Most of these are corrected before the onset of anaphase by a mechanism involving the protein 

kinase Aurora B which destabilizes kinetochore microtubules in the absence of tension between 

sister chromatids. Here, we describe a minimal mathematical model of mitotic chromosome 

segregation based on stochastic attachment and detachment of kinetochore microtubules. The 

model accurately reproduces the process and timing of correct chromosome bi-orientation and 

segregation seen in fission yeast. Prevention of attachment defects requires both appropriate 

kinetochore orientation and an Aurora B-like activity. The model also reproduces abnormal 

chromosome segregation behavior (due, for example, to inhibition of Aurora B). It predicts that, 

in metaphase, merotelic attachment is prevented by a kinetochore orientation effect and 

corrected by an Aurora B-like activity whereas in anaphase it is corrected through unbalanced 

forces applied to the kinetochore. These unbalanced forces are sufficient to prevent aneuploidy 

and the ‘cut’ phenotype. 

2265 

High Throughput imaging of cell based assays and cellular models using microplate 

cytometry. 

P. Wylie 1 , D. Caracino 2 , D. Onley 1 , W. Gaisford 1 ; 1 TTP Labtech Ltd, Royston, United Kingdom, 

2 TTP Labtech, Cambridge, MA 

Microscope-based, high-content instruments are used for many cell based assays in high 

content screening (HCS). Using Acumen eX3 ® with its wide field objective lens and “on-the-fly” 

laser scanning capabilities, it is possible to rapidly scan entire wells in a wide range of cell 

based assays relevant to the drug discovery industry including cell cycle analysis, cell surface 

markers and cell migration. 

In this poster, data is shown demonstrating the use of Acumen to assess the effect of inhibitory 

compounds on the mitotic index and cell cycle. In addition, with its high depth of field, it has also 

shown to be capable of scanning larger organisms such as C.elegans and even Zebrafish to 

provide information on multi-cellular drug interactions at an early screening stage. For efficient 

and rapid resolution most assays require the use of higher resolutions which entail lengthy read 

times, using single colours and analyzing only a small percentage of the total number of cells in 

a well to keep plate read times at a minimum. Recent studies also demonstrate that Acumen is 

capable of rapidly analysing complex cellular or animal models, such as angiogenic tube 

formation, C. elegans or drosophila larvae, thus enabling the development of whole organism or 

tissue based assays models for HCS. 

Built in software offers the flexibility of exporting whole well open source TIFF images for batch 

processing by third party image analysis software packages. This new screening paradigm 

represents a major breakthrough in how microplate cytometry can be applied to complex cellular 

models since rapid cytometric analysis can now be combined with image-processing 

methodology.

2266 

Condensin structures chromosomal DNA through topological links. 

S. Cuylen 1 , J. Metz 1 , C. H. Haering 1 ; 1 Cell Biology & Biophysics Unit, EMBL, Heidelberg, 

Germany 

TUESDAY 

The multisubunit condensin complex is essential for the structural organization of eukaryotic 

chromosomes during their segregation by the mitotic spindle, but the mechanistic basis for its 

function is not understood. To address how condensin binds to and structures chromosomes, 

we have isolated from Saccharomyces cerevisiae cells circular minichromosomes linked to 

condensin. We find that either linearization of minichromosome DNA or proteolytic opening of 

the ring-like structure formed through the connection of the two ATPase heads of condensin’s 

structural maintenance of chromosomes (SMC) heterodimer by its kleisin subunit eliminates 

their association. This suggests that condensin rings encircle chromosomal DNA. We further 

show that release of condensin from chromosomes by ring opening in dividing cells 

compromises the partitioning of chromosome regions distal to centromeres. Condensin hence 

forms topological links within chromatid arms that provide the arms with the structural rigidity 

necessary for their segregation. 

2267 

Antioxidants rescue carcinogen induced mitotic defects in both chromosomally stable 

and unstable cells. 

I. S. Griffin 1 , T. J. Yates 1 , N. J. Quintyne 1 ; 1 Honors College, Florida Atlantic University, Jupiter, 

FL 

Tumor cells are characterized by an increase in genomic instability, brought about by both 

chromosomal rearrangement and chromosomal instability. Both of these broad changes can be 

induced by exposure to carcinogens. During mitosis, cells can exhibit lagging chromosomes, 

multipolar spindles or anaphase bridges, all of which contribute to genomic rearrangement. We 

have studied the link betweenexposure to carcinogen and prevalence of mitotic defect in both 

chromosomally stable and unstable cell lines. We have exposed UPCI:SCC103 oral cancer 

cells, MES-SA uterine cancer cells and RPE normal retinal cells to vinyl chloride,tamoxifen and 

aflatoxin at varying concentrations. When treated with carcinogens, we see a preferential 

increase in the frequency of lagging chromosomes, although rates of other mitotic defects are 

also higher. In addition, we have examined the effects of antioxidants on decreasing the 

frequency of mitotic defect: analysis on chromosomally unstable cells with or without carcinogen 

treatment as well as chromosomally stable cells after carcinogen treatment indicates that Betacarotene, 

vitamin C and vitamin E are all effective at reducingrates of mitotic defect. We are 

also investigating whether pre-treatment with the antioxidants will weaken the effects of 

carcinogen exposure in these cell lines. 

2268 

Bod1 inhibits Protein Phosphatase 2A-B56 during mitosis. 

I. M. Porter 1 , K. Schleicher 1 , J. R. Swedlow 1 ; 1 Wellcome Trust Centre for Gene Regulation and 

Expression, University of Dundee, Dundee, UK 

Bod1 is a small, 22 kDa, protein that is required for resolution of syntelic attachments and 

proper chromosome alignment during mitosis and proper phosphorylation of MCAK by Aurora B 

in mitosis (Porter et al., 2007). Here we demonstrate that Bod1 has sequence similarity to Ensa 

and Arpp-19, two proteins recently identified as inhibitors of PP2A-B55, which are required for 

proper entry into mitosis (Mochida et al., 2010; Gharbi-Ayachi et al., 2010). We have now shown 

that Bod1 binds to the PP2A-B56 holo-enzyme during mitosis and is required for mitotic

TUESDAY 

progression. Binding of Ensa and Arpp-19 to PP2A-B55 requires a conserved Asp residue and 

phosphorylation by Greatwall kinase at a conserved Ser adjacent to the Asp. The Asp residue is 

conserved in Bod1 (D98), whereas the Ser is not, but a consensus CDK1 site (T95) is present 

immediately upstream. We show that Bod1 is phosphorylated in vitro by CDK1 kinase. 

Expression of Bod1 D98A or T95A mutants fail to rescue Bod1 depletion whereas the T95E 

mutations rescues as well as expression of wt Bod1. siRNA depletion of Bod1 from HeLa cells 

increases localisation of PP2A-B56 to kinetochores and loss of Sgo1 and Plk1 from 

kinetochores. Overall, these data show that when phosphorylated by CDK1, Bod1 binds PP2A- 

B56 and suggest that this binding inhibits the activity of PP2A. In the presence of hyper-active 

kinetochore-associated PP2A, Plk1 activity is compromised and phosphorylation of downstream 

targets is decreased. Together Bod1, Ensa and Arpp-19 form a new class of PP2A inhibitors. 

2269 

Daxx and USP7: novel regulators of genomic stability, mitosis and taxanes sensitivity. 

S. Giovinazzi 1 , V. Morozov 1 , W. C. Reinhold 2 , A. M. Ishov 1,3 ; 1 Anatomy and Cell Biology, 

University of Florida, Gainesville, FL, 2 Genomics & Bioinformatics Group, National Cancer 

Institute, NIH, Bethesda, MD, 3 Shands Cancer Center, University of Florida, Gainesville, FL 

Daxx is a multifunctional protein that plays a pivotal role in both physiological and pathological 

cellular processes. We previously demonstrated that cells with low levels of Daxx have reduced 

sensitivity to taxanes, powerful chemotherapeutic agents, by persisting in a pro-metaphase 

block that allows cells to escape taxane-induced cell death. In this study we dissected the 

mechanisms of Daxx-dependent taxanes resistance that also suggests function of this protein in 

mitotic progression. We show that Daxx interacts and cooperates with Ubiquitin Specific 

processing Protease-7 (USP7) to regulate mitosis. We demonstrate that depletion of USP7 

promotes stabilization of cyclin B, aneuploidy and mitotic anomalies, as it was previously 

observed for Daxx. We further demonstrate that USP7 depletion results in reduced stability of 

the mitotic E3 ubiquitin ligase Checkpoint with Forkhead and RING finger (CHFR). 

Consequently cells depleted by USP7 accumulate CHFR substrate, Aurora A kinase that has a 

crucial role in mitotic progression. We conclude that Daxx and USP7 are necessary to regulate 

proper execution of mitosis and their effects are at least partially mediated by CHFR and Aurora 

A kinase. Results from colony formation assay and in silico analysis show that USP7 expression 

negatively correlates with response to taxanes in cancer cell lines indicating that this protein can 

be used as predictive factor for taxanes response in cancer patients. 

2270 

Effects of actin and myosin inhibitors on PtK2 spindle length changes induced by laser 

microbeam irradiations across the spindle. 

R. Sheykhani 1 , M. W. Berns 2 , A. Forer 1 ; 1 York University, Toronto, ON, Canada, 2 University of 

California San Diego, San Diego, CA 

Cutting spindle microtubules across the entire half spindle using a 532 nm pulsed picosecond 

laser microbeam causes the irradiated spindle pole to move inward (N. Baker PhD thesis, 

University of California, San Diego 2010). This usually is followed by the inward movement of 

the un-irradiated pole. We tested whether the inward movement of the poles might be due to the 

function of actin and myosin: we pharmacologically poisoned the actin-myosin system using 

anti-actin drugs Cytochalasin D (CD), Latrunculin B (LatB), or Jasplakinolide (JASP), or antimyosin 

drugs BDM and Y-27632, and then irradiated across the entire half spindle. All these 

drugs altered the inwards movements of both poles. In some cells neither pole moved, in some 

cells only one pole moved, in some cells movements of the poles were delayed, and in only a 

few cells did the poles move as far as in non-treated cells. This suggests that, as in Xenopus

TUESDAY 

cells (Woolner et al., 2008), there is an interaction between actin, myosin and microtubules to 

control spindle length, though in our case it might be that actin/myosin forces are antagonistic to 

microtubule forces. 

2271 

HDAC3 regulates NudC acetylation in mitosis. 

C. Chuang 1 , J. Pan 2 , D. H. Hawke 3 , S-H. Lin 3 , L-Y. Yu-Lee 1,2 ; 1 Molecular and Cellular Biology, 

Baylor College of Medicine, Houston, TX, 2 Medicine, Baylor College of Medicine, Houston, TX, 

3 Department of Molecular Pathology, MD Anderson Cancer Center, Houston, TX 

Mitosis is a highly regulated process in which errors can lead to genomic instability, a hallmark 

of cancer. During this phase of the cell cycle, transcription is silent and RNA translation is 

globally inhibited. Thus, mitosis is largely driven by post-translational modification of proteins, 

including phosphorylation, methylation, ubiquitination, and sumoylation. Recent proteomics 

studies suggest that protein acetylation is as prevalent as protein phosphorylation. However, 

very little is known about the role of acetylation in mitotic progression. 

Using anti-acetyl-lysine immunoprecipitation of mitotic HeLa cell lysates followed by mass 

spectrometry, we identified 51 unique non-histone proteins, including proteins involved in RNA 

binding and processing, and cell cycle regulation (1). One cell cycle protein identified is NudC, a 

highly-conserved dynein/dynactin associated factor that plays a role in mitosis and cytokinesis. I 

confirmed that NudC is acetylated in mitosis by immunoprecipitation followed by immunoblot. 

Moreover, NudC acetylation in mitosis increased upon treatment with the HDAC inhibitor 

apicidin that shows specificity towards HDAC3, suggesting that NudC acetylation is regulated by 

HDAC3. 

Interestingly, comparing unperturbed HeLa cells to mitotically-enriched HeLa cells, NudC is 

more acetylated outside of mitosis. Utilizing a double thymidine block and release protocol, I 

enriched for HeLa cells in S, G2, early mitosis (prometaphase-like), late mitosis (anaphase), and 

G1. I found that NudC associates with HDAC3 throughout mitosis, which may explain its lower 

level of acetylation during mitosis. Immunofluorecense staining confirmed that NudC and 

HDAC3 co-localize during early mitosis. 

Mass spectrometry analysis of NudC immunoprecipitated from unperturbed HeLa cell lysate 

identified one acetylation site on NudC at K39. I generated NudC K39 acetyl-deficient (K39R) 

and acetyl-mimetic (K39Q) mutants and will decipher the role of NudC K39 acetylation in mitotic 

progression. Additionally, how NudC acetylation/deacetylation regulates its association with 

various mitotic regulators to promote mitotic progression are under analysis. 

These studies provide a framework from which to address how dynamic 

acetylation/deacetylation regulates the function and/or localization of proteins that drive mitotic 

progression and cell division. Understanding acetylation of cell cycle protein networks in cell 

cycle regulation is likely to reveal new paradigms for anti-cancer therapy. 

1. Chuang et al. J. Proteome Res. 2010, 9, 4554-4564.

2272 

Attenuation of mitotic RanGTP gradient in normal somatic cells. 

K. Hasegawa 1 , S. Ryu 1 , P. Kalab 1 ; 1 LCMB, National Cancer Institute, NIH, Bethesda, MD 

TUESDAY 

Studies in variety of organisms showed that mitotic segregation of chromosomes requires 

regulation of spindle assembly factors (SAFs) by small GTPase Ran. Essential feature of such 

regulation is thought to be the chromosome-centered concentration gradient of free RanGTP 

which arises in mitotic cells due to chromosome binding of RCC1, the guanine nucleotide 

exchange factor for Ran and cytoplasmic localization of RanGAP1. Close to chromosomes, 

RanGTP releases spindle assembly factors (SAFs) from their inhibitory complexes with 

importins, thus providing a spatial clue to mitotic spindle assembly. Using live cell microscopy 

with Forster resonance energy transfer (FRET) biosensors, we observed that in contrast to 

HeLa, normal human primary cells divide in the absence of a steep mitotic RanGTP gradient or 

a gradient of activated SAFs. Consistent with proposed Ran functions in mitotic spindle 

assembly, the absence of a steep mitotic RanGTP gradient in primary cells correlated with 

extended prometaphase, although no mitotic defects were observed in such cells. As expected, 

we found that RCC1 abundance and its binding to chromatin were among the key factors 

controlling the steepness of mitotic RanGTP gradient. These results show that a steep RanGTP 

gradient is not required for mitosis in all normal cells and suggest that attenuated mitotic Ran 

function may be characteristic to homeostatic cell proliferation of normal tissues in vivo. 

2273 

Molecular mechanisms governing extrinsic forces in mitotic spindle organization. 

M. Kwon 1,2 , M. Bagonis 2 , G. Danuser 2 , D. Pellman 1,2 ; 1 HHMI/Pediatric Oncology, Dana-Farber 

Cancer Institute, Boston, MA, 2 Cell Biology, Harvard Medical School, Boston, MA 

Precise control of centrosome number is crucial for bipolar spindle assembly and accurate 

segregation of chromosomes to daughter cells. Cancer cells often contain supernumerary 

centrosomes that create the potential for catastrophic multipolar divisions. Despite the presence 

of extra centrosomes, many cancer cells successfully divide because of mechanisms that 

suppress multipolar mitoses by clustering their extra centrosomes. Using functional genomics 

and follow-up studies, we have previously identified key pathways contributing to centrosome 

organization in cancer cells. In addition to spindle intrinsic forces, we found that cell adhesion 

patterns can determine the fate of mitosis whether they divide into two (bipolar) or more 

(multipolar). Yet, the mechanism by which actin and adhesion-dependent forces to control the 

fidelity of mitosis remain unclear at the molecular level. One model proposes that sites of strong 

cell matrix adhesion are imprinted as an actin rich structure called retraction fibers during 

mitosis, and the retraction fibers serve as cortical cues to concentrate force generators and 

regulators to pull centrosomes. By imaging cancer cells plated on fibronectin micropatterns, 

here we demonstrate that Myo10, an unconventional myosin identified from our genome-wide 

screen, is an essential component linking retraction fiber-mediated forces to astral microtubules 

(MTs) during spindle organization. Myo10 specifically localizes along and to the tips of the 

retraction fibers. When cells with extra centrosomes were plated on Y-shaped fibronectin 

micropatterns, control cells divide tripolar toward Y-adhesion axis; however, cells depleted of 

Myo10 undergo bipolar division independent of adhesion and retraction fiber positions, 

suggesting that Myo10 is a force coupler from retraction fibers to centrosomes. We have 

hypothesized that intrinsic properties of MTs adjacent to retraction fibers are altered to favor 

stable interaction between astral MTs and cell cortex. To uncover heterogeneity of MT dynamics 

in relation to retraction fibers, we have performed live cell imaging of GFP-EB3 with high spatiotemporal 

resolution in cells plated on fibronectin micropatterns where retraction fiber positions 

are precisely manipulated. We have developed a system that quantifies and visualize MT

TUESDAY 

dynamics in different subregions of cells by an automated computer program capable of 

tracking GFP-EB3 comets. This approach uncovered heterogeneity of MT dynamics around 

retraction fiber regions. Interestingly, our analyses have revealed that Myo10 is in part 

responsible for promoting the long-lived and long distance travelling MTs preferentially at 

adhesion sites. Taken together, these results suggest that Myo10 is a key adhesion-dependent 

regulator that confers heterogeneity of mitotic cortex by coupling cell geometry/adhesion to 

mitotic fidelity and genome stability. 

2274 

Terminating Mitotic Checkpoint Surveillance at the Anaphase Onset. 

M. D. Vazquez Novelle 1 , M. Petronczki 1 ; 1 Cancer Research UK (Clare Hall Labs), Potters Bar, 

England 

The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays 

anaphase onset until all sister kinetochores have become attached to opposite poles. Correct 

bipolar attachment leads to kinetochore deformation and tension that silences the checkpoint. 

What prevents mitotic checkpoint reactivation when sister centromeres are split and tension is 

lost at anaphase onset? Aurora-B kinase, the catalytic subunit of the chromosomal passenger 

protein complex (CPC), acts as a sensor at inner centromeres for the status of attachment. 

Phosphorylation of Aurora-B targets at erroneously attached kinetochores elicits the correction 

of these attachments and the activation of the mitotic checkpoint. At anaphase the CPC leaves 

the centromeres and relocates to the spindle midzone. This Cdk1-controlled iconic translocation 

might prevent the checkpoint from re-engaging after anaphase onset. To test this hypothesis we 

experimentally retained Aurora-B and the CPC at the centromere throughout anaphase in 

human cells. Preventing CPC translocation caused the untimely recruitment of mitotic 

checkpoint proteins (Bub1, BubR1 and Mps1) to kinetochores at anaphase in an Aurora-B 

kinase activity dependent manner. Our results suggest that the relocalization of the CPC, an 

evolutionarily conserved event in eukaryotes, is a key mechanism that prevents mitotic 

checkpoint activation at anaphase. However, the retention of Aurora-B at centromeres after 

anaphase onset does not suffice to trigger a full mitotic checkpoint engagement in response to 

the loss of tension due to sister chromatid splitting. Mad1 and Mad2 recruitment to kinetochores 

is suppressed after anaphase onset independently of CPC relocalization. What regulates these 

events at the metaphase-to-anaphase transition in human cells? Experiments in yeast, flies and 

frogs suggest that Cdk1 activity is required for mitotic checkpoint activity and the error correction 

pathway. Thus, we are investigating whether inactivation of Cdk1 at the metaphase-toanaphase 

transition could play an important role in terminating mitotic checkpoint surveillance. 

Our preliminary data are consistent with the notion that Cdk1 is a master regulator of the mitotic 

checkpoint in human cells. In conclusion, together our results suggest that Cdk1 inactivation 

prevents mitotic checkpoint re-engagement at anaphase onset, through the translocation of 

CPC and other events that control Mad1/2 recruitment to kinetochores. 

2275 

A mechanism linking microtubule destabilisation to maturation of chromosomemicrotubule 

attachments. 

R. L. Shrestha 1 , N. Tamura 1 , I. Zulkipli 1 , V. M. Draviam 1 ; 1 Department of Genetics, University of 

Cambridge, Cambridge, United Kingdom 

Selective destabilization of erroneous chromosome-microtubule attachments is critical to 

prevent the mis-segregation of chromosomes. Initially, chromosomes attach to lateral walls of 

microtubules and then convert this attachment to the ends of microtubules through a poorly 

understood maturation process. We demonstrate here a role for microtubule destabilization in

TUESDAY 

the maturation of attachments made to microtubule walls, and describe the consequence of 

persistent immature attachments. TAO1 kinase mediates microtubule destabilisation during 

mitosis and cells depleted of TAO1 display persistent laterally attached kinetochores that fail to 

congress properly. By briefly exposing cells to mild microtubule stabilizing drugs, we 

demonstrate that suppressing microtubule dynamics is sufficient to deter the maturation of 

lateral attachments. Laterally attached kinetochores, under microtubule stabilizing conditions, 

fail to retain Mad2 or MPS1 and compromise error-correction in cells released from monastrol 

treatment. Together our findings provide first insight into the fate of unresolved lateral 

attachments, and highlight the importance of maturation in ensuring the accurate segregation of 

chromosomes. 

Signal Transduction and Signaling Networks II 

2276/L50 

Regulation of Notch1 signaling by Jagged-1 intracellular domain. 

J-S. Ahn 1 , E-J. Ann 1 , J-H. Yoon 1 , H-S. Park 1 ; 1 School of Biological Sciences and Technology, 

Chonnam national university, Bukku, Gwangju, Korea 

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after 

binding to its transmembrane ligands. Jagged-1 also undergoes proteolytic cleavage by 

gamma-secretase and releases an intracellular fragment. In this study, we have demonstrated 

that the Jagged-1 intracellular domain (JICD) inhibits Notch1 signaling via a reduction in the 

protein stability of the Notch1 intracellular domain (Notch1-IC). The formation of the Notch1-IC- 

RBP-Jk-Mastermind complex is prevented in the presence of JICD, via a physical interaction. 

Furthermore, JICD accelerates the protein degradation of Notch1-IC via Fbw7-dependent 

proteasomal pathway. These results indicate that JICD functions as a negative regulator in 

Notch1 signaling via the promotion of Notch1-IC degradation. 

2277 

Role of R-Ras GTPases in the Wnt-Planar Cell Polarity pathway. 

N. Hartig 1 , M. Muñoz-Alegre 1 , L. Young 1 , P. Rodriguez-Viciana 1 ; 1 UCL Cancer Institute, London, 

United Kingdom 

Ras genes are mutated in about 30% of human tumours and likely play a role by indirect 

activation (e.g. receptor tyrosine kinases) in an even larger number of cancers. The closely 

related R-Ras GTPases share many of the properties of Ras genes including the ability to 

behave as oncogenes. Despite sharing biochemical and biological properties, the R-Ras 

subgroup also have distinct functions. In order to identify novel R-Ras effector proteins 

potentially accounting for these differences, we employed a Tandem Affinity Approach (TAP) 

using activated R-Ras as bait. This approach lead to the identification of Wnt-Planar Cell 

Polarity (PCP) protein Vangl1 as a novel R-Ras interacting protein. 

The Wnt pathway plays a key role in development and disease. In addition to the better studied 

ß-catenin dependent pathway, Wnt ligands can also activate the separate ‘non-canonical’ or 

PCP pathway, which coordinates complex cell migration during development. Perturbations in 

the PCP pathway contribute to the pathogenesis of a variety of diseases including cardiac and 

neuronal tube defects, cilia based disorders and to the invasiveness of cancer cells. 

We have shown that Vangl1 functions as a true R-Ras effector and mapped the interaction to 

the Vangl1 C-terminus. We have identified several Vangl1 interacting proteins that confirm it’s

TUESDAY 

conserved role in polarity and conducted biological assays that show that Vangl1 is critically 

required in migration. We have also shown that certain Frizzled receptors cooperate with the 

Ror2 tyrosine kinase to selectively stimulate Vangl1 function and possibly activation of R-Ras 

GTPases. 

In conclusion, the identification of Vangl proteins as novel effectors of R-Ras GTPases provides 

an exciting new link between Ras signalling and the Wnt-PCP pathway. 

2278 

Loss of serine protease HtrA1 potentiates transforming growth factor-β signaling and 

bone formation. 

J. R. Graham 1 , A. Chamberland 1 , X. Li 1 , D. Dai 1 , Q. Lin 2 , M. S. Ryan 3 , W. Zeng 1 , T. Blanchet 1 , 

M. A. Rivéra-Bermúdez 1 , C. R. Flannery 1 , Z. Yang 1 ; 1 Inflammation & Remodeling Research 

Unit, Pfizer Inc, Cambridge, MA, 2 Global Biotherapeutics Technologies, Pfizer Inc, Cambridge, 

MA, 3 Immunology & Autoimmunity Research Unit, Pfizer Inc, Cambridge, MA 

HtrA1 is a member of the High Temperature Requirement (HtrA) family of serine proteases. 

Genetic and gene expression studies have implicated HtrA1 in the progression of several 

pathologies, most notably in conditions with aberrant deposition of extracellular matrix. The 

inhibitory activity of HtrA1 on the transforming growth factor (TGF)-β family pathway has been 

reported, partially explaining the function of HtrA1 in physiological or disease processes. This 

study characterized the molecular mechanism by which HtrA1 regulates TGF-β signaling and 

examined the effect of HtrA1 gene deletion on musculoskeletal tissues in mice. 

Overexpression of HtrA1 inhibited the TGF-β-mediated transcription of a Smad-responsive 

promoter. Incubation of HtrA1 with several candidate substrates in the TGF-β pathway revealed 

that this protease was capable of efficiently cleaving type II and III TGF-β receptors (TβRII and 

TβRIII), but not TβRI or TGF-β itself. Furthermore, cleavage increased with the addition of CPII, 

an HtrA1 agonist. Overexpression of HtrA1 decreased the cell surface levels of both TβRII and 

III, but not TβRI. Conversely, silencing HtrA1 expression resulted in significantly more TGF-β 

cell surface binding, increased Smad2 phosphorylation, and enhanced TGF-β-regulated gene 

expression. To better characterize the role of HtrA1, we generated mice with targeted gene 

deletion. MicroCT analysis showed that deletion of the HtrA1 gene resulted in a marked 

increase in bone volume in the distal femur and lumbar vertebrae (48% and 20%, respectively). 

Embryonic fibroblasts from HtrA1 knockout mice exhibited increased TGF-β-induced expression 

of three genes known to promote bone formation, Runx2, CTGF and PAI-1. 

Our results reveal that the cleavage of two TGF-β receptors (TβRII and III) is a novel 

mechanism for HtrA1 inhibition of TGF-β signaling. Deletion of HtrA1 in mice leads to a dramatic 

increase in bone formation, most likely due to enhanced signaling by TGF-β or TGF-β family 

proteins.

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2279 

Cellular and Biochemical Activities of the Non-Canonical Wnt Receptors ROR1 and 

ROR2. 

T. W. Bainbridge 1 , V. De Almeida 2 , C. Chalouni 3 , R. Kelly 1,4 , J. Goldsmith 2 , A. Majeed 1,5 , B. 

Pan 6 , G. Quinones 1 , J. Lill 1 , W. Sandoval 1 , A. Izrael-Tomasevic 1 , D. Arnott 1 , M. Costa 2 , P. 

Polakis 2 , B. Rubinfeld 2 , J. A. Ernst 1,7 ; 1 Protein Chemistry, Genentech, South San Francisco, CA, 

2 Cancer Pathways, Genentech, South San Francisco, CA, 3 Center for Advanced Light 

Microscopy, Genentech, South San Francisco, CA, 4 University of California, Los Angeles, CA, 

5 Materials Science & Engineering, Stanford University,Palo Alto, CA, 6 Structural Biology, 

Genentech, South San Francisco, CA, 7 Early Discovery Biochemistry, Genentech, South San 

Francisco, CA 

The receptor tyrosine kinases, ROR1 and ROR2, activate non-canonical Wnt signaling and 

planar cell polarity pathways in response to Wnt5a. Various mutations within the intracellular 

domain (ICD) of ROR2 have been linked to Robinow syndrome and brachydactyly type B, while 

increased expression of ROR1 has been associated with acute lymphoblastic leukemia, chronic 

lymphocytic leukemia and mantle cell lymphoma. Previous studies have shown phosphorylation 

of ROR2 in response to Wnt5a stimulation, although autophosphorylation has not been 

demonstrated. The human ROR kinase domains lack conservation of the P-loop consensus 

sequence and are expected to be deficient in phosphotransferase activity, but studies with 

isolated ROR have provided conflicting results in kinase activity assays. CAM-1, the C. elegans 

ortholog of ROR, retains the P-loop consensus sequence, but the kinase activity has not been 

evaluated in vitro. In this work, we investigate the cellular role of the ROR ICD and compare the 

in vitro kinase activities of the isolated ICDs of ROR1, ROR2, CAM-1, and the most closely 

related human kinase, MuSK. 

2280 

Serotonin Receptor Signaling in JC Virus Infection. 

M. Maginnis 1 , W. Atwood 1 ; 1 Molecular Biology, Cell Biology & Biochemistry, Brown University, 

Providence, RI 

The JC polyomavirus (JCV) is a prevalent human pathogen and the causative agent of the fatal, 

demyelinating disease Progressive Multifocal Leukoencephalopathy (PML). JCV infection is 

initiated by interactions with cell-surface alpha 2,6-linked sialic acid on lactoseries 

tetrasaccharide c (LSTc) and serotonin 5-hydroxytryptamine2A receptor (5-HT2AR). Engagement 

of viral receptors can activate intracellular signaling pathways that regulate key steps in the 

virus life cycle. The objective of this study is to define whether cellular signaling events emanate 

from the G-protein coupled-receptor (GPCR) 5-HT2AR in JCV infection. 5-HT2AR activates the 

Ca 2+ /calmodulin pathway leading to activation of the ERK/MAPK signaling pathway and 

phosphorylation of ERK. To determine whether ERK is phosphorylated following engagement of 

5-HT2AR, cell lysates were harvested at early time points following JCV infection and analyzed 

by immunoblot analysis for ERK phosphorylation. ERK was phosphorylated as early as 5 

minutes post-infection consistent with receptor engagement. Furthermore, treatment of cells 

with inhibitors of MEK and ERK abolished JCV infection in a dose-dependent manner 

suggesting that activation of the ERK/MAPK signaling pathway is critical for JCV infection. To 

define whether Ca 2+ /calmodulin signaling is activated during JCV infection, cells were treated 

with inhibitors of calcium release and calmodulin kinase, both of which decreased JCV infection. 

5-HT2AR contains two calmodulin binding sites expressed in the second transmembrane loop 

and carboxyl terminus. Mutations in key hydrophobic residues in the calmodulin binding sites 

were generated in a 5-HT2AR construct and expressed in poorly permissive HEK cells. 

Expression of a wild-type 5-HT2AR rescues infection in HEK cells, but a mutation in the

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calmodulin-binding site in the carboxyl terminus resulted in a decrease in JCV infection. These 

findings provide new information regarding the role 5-HT2AR in JCV infection, and suggest that 

signals initiated upon engagement of the 5-HT2AR are essential for a productive viral infection. 

2281 

A genetic approach to identify new regulators for Smoothened. 

H-T. Shih 1 , W-Y. Chen 1 , J-T. Wu 1 ; 1 Institute of Molecular Medicine, National Taiwan University, 

Taipei City, Taiwan 

G protein-coupled receptors (GPCRs) are seven-transmembrane receptors that initiate signaling 

processes of many physiological responses upon ligand stimulation. In Drosophila, Smoothened 

(Smo) belongs to GPCR family and transduces Hedgehog signaling that is critical in embryonic 

and larval development. Whereas the strength of GPCR-mediated signaling could be modulated 

at the level of GPCR by desensitization and internalization of the GPCRs, it is not clear whether 

the level of Smo is also subjective to any regulation that eventually changes the strength of 

Hedgehog signaling. To test this possibility, we performed an in vivo RNAi screen in a smo lossof-function 

mutant background to identify regulators for Smo expression that also modulate the 

strength of Hedgehog signaling. First, we used a MS1096-Gal4 driven USA-smo5A to generate 

a fused L2 and L3 wing vein, the characteristic smo deficient phenotype in adult wing. Next, we 

crossed MS1096>smo5A to RNAi or dominant negative stocks for small G proteins, Rac1, 

Cdc42, RhoA, and Rab, and GTPase-activating proteins (GAPs). Our preliminary data showed 

that ectopic expression of small G proteins caused a marked wing shrinkage or lethality. But 

dominant negative form of small G proteins enhanced Smo deficient phenotype. Interestingly, 

most GAPs had no obvious effect on the smo deficient phenotype, suggesting a functional 

redundancy among GAPs. Our long term goal is to understand the distinct regulation impinged 

on Smo as oppose to those on conventional GPCRs. 

2282 

Dihydrotestosterone activates Src to increase EGF-induced Erk activation in fetal lung 

fibroblasts. 

M. K. Lee 1 , S. M. Smith 1 , S. Murray 2 , L. D. Pham 2 , H. C. Nielsen 2 ; 1 Center for Craniofacial 

Molecular Biology, University of Southern California, Los Angeles, CA, 2 Newborn Medicine, 

Tufts Medical Center, Boston, MA 

Neonatal respiratory distress syndrome has long been recognized as being more severe in 

premature male infants. This observation has been ascribed to differences in lung maturation, 

which is regulated by interactions between mesenchymal and epithelial cell populations. 

Fibroblast-type II cell communications are, in turn, modulated by epidermal growth factor (EGF)mediated 

Erk1/2 activation. Because dihydrotestosterone (DHT) has been implicated in delayed 

lung maturation, we hypothesized that androgens could affect tyrosine kinase signaling in fetal 

lung fibroblasts. Androgens act by both translation-dependent and independent mechanisms, 

and we further hypothesized that DHT regulates fetal lung fibroblast signaling by inducing 

cytosolic Src to phosphorylate EGF receptors, thereby enhancing their subsequent activation by 

EGF. To assess these postulates, fetal lung fibroblasts were isolated from either pregnant mice 

treated with subcutaneous timed-release dihydrotestosterone (DHT) pellets or from untreated 

timed-gestation mice at 16 days gestation. Cells were maintained in culture containing 100 nM 

DHT for up to 4 d prior to overnight serum starvation and stimulation with 100 ng/ml EGF for up 

to 30 m. Cells were lysed in RIPA buffer, resolved by SDS-PAGE, and transferred to 

nitrocellulose for Western analysis. Membranes were probed with antibodies against 

phosphoErk1/2, phosphoEGF receptor, and phosphoSrc. To attenuate Src activity, selected 

cells were transfected with siRNA against Src, treated with the pharmacological Src inhibitor

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PP2, or infected with adenoviruses expressing a dominant negative Src. Both long-term (in 

utero plus in culture) and short-term (in culture only) DHT exposure increased peak Erk 

activation by EGF, and this increase was evident with as little as 60 min of DHT exposure. The 

phosphorylation of Src and EGF receptor was increased by DHT. EGF-induced Shc 

phosphorylation and Grb2 association with Shc were similarly increased by prior DHT exposure, 

confirming pathway activation. Finally, Src silencing, PP2 treatment, and dominant negative Src 

adenoviruses all prevented DHT from increasing EGF-mediated Erk1/2 phosphorylation. These 

results suggest that DHT activates Src to increase the activity of EGF receptors and, hence, the 

EGF signaling pathway leading to Erk activation in primary fetal lung fibroblasts. 

2283 

Regulation of calcium-dependent calcineurin activity by inositol sphingolipids. 

S. A. Jesch 1 , S. A. Henry 1 ; 1 Molecular Biology and Genetics, Cornell University, Ithaca, NY 

Inositol-containing sphingolipids and phosphoinositides at the plasma membrane play important 

roles in regulating stress response pathways, which are critical for lipid homeostasis and cell 

viability. We previously showed that inhibiting inositol sphingolipid metabolism triggers protein 

kinase C signaling and subsequent activation of the MAP kinase, Slt2p/Mpk1p, by regulating the 

Stt4p-dependent pool of phosphatidylinositol-4-phosphate (PI4P) at the plasma membrane. To 

identify additional lipid-mediated signaling networks that are activated by a similar mechanism, 

we carried out gene expression profiling following interruption of inositol sphingolipid synthesis 

by starving cells for inositol. We found that calcineurin-dependent genes are highly up-regulated 

following inositol starvation. Previous work has demonstrated that calcineurin, a calciumdependent 

protein phosphatase, regulates the activities of the PH-domain proteins Slm1p and 

Slm2p, which are necessary for normal sphingolipid metabolism. Consistent with these data, 

calcineurin, stt4, and slm mutants exhibit inositol auxotrophy. Moreover, cells lacking Csg2p, a 

calcium binding regulator of mannosyl-inositolphosphorylceramide synthesis, constitutively 

activates calcium-dependent calcineurin signaling. This activation requires stt4, slt2/mpk1, and 

the plasma membrane-localized calcium channel Cch1p, which is a known target of 

Slt2p/Mpk1p. Together, these results suggest a feedback mechanism for regulating sphingolipid 

mediated signaling by regulating plasma membrane pools of PI4P and intracellular calcium 

levels. 

2284 

Anti-Inflammatory Effect of Benzylideneacetophenone Derivative on Thyroid-Associated 

Ophthalmopathy through Suppression CXCL10/IP-10 Production. 

S. Lee 1 , Y-H. Choi 1 ; 1 Ewha Woman's University, Seoul, Korea 

Thyroid-associated ophthalmopathy (TAO) is an autoimmune inflammatory disorder of the 

orbital fatty tissue/connective tissue and the extra ocular muscles that is closely associated with 

Graves’ hyperthyroidism. Recent experimental evidence has shown that CXCL10/IP-10 is 

elevated in patients with TAO and plays an important role in the initial phases of TAO. 

Yakuchinone B from Alpinia oxyphylla,that belongs to ginger family has been used in oriental 

herbal medicine. It has been reported as one of anti-inflammatory phytochemicals which 

regulate COX-2 expression. To search for new candidate of anti-inflammatory compound at the 

inflammatory stage of TAO, benzylideneacetophenone compound JC3, (2E)-3-(4-hydroxy-3methoxyphenyl)phenylpro-2-en-l-one), 

was synthesized from Yakuchinone B and their potential 

to suppress CXCL10/IP-10 production was evaluated. IFN-γ strongly increased the level of 

CXCL10/IP-10 in orbital fibroblasts with TAO and normal. JC3 compound revealed the potent 

inhibitory effect on IFN-γ-induced CXCL10/IP-10 protein expression. JC3 compound effectively 

decreased mRNA expression level of CXCL10/IP-10 and exhibited no toxicity in orbital

TUESDAY 

fibroblasts at the 10μM concentration. Moreover, the result obtained from gel shift assay 

showed that JC3 suppressed IFN-γ-induced DNA binding of STAT-1. These results suggest that 

JC3 modulates high levels of CXCL10/IP-10 protein in response to IFN-γ, indicating that JC3 

may be an attractive therapeutic agent to reduce the inflammatory stage of TAO. 

2285 

IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells. 

T-D. Kim 1 , J. Park 1 , I. Choi 1 ; 1 Korea Research Institute of Bioscience and Biotechnology, 

Daejeon, Korea 

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune 

cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the 

function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in 

human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a 

maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be 

expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively 

increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 

antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no 

effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are 

implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on 

NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 

or IL-15. 

2286 

Anthrax Toxin Receptor 2 Carboxyl-terminal 12-residues are Important for Toxin Entry 

into Cells. 

R. Jimenez 1 , G. J. Chaudry 1 , S. Manam 1 ; 1 South Texas Center for Emerging Infectious Disease, 

Department of Biology, Univeristy of Texas at San Antonio, San Antonio, TX 

Anthrax toxin is secreted by the Gram-positive, spore-forming, rod-shaped bacterium Bacillus 

anthracis. The toxin is one of two virulence factors of the organism, the other being its 

antiphagocytic poly-D-glutamate capsule. Three secreted proteins make up anthrax toxin: 

protective antigen (PA), edema factor (EF), and lethal factor (LF). EF is an adenylyl cyclase that 

harms cells by producing excessive amounts of cAMP. LF is a Zn-dependent metalloprotease 

that cleaves MAP kinase kinases. PA delivers EF and LF to cytosol, but itself is without any 

toxic catalytic activity. Thus, active anthrax toxins are PA+EF and PA+LF. The toxins enter cells 

by receptor-mediated endocytosis. PA utilizes two receptors for entry, tumor endothelial marker 

8 (TEM8; ANTXR1) and capillary morphogenesis gene 2 protein (CMG2, ANTXR2). The two 

membrane-bound forms of CMG2 that function as PA receptors comprise 488 and 489 

residues, and they differ only in the last 12 residues. Our analysis shows that absence of these 

residues results in a receptor that affords PA binding and its cleavage by furin, but does not 

support intoxication by the toxin. The block to intoxication appears at the level of the SDSresistant 

heptamer, a structure that forms upon exposure to acidic pH in endosomes. Functional 

analysis by alanine-scanning mutagenesis revealed that essentially all 12 residues are 

important for receptor function. Surprisingly, deletion of the entire cytoplasmic portion of CMG2 

left the truncated receptor fully functional for anthrax toxin. Thus, the 12-residue C-terminal 

segment must remain intact for the full-length receptor to function, but the segment is 

dispensable when the rest of the cytoplasmic domain is also absent. The results also suggest 

that different mechanisms underlie toxin entry via the full-length CMG2 and the receptor lacking 

the cytoplasmic domain.

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2287 

Rho A signaling contributes to statin-induced osteogenesis in bone marrow 

mesenchymal stem cells. 

I-C. TAI 1,2 , M-L. Ho 2,3 , J-K. Chang 2,4 ; 1 Graduate Institute of Medicine, Kaohsiung Medical 

University, Kaohsiung, Taiwan, 2 Orthopaedic Research Center, Kaohsiung Medical University, 

Kaohsiung, Taiwan, 3 Department of Physiology, Kaohsiung Medical University, Taiwan, 

4 Department of Orthopedic Surgery, Kaohsiung Medical UniversityHospital, Taiwan 

Statins, 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, reduce 

cholesterol synthesis and prevent cardiovascular disease. Previous in vitro and in vivo studies 

showed that statin stimulated bone formation. They also have been found to inhibit prenylation 

of Rho proteins in recent decade. Previous reports showed that statins inhibited protein 

prenylation and decreased the active form of Rho A in osteoclasts. Others reports indicated that 

statins inhibited protein prenylation but increased the active form of Rho A in human 

erythroleukemia cells. Therefore, the role of statins regulate Rho A activity remains unclear. Rho 

GTPases act as molecular switches to regulate mesenchymal stem cell differentiation. Previous 

study showed that transfected constitutively active-form of RhoA into human mensenchymal 

stem cells (hMSCs) which leaded differentiation of hMSCs into osteoblasts. On the other hand, 

dominant negative RhoA leaded differentiation of hMSCs into adipocytes. According to the 

description above, we want to investigate whether Rho A signaling contributes to statin-induced 

osteogenesis in BMSCs. Pluripotent mesenchymal cells, D1, which were cloned from Balb/c 

mouse bone marrow cells and purchased from ATCC. For all experiments, cells were seeded at 

a density of 80% confluence and treated with or without simvastatin. The mRNA expression of 

Rho A was detected and quantified by real time PCR. And the mineralization effect on rat bone 

mesenchymal stem cells (rBMSCs) was tested by Alizarin Red S Staining. The Alizrin red S 

staining assay showed that the simvastatin were potentially enhanced the cell mineralization on 

BMSCs. The data of mRNA expression of Rho A showed that simvastatin 1uM significantly 

increased Rho A gene expression on first day. However, there were no significantly different 

between control and treatment group on third and fifth days. The protein level of Rho A 

increased with 0.5uM and 1uM but not 0.1uM simvastatin treatment on first day. From these 

results, we suggest Rho A signaling may contribute to simvastatin-enhanced osteogenesis in rat 

bone mesenchymal stem cells (rBMSCs). 

2288 

Magnetic Manipulation of signaling “hotspots” inside living cells shows contextdependent 

amplification of the Rac pathway. 

F. Etoc 1 , M. Coppey 1 , D. Lisse 2 , Y. Bellaiche 3 , J. Piehler 2 , M. Dahan 1 ; 1 LKB-IBENS, Paris, 

France, 2 biophysics, Osnabruck University, Germany, 3 Curie Institute, France 

In vivo imaging has shown how the establishment and maintenance of a polarized cellular state 

relies on complex mechanisms by which signaling cascades become activated and regulated at 

sub-cellular levels. It has recently led to the concept of spatially-restricted signaling modules. 

Yet, it remains unclear which ingredients and processes specify a module. In this context, we 

used a new tool to locally perturb and probe signaling pathways associated to the small Rho- 

GTPases. 

First, magnetic nanoparticles (500nm), functionalized with constitutively active Rho-GTPases 

(Rac1 and cdc42) were microinjected inside living cells. We observed the induction of 

downstream signaling at the particle surface by visualization of effectors binding (Pak and N- 

WASP) and the local polymerization of actin structures, thereby demonstrating that these 

particles behave in the cytosol as signaling "hotspots". Using FRAP, we further measured in

TUESDAY 

vivo dissociation rates between the immobilized GTPase and its direct effector. Next, the 

nanoparticles were manipulated with a magnetic tip to position their signaling activity at different 

subcellular locations. With nanoparticles functionalized in situ with Tiam (a known Rac1 

activator), we were able to specifically activates Rac1 signaling by bringing the nanoparticles in 

contact with the plasma membrane. The Rac1 activation occurred regardless of the subcellular 

localization at the membrane. However, the signal was non-linearly amplified only when the 

signaling activity was carried to protrusive areas of the cell. This result reinforces the concept of 

subcellular signaling modules and shows that a module could be specified by its ability to 

amplify rather than just linearly transduce incoming signals. 

2289 

Implications of RhoA regulation in Hela cells exposed to stress radiation. 

J. H. Osaki 1 , G. E. Silva 1 , F. L. Forti 2 ; 1 Biochemistry, Institute of Chemistry, University of Sao 

Paulo, 2 Biochemistry, Institute of Chemistry, University of Sao Paulo, Sao Paulo, Brazil 

The protein RhoA GTPase belongs to the superfamily of small GTPases related to Ras. RhoA 

are enzymes acting as molecular switches to control a wide variety of signal transduction 

pathways in eukaryotic cells such as remodeling of actin cytoskeleton, cell cycle, and cell death. 

RhoA overexpression is found in aggressive breast cancer and cervix carcinomas, where the 

enzyme is usually constitutively activated. Ionizing radiation (gamma and UV) causes single and 

double strand breaks in DNA, and also pyrimidine dimers lesions, damages usually repaired by 

homolog recombination, non-homologous end-joining and nucleotide excision repair 

mechanisms. In this study, using HeLa cells and mutants expressing dominant-negative RhoA 

(HeLa-RhoA-N19) and constitutively-active RhoA (HeLa-RhoA-V14), the RhoA protein was 

analyzed as a possible player in survival, senescence, migration, DNA damage response and 

repair after irradiation of cells with low doses of gamma and UV light. Results show that RhoA 

activity was downregulated by the highest energy ionizing. The HeLa-RhoA-V14 presents an 

increase in cell proliferation, survival and migration rate compared to HeLa-RhoA-N19. 

Furthermore, in RhoA-V14 clones either the DNA damage is decreased or the repair 

mechanisms are increased resulting in attenuated cellular senescence after the different 

ionizing treatments. In conclusion, these data confirm the essential role of RhoA protein in 

proliferation and migration and more intriguingly point to possible involvements of this enzyme in 

mechanisms of DNA repair and cell senescence after genotoxic stress induced by ionizing 

radiation. 

Supported by Fapesp: Project # 2008/58264-5. 

2290 

Rho small GTPases activates PI3K via a cooperative positive feedback loop. 

H. Lee 1 , H. Yang 1 , M-G. Shin 1 , S. Lee 1 , W. Park 1 , J-R. Kim 1 , K-H. Cho 1 , T. Meyer 2 , W. Heo 1 ; 

1 Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2 Stanford University 

Phosphoinositide3-kinases (PI3Ks) and Ras and Rho family small GTPases are key regulators 

of processes such as cell polarization, motility and chemotaxis. These signaling components 

may control each other¡¯s activities by direct and indirect feedback processes that are only 

partially understood. Here, we used a live cell screen to evaluate the role of 100 human small 

GTPases in the regulation of PI3K and found that 21 small GTPase homologs activate PI3K. 

Using a protein-protein interaction assay that we recently developed, we found that K-Ras, H- 

Ras and 5 other homologs function upstream of PI3K and directly bind to the PI3K catalytic 

subunit, p110, while three Rac isoforms and 11 other small GTPase homologs activate PI3K by 

indirect feedback mechanisms. Furthermore, live cell experiments and in silico analysis 

revealed that significant level of PI3K activation can be generated by cooperation of multiple

TUESDAY 

activators in endogenous level. We demonstrate two modes of PI3K activation that is initiated by 

upstream activators and is further enhanced by the cooperation of PI3K activators. 

2291 

Arl4A complexes with ELMO to promote actin cytoskeleton remodeling. 

T-C. Chiang 1 , M. Patel 2,3 , V. Tran 2 , F-J. Lee 1 , J-F. Cote 2,3 ; 1 Institute of Molecular Medicine, 

Taipei, Taiwan, 2 Institut de Recherches Cliniques de Montreal, 3 Departement de Medicine, 

Université de Montréal 

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is 

indispensable during processes such as cell migration and myoblast fusion. The biological 

activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that 

auto-inhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate 

the ELMO/DOCK180 complex appears to be the recruitment of this complex to the membrane 

via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel 

ELMO-interacting proteins to further define the molecular events capable of controling ELMO 

recruitment to the membrane. To do so, we performed two independent interaction screens: one 

specifically interrogated an active GTPase library while the other probed a brain cDNA library. 

Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. 

Biochemically, Arl4A is constitutively GTP-loaded and our binding assays confirm that both wildtype 

and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we 

report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In 

addition, we report that membrane targeting of ELMO via Arl4A promotes cytoskeletal 

reorganization including membrane ruffling and stress fiber disassembly via an 

ELMO/DOCK180/Rac signaling pathway. We conclude that ELMO is capable of interacting with 

GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile 

RBD. Furthermore, via binding of an Arf family GTPase, the ELMO/DOCK180 is uniquely 

positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton. 

2292 

New biosensor for Cdc42-GDI complexation reveals dynamic regulation of GDI-Cdc42 

binding by Src-kinase. 

D. Spiering 1 , L. Hodgson 1 , M. Sabouri 2 , C. der Mardirossian 2 , G. Danuser 3 , K. Hahn 4 ; 1 Albert 

Einstein College of Medicine, Bronx, NY, 2 The Scripps Research Institute, La Jolla, CA, 

3 Harvard Medical School, Boston, MA, 4 University of North Carolina at Chapel Hill, Chapel Hill, 

NC 

Signaling by the p21 Rho family GTPases is critically important in a broad range of cell 

behaviors. They have been most extensively studied in motility and polarization, where their 

activation (GTP binding) is tightly coordinated in space and time to generate cell morphological 

changes. Guanosine Dissociation Inhibitors (GDI) sequester inactive (GDP bound) GTPases in 

the cytosol, and mediate localization of the activated GTPase in the plasma membrane. The 

role of GDI in controlling the precise placement and timing of GTPase activation remains poorly 

understood. To investigate this, we constructed fluorescent biosensors reporting the interaction 

of endogenous RhoGDI with either Cdc42, Rac1 or RhoA. Using the Cdc42-GDI biosensor 

together with the meroCBD biosensor detecting the activation of endogenous Cdc42 in MEFs, 

we imaged simultaneously the Cdc42–endogenous GDI complexation dynamics and the 

endogenous Cdc42 activation dynamics in single living cells. Using the morphodynamics 

mapping and computational multiplexing technology, we show directly for the first time that the 

Cdc42 uptake rate by the GDI is regulated through Src-kinase-mediated phosphorylation.

Proteostasis, Cell Stress, and Aging 

TUESDAY 

2293 

Is the ubiquitin ligase itch phosphorylation modulating its binding and ligase activity 

towards all substrates? 

R. Forget 1 , A. Angers 1 ; 1 Biological Sciences, University of Montreal, Montréal, Québec, Canada 

Itch is an ubiquitin ligase closely related to Nedd4. These enzymes bind to their substrate 

proteins with their central region and transfer ubiquitin through their C-terminal catalytic HECT 

domain. By promoting the proteasomal degradation of substrate proteins, ubiquitin ligases are 

involved in a variety of process like endocytosis, apoptosis, inflammation, cell signaling, etc. 

Recently, the regulation of the activity of ubiquitin ligases themselves has received a lot of 

attention. Itch is the target of the cJun N-terminal kinase (JNK) which phosphorylates three 

residues in a region overlapping with a proline-rich region of Itch that mediates recognition by 

SH3-domain proteins. Phosphorylation of Itch by JNK has been shown to regulate its activity 

towards itself and substrates cJun and JunB. An inhibitory intramolecular interaction abrogated 

by JNK-phosphorylation has been proposed to mediate this effect. Here, we evaluate if this 

model can be generalized to other Itch substrates, most specifically endophilin that has been 

shown to interact with Itch through its SH3 domain, distinct from most Itch known substrates. 

Using site-directed mutagenesis, we created mutants of Itch that could not be phosphorylated 

by JNK, and a phosphomimic mutant. These mutants were tested for their ability to interact with 

endophilin, and to mediate endophilin ubiquitylation and proteasomal degradation. We found 

that Itch phosphorylation by JNK has no effect on endophilin recognition by Itch, nor on the 

ligase ability to promote endophilin ubiquitylation. Although Itch phosphorylation is not required 

for proteasomal degradation of endophilin, a small facilitating effect of Itch phosphorylation 

could be observed. In conclusion, if JNK- mediated phosphorylation of Itch can slightly 

potentiate its ubiquitin- ligase activity, it has no incidence on the interaction between Itch and 

SH3 proteins like endophilin. Therefore, the regulation of Itch activity seems to differ according 

to the selected substrate. 

2294 

Potentiation of ER stress-mediated apoptotic pathway by protein tyrosine kinase p56lck 

in proteasome inhibitor MG132-induced apoptosis human Jurkat T cells. 

C. Han 1 , H. Park 1 , D. Jun 1 , H. Woo 1 , J. Lee 1 , Y. Kim 1 ; 1 School of Life Science and 

Biotechnology, Kyungpook National University, Daegu, Korea 

Exposure of human Jurkat T cells to MG132 caused apoptosis along with upregulation of 

Grp78/BiP and CHOP/GADD153, activation of JNK and p38MAPK, activation of Bak, 

mitochondrial membrane potential (¥Ä¥÷m) loss, cytochrome c release, activation of caspase- 

12, -9, -3, -7, and -8, cleavage of Bid and PARP, and DNA fragmentation. However, these 

MG132-induced apoptotic events, with the exceptions of upregulation of Grp78/BiP and 

CHOP/GADD153 and activation of JNK and p38MAPK, were abrogated by Bcl-xL 

overexpression. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented MG132induced 

apoptotic caspase cascade, but allowed upregulation of Grp78/BiP and 

CHOP/GADD153 levels, activation of JNK and p38MAPK, ¥Ä¥÷m loss, and cleavage of 

procaspase-9 (47 kDa) to active form (35 kDa). Further analysis using selective caspase 

inhibitors revealed that caspase-12 activation was required for activation of caspase-9 and -3 to 

the sufficient level for subsequent activation of caspase-7 and -8. MG132-induced cytotoxicity, 

apoptotic sub-G1 peak, Bak activation, and ¥Ä¥÷m loss were reduced in part by p38MAPK 

inhibitor, but not by JNK inhibitor. MG132-induced apoptotic changes, including upregulation of 

Grp78/BiP and CHOP/GADD153 levels, activation of caspase-12, p38MAPK and Bak, and

TUESDAY 

mitochondria-dependent activation of caspase cascade were more significant in p56lck-stable 

transfectant JCaM1.6/lck than in p56lck-dificient JCaM1.6/vector. The cytotoxicity of MG132 

toward p56lck-positive Jurkat T cell clone was not affected by the Src-like kinase inhibitor PP2. 

These results demonstrated that MG132-induced apoptosis was caused by ER stress and 

subsequent activation of mitochondria-dependent caspase cascade, and that the presence of 

p56lck enhances MG132-induced apoptosis by augmenting ER stress-mediated apoptotic 

events in Jurkat T cells. 

Key words: proteasome inhibitor, apoptosis, ER stress, mitochondria-dependent caspase 

cascade, 

p56lck 

2295 

Translation elongation factor eEF3 and translation termination factor eRF3 are novel 

constituents of heat-induced stress granules in Saccharomyces cerevisiae. 

T. Grousl 1 , P. Ivanov 2 , I. Malcova 1 , I. Frydlova 1 , P. Pompach 1 , R. Slaba 1 , P. Vasicova 1 , L. 

Novakova 1 , J. Hasek 1 ; 1 Inst. of Microbiology of ASCR, v.v.i., Prague 4, Czech Republic, 2 Faculty 

of Biology, Moscow State University, Moscow, Russia 

In response to severe environmental stresses eukaryotic cells shut down translation and form 

transient mRNA assemblies called stress granules (SGs). Stress granules are often referred to 

as dominant accumulations of stalled translation preinitiation complexes since their typical 

components are the translation initiation factor eIF3 and the small ribosomal subunit 40S. We 

have shown previously that in the budding yeast Saccharomyces cerevisiae the SGs containing 

the eIF3 complex and the 40S ribosomal subunit are formed upon the robust heat shock at 46°C 

only. The formation of these SGs is independent of phosphorylation of eIF2alpha by Gcn2 

kinase and also of the scaffolding proteins of P bodies Edc3 and Lsm4. Here we present the 

evidence that the robust heat shock-induced SGs of S. cerevisiae contain also the translation 

elongation factor eEF3 (Yef3). In addition, distinct accumulations of Yef3GFP were observed in 

the edc3lsm4C mutant even upon a mild heat shock at 42°C and they pre-determined 

accumulations of eIF3a upon the robust heat shock at 46°C. Furthermore, some of the robust 

heat shock-induced stress granules co-localized with accumulations of Sup35 suggesting that 

also translation termination factor eRF3 is a constituent of SGs. While the N-terminal domain of 

Sup35 is essential for [PSI + ] prion formation its absence did not affect assembly of SGs. Our 

data indicate that SGs containing translation preinitiation complexes and termination factors are 

successively assembled on accumulations of the elongation factor eEF3 (Yef3) that was 

released from translation during heat shock. This work was financed by grants CSF 

204/09/1924, LC545 and IRCAV0Z50200510. 

2296 

Pathogenic missense mutations located in distinct domains affect the folding and 

function of dystrophin. 

D. M. Strandjord 1 , D. M. Henderson 1 , B. Li 1 , J. M. Ervasti 1 ; 1 University of Minnesota, 


Dystrophin is a 427-kDa protein encoded by the DMD gene on the X chromosome. This 

structural protein localizes to the plasma membrane of muscle cells, where it establishes a 

mechanical link between the cytoskeleton and the extracellular matrix. The N-terminus and a 

segment of the middle rod domain in dystrophin interact with cytoplasmic actin filaments while 

modules near the C-terminus interact with the transmembrane protein �-dystroglycan. 

Dystrophin and �-dystroglycan, along with several other proteins, function as a complex known

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as the dystrophin-glycoprotein complex, or DGC, which is thought to stabilize the sarcolemma 

against mechanical stress. 

Males with a mutation in their only copy of the gene encoding dystrophin are diagnosed 

as having either Duchene Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD). 

Many patients have mutations that cause a dystrophin-deficiency in cells and develop extreme 

muscle deterioration in their first decade of life. More recently, sequencing has revealed that 

some patients have mutations in the dystrophin gene that result in a single amino acid change 

in the protein. Less is known about the molecular pathomechanism of these missense 

mutations. We recently demonstrated that missense mutations in the N-terminal actin-binding 

domain (ABD1) of dystrophin had minimal effect on actin binding activity, but caused thermal 

instability and aggregation in vitro (D.M. Henderson et al., 2010 PNAS). Additional pathogenic 

missense mutations have been identified in the �-dystroglycan binding domain (DgBD) of 

dystrophin. To determine the effect of these pathogenic changes in a location distinct from 

ABD1, we engineered the disease-causing missense mutations D3187G, C3207R, F3228L, 

A3311P, C3313F, D3335Y into dystrophin. Both wild-type dystrophin and E3032K, a nonsynonomous 

Single Nucleotide Polymorphism (SNP) found in the population, served as 

controls. When expressed in insect cell culture, quantification of the protein in the soluble versus 

insoluble fractions of the cell lysate revealed that some missense mutations significantly 

decreased the solubility of dystrophin. Circular Dichroism melt curves of purified proteins 

revealed that missense mutations decreased the cooperative unfolding transition which is 

indicative of a misfolded, heterogeneous population of protein. Supported by the Torske 

Klubben Graduate Fellowship and a grant from the National Institutes of Health (AR042423). 

2297 

Two distinguishable aging states found in an isogenic population at a single 

chronological age. 

D. M. Eckley 1 , S. Rahimi 1 , N. V. Orlov 1 , S. Mantilla 1 , C. E. Coletta 1 , J. D. Delaney 1 , Y. Zhang 2 , 

K. Becker 2 , W. Iser 3 , M. Wilson 3 , I. G. Goldberg 1 ; 1 IICBU, Laboratory of Genetics, National 

Institute on Aging (NIA), National Institutes of Health, Baltimore, MD, 2 Research Resources 

Branch, NIA-Intramural Research Program, 3 IMGU, Laboratory of Neuroscience, NIA 

We present the initial molecular characterization of a transition between two early aging states. 

An age score reflecting physiological age was developed using a machine classifier. Images of 

C. elegans populations at fixed chronological ages throughout their lifespan were used for 

training. The distribution of age scores identified three stable post-developmental states and 

transitions. We examined the transition that occurs at day 5 post L4 molt, because a significant 

percentage of that population exists in both State I and State II. We measured the temperature 

dependence of the timing of the transition (Q10 ~ 1.17). This value is too low to be explained by 

a step-wise process with an enzymatic or chemical rate-limiting step, implicating a more 

complex timing mechanism. Individual animals at day 5 were sorted into State I and State II 

groups by the machine classifier and analyzed by microarray expression profiling. Despite being 

isogenic, grown for the same amount of time, and indistinguishable by eye, these two 

morphological states were confirmed to be molecularly distinct by hierarchical clustering and 

principal component analysis. The distinction demonstrates that pharynx morphology must 

reflect the aging state of the whole organism. Our expression profiling also yielded a gene set 

that showed significant overlap with those from three previous age-related studies and identified 

several genes not previously implicated in aging. A highly represented group of genes unique to 

this study is involved in the ubiquitination pathway, including Skip1-related (SKR) as well as Fbox-containing 

adaptors which target specific proteins for degradation.

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2298 

Glucose Stress Reduces Fertility and Impairs Mating in Caenorhabditis elegans. 

A. K. Engstrom 1 , M. R. Liggett 1 , M. A. Mondoux 1 ; 1 Department of Biology, College of the Holy 

Cross, Worcester, MA 

Diabetes is a chronic disease marked by insulin resistance and the inability of cells to correctly 

respond to high levels of blood glucose. Diabetes currently affects 25.8 million people in the 

United States, and has been shown to lead to increased potential for birth defects, miscarriages, 

and infertility. Caenorhadiditis elegans has a conserved insulin-signaling pathway, making it an 

ideal model organism for studying the effects of excess glucose exposure. In previous studies, 

glucose stress in C. elegans was shown to lead to both a developmental delay and a reduction 

in hermaphrodite fertility; however the cause(s) of these defects is unknown. By exposing 

worms to high glucose during either development or adulthood, and then studying their 

reproductive profile and brood size, we found that the developmental delay is separable from 

the decrease in fertility. Feeding high glucose only during development led to a two-day 

reproductive delay but had no effect on fertility. Likewise, glucose stress during adulthood 

reduced fertility as much as lifetime exposure to high glucose. We assayed post-fertilization 

phenotypes under high glucose conditions and saw no change in external or internal hatching, 

suggesting that glucose stress causes defects in the adult germline. We also tested whether 

glucose stress affects male fertility and mating and find that glucose stress interferes with 

mating, leading to decreased brood sizes and decreased numbers of male offspring. Preliminary 

evidence suggests that male fertility is also decreased by glucose stress. We are currently 

investigating the roles of the insulin-signaling pathway and apoptosis in the response to glucose 

stress in hermaphrodites and determining which components of the male fertility and mating 

pathways respond to glucose stress. 

2299 

Differences in neuropeptide Y and Tirosine Hydroxylase levels related to ageing and 

gender in Locus Coeruleus. 

K. L. Farizatto 1 , M. F. Ferrari 1 , D. R. Chadi 2 ; 1 Faculdade de Medicina, Fisiopatologia 

Experimental, Universidade de Sao Paulo, Sao Paulo, Brazil, 2 Universidade de Sao Paulo, Sao 

Paulo, Brazil 

Age is a important factor for emerging of some diseases, as neurodegeneration and 

hypertension. Locus Coeruleus (LC) is a brain area involved with autonomic functions and 

behavior wich play a role in differences of blood pressure between males and females during 

lifespan. The goal of this study was to analyze the NPY and TH levels in the LC of 

spontaneously hypertensive (SHR) rats during aging in males and females. Methods: All the 

procedures and protocols were performed in accordance with the Institutional and International 

Guidelines for Animal Experimentation. We have employed SHR rats of: 3 months, 1 and 2 

years (N=6) old. Animals were decapitated, the brains were frozen to achieve coronal sections 

from pons for the analysis of LC, through in situ hybridization and imunohistochemistry 

techniques. Afterwards, sections were analyzed by means of a semiquantitative 

microdensitometric procedure. Results are showed as mean ± standard error in arbitrary units. 

Two-way ANOVA was used for statistical analysis. Results: NPY mRNA expression in the LC is 

increased at 2 years females (0,056 ± 0,005) as compared to 1 year (0.036 ± 0.004) and 

decreased at 1 year (0.036 ± 0.004) as compared to 3 months old female rats (0.090 ± 0.010). 

In males group is decreased at 2 years (0.031 ± 0.002) as compared to 3 months old male rats 

(0.090 ± 0.016), (p

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peptide is increased at 2 years females (80.5 ± 2.5) as compared to 1 year (68.0 ± 2.5) and 3 

months old female rats (60.0 ± 1.5). In males group is decreased at 2 years (54.5 ± 3.0) as 

compared to 3 months old male rats (65.5 ± 2.0), (p

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2301 

Depletion of ARMS enhances reactive oxygen species-induced autophagy. 

Y-H. Liao 1,2 , P-H. Huang 3,4 ; 1 Department of Dermatology, National Taiwan University College of 

Medicine, Taipei, Taiwan, 2 Department of Dermatology, National Taiwan University Hospital, 

Taipei, Taiwan, 3 Institute of Pathology, National Taiwan University College of Medicine, Taipei, 

Taiwan, 4 Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan 

Increased resistance to oxidative stress in melanoma cells plays an important role for 

melanoma development and contributes to the ineffectiveness of treatment aimed at oxidative 

stress-induced cell death. Autophagy is a self-digestion process in response to stimuli including 

oxidative stress. We have previously found that depletion of ankyrin repeat-rich membrane 

spanning (ARMS) protein in melanoma cells caused significantly increased autophagosomes. 

To determine the role of ARMS in oxidative stress-induced autophagy, we established siRNAmediated 

ARMS-knockdown and control B16F0 stable melanoma cell lines. We found that 

depletion of ARMS significantly enhanced autophagosome formation, as evidenced by 

autophagic vacuolization examined by electron microscopy, punctate translocation of GFP-LC3 

(microtubule-associated protein light chain 3) by immunofluorescence, and increased LC3-II by 

Western blotting. Significantly increased intracellular reactive oxygen species production in 

ARMS-RNAi cells at baseline and in time course of hydrogen peroxide treatment was revealed 

by DCHF-DA flow cytometry. Moreover, the percentage of cells with GFP-LC3 puncta was 

suppressed by treatment with antioxidant N-acetyl-L-cysteine or tiron in ARMS-RNAi cells. In 

vivo knockdown of ARMS in Drosophila imaginal eye discs also showed robust autophagy. In 

conclusion, these results suggested that ARMS silencing increased ROS production, which led 

to enhanced autophagy. 

2302 

Physiological role of the membrane contact site:Nucleus-Vacuole (NV) junctions. 

C-C. Lin 1 , D. S. Goldfarb 1 ; 1 Biology, University of Rochester, Rochester, NY 

Nucleus-Vacuole (NV) junctions are membrane contact sites (MCSs) where perinuclear ER 

(nuclear envelope) and vacuole membrane come into close apposition. NV junctions are formed 

by the direct interaction between outer nuclear membrane protein Nvj1 and the vacuole 

membrane protein Vac8. A unique autophagic process in yeast Saccharomyces cerevisiae 

called ‘Pieacemeal microautophagy of the nucleus (PMN)’ emanates from NV junctions. Osh1 

and Tsc13, which are involved in lipid homeostasis, are recruited to NV junctions and are 

essential for normal PMN biogenesis. Here, we report another protein that is sequestered to NV 

junctions-Nvj2. Unlike Osh1 and Tsc13, Nvj2 is neither required for NV junctions formation nor 

PMN process. Nvj2 localizes on ER, and is enriched on NV junctions upon Nvj1 expression is 

up regulated. Nvj1-Vac8 complex and the Y321 of Nvj1 are essential but not sufficient for the 

enrichment of Nvj2 on NV junctions. The absence of NVJ2 confers fungicide resistance, and 

overexpression of Nvj1, which sequesters Nvj2 to NV junctions, phenocopy the deletion of 

NVJ2. These results suggest an alternative role of NV junctions for the fungicide resistance 

other than PMN process. 

2303 

Premature aging of yeast Saccharomyces cerevisiae RAS2 val19 mutant. 

A. Pichová 1 , K. Sigler 1 ; 1 Institute of Microbiology, Academy of Sciences of the Czech Republic, 

Praha 4, Czech Republic 

Aging and apoptosis, multifactorial processes involving a complex network of regulations, are 

conveniently studied on single cell models. Yeast, especially S. cerevisiae, is suitable for this

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purpose because it exhibits mother cell specific aging, where the cell divides asymmetrically, 

giving rise to a next-generation large mother cell and a smaller daughter cell that resets the 

clock to zero while mother cell accumulates all aging-connected changes. This asymmetry is 

disturbed in the oncogene mutant RAS2 val19 . This strain, known as a short-lived mutant, shows 

also a higher sensitivity to starvation and heat shocks. Micromanipulations revealed a high 

percentage of virgins unable to form micro- and macro-colonies. Young cells, mostly virgins, 

separated by centrifugal elutriation show markers of apoptosis, such as having a high load of 

ROS (reactive oxygen species) detected by DHR (dihydrorhodamine); most cells appeared with 

DNA breaks stained by the TUNEL test and their nuclei also appeared to have aberrant 

morphology. Quantitative comparison of the amount of extrachromosomal DNA rings (ECR) in 

separated young and old cells of the wild type, RAS2 val19 mutant and a strain with delayed aging 

together with other data point to the premature aging being connected with symmetric aging, 

where the daughter cell is already inheriting some features of aging. 

2304 

The effects of OYE2 and ZWF1 overexpression in furfural stressed Saccharomyces 

cerevisiae. 

N. Carlson 1 , R. McRae 1 , S. Raehtz 1 , S. W. Gorsich 1 ; 1 Biology, Central Michigan University, 

Mount Pleasant, MI 

A renewable fuel source is needed as economical, environmental, and security concerns 

continue to rise regarding the use of fossil fuels. Bio-ethanol is one potential solution. In the U.S. 

bio-ethanol is mainly produced by fermenting agricultural products such as corn starch. 

However, the use of corn starch is problematic due to concerns with quantity of of corn supply 

and the competing use of corn as a food source. Alternatively, biomass waste products such as 

lignocellulose (e.g. corn stover, wood chips) could be used. Currently, lignocellulose has not 

been optimized for fermentation. One major barrier to using lignocellulose is that during its 

hydrolysis many growth inhibitors are produced. Furfural is a major inhibitor generated. Furfural 

has been shown to inhibit growth and metabolic enzymes, reduce ethanol yield, and increase 

reactive oxygen species (ROS) that go onto damage internal membranes, actin filaments, and 

chromatin. Furfural can be removed using various chemical and physical methods, but at the 

expense of increased cost and environmental waste production. Our method to reduce the 

effect of furfural is to genetically engineer yeast that are robust enough to tolerate high 

concentrations of furfural while optimizing ethanol yield. For instance, overexpressing either 

ZWF1 (Zwf1p produces NADPH from the pentose phosphate pathway) or OYE2 (Oye2p is an 

NADPH-dependent reductase linked to oxidative stress tolerance) individually improves 

tolerance to furfural and reduces ROS accumulation and damage to cellular structures. In the 

present study we show how overexpressing both OYE2 (pRS425-MET25-OYE2) and ZWF1 

(pRS426-ZWF1) in the same yeast strain effects overall furfural tolerance as assayed by growth 

(absorbance A600), reactive oxygen species (ROS) accumulation (dihydroethidium stain), 

membrane damage (mitochondrial targeted-GFP and FM 4-64), and chromatin damage (DAPI 

stain). Our strategy of genetic engineering fermenting microorganisms is valuable in improving 

the technology needed to optimize bio-ethanol fermentation from lignocellulose. 

2305/L80 

Vitamin C recycling and glutamate effects in astrocytes. 

P. Cisternas 1 , K. Oyarce 1 , P. Llanos 1 , C. Silva-Alvarez 1 , F. Nualart 1 ; 1 Department of Cell 

Biology, University of Concepcion, Concepcion, Chile 

Specialized cells transport vitamin C in its reduced form using sodium-dependent cotransporters 

(SVCT1 and SVCT2). Additionally, different cells transport the oxidized form of vitamin C,

TUESDAY 

dehydroascorbic acid (DHA), through glucose transporters (GLUTs). Vitamin C recycling 

associated with neuron-astrocyte interactions needs specific AA uptake in neurons and DHA 

uptake in astrocytes. SVCT2 is expressed principally in neurons mediating temperature- and 

sodium- dependent AA uptake. GLUT1 is expressed in astrocytes mediating glucose-DHA 

uptake. It has been suggested that glutamate increases vitamin C recycling. However, the 

specific mechanism has not yet been defined. In this work we analyzed the expression of 

GLUT1 and GLUT3 primary cultures of astrocytes and neurons obtained from rat brain, and 

assessed their differential capacities to incorporate AA or DHA. Using HPLC and radioactive 

pulse tracing we defined the effect of glutamate in the regeneration of AA in neurons and 

astrocytes. Our results confirmed that, in vitro, astrocytes only express GLUT1 and neurons 

preferentially express GLUT3. Functional data indicate that astrocytes transport mainly 

dehydroascorbic acid, using the glucose transporter GLUT1. Additionally, we defined that 

glutamate stimulates intracellular reduction of DHA in astrocytes of 15 and 30 DIV. In parallel, 

we observed that neurons have a lower reductive capacity, which increases only in the 

presence of high concentrations of glutamate. In this study we determined that glutamate is 

directly involved in the recycling of vitamin C in the CNS. This recycling model may work as an 

efficient system for the salvage of vitamin C by avoiding the hydrolisis of DHA produced by 

antioxidative protection. 

Grants Support by FONDECYT 1100396. 

2306 

ER Calcium Depletion and ER Stress by Palmitate in Mouse Podocyte. 

S. Xu 1 , J-H. Kim 1 , R. Das 1 , X. Quan 1 , S-K. Cha 1,2 , K-S. Park 1,2 ; 1 Physiology, Yonsei University 

Wonju College of Medicine, Wonju, Korea, 2 Institute of Lifestyle Medicine 

Podocytes play a major role in glomerular filtration barrier, but also participate in pathogenic 

process of diabetic nephropathy. Diabetic patients have elevated plasma levels of saturated free 

fatty acid (FFA) that induces ER stress and apoptosis in different cell types. In this study, we 

investigated the cytotoxic mechanism of FFA in immortalized mouse podocyte. Incubation with 

palmitate, a saturated FFA, elicited apoptotic cell death in a dose dependent manner 

(50~500�M). Palmitate elevated cytosolic or mitochondrial reactive oxygen species (ROS) 

production measured by fluorescence imaging system, which was blocked by N-acetyl-cystein 

(NAC). However, a mono-unsaturated FFA oleate itself or combination with palmitate did not 

increase the ROS level in podocyte. ER stress proteins such as GRP78/Bip, spliced Xbp1 and 

CHOP were strongly upregulated by palmitate, but not by oleate or oleate with palmitate. 

Palmitate decreased the luminal Ca 2+ level in ER measured with ER Ca 2+ sensing protein 

D1ER. In the absence of extracellular Ca 2+ , cytosolic Ca 2+ increase induced by blocking ER 

Ca 2+ uptake with cyclopiazonic acid (CPA) was markedly reduced in palmitate-treated cells, 

which could be recovered by NAC pretreatment. These data suggest that palmitate as the 

predominant circulating saturated FFA leads to ER stress and apoptosis in podocyte, which 

might be mediated by ROS-triggered ER Ca 2+ depletion. 

2307 

Involvement of yeast homolog MOH1 of human YPEL5 gene in DNA damage-induced 

apoptosis in the budding yeast Saccharomyces cerevisiae. 

J. Lee 1 , D. Jun 1 , Y. Kim 1 ; 1 School of Life Science and Biotechnology, Kyungpook National 

University, Daegu, Korea 

The homolog of Drosophila yippee protein is known to be highly conserved among eukaryotes 

from yeast to human. For five members of the human YPEL family, YPEL5 is evolutionarily

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distinct from other four members, YPEL1 through YPEL4, especially. However, their functional 

roles are still poorly understood. To understand the role of yippee protein, we have employed 

the budding yeast (Saccharomyces cerevisiae) mutant possessing disrupted yeast homolog 

(MOH1; YBL049W) of yippee protein gene, and compared its physiological characteristics with 

those of the wild-type strain. The mutant for the MOH1 gene exhibited an enhanced resistance 

to UVC (254 nm) irradiation and genotoxic agents as compared to the wild-type. This 

phenomenon was abrogated when the disrupted MOH1 gene in the mutant was restored by 

transformation of the functional gene. The level of MOH1 mRNA in the wild-type was 

upregulated in response to UVC irradiation and genotoxic agents. And exposed 

phosphatidylserine was detected by reaction with annexin V-FITC and PI staining after UVC 

treated. Our findings provide evidence for functional conservation of YPEL5 protein between 

lower and higher eukaryotes, which can confer apoptotic cell death to DNA damage in S. 

cerevisieae. 

Key word: MOH1, YBL049W, Saccharomyces cerevisiae, complementation test, YPEL5, UVC 

irradiation, genotoxic agent 

Development and Morphogenesis II 

2308 

Two periods of testicular regression, not directly linked to apoptosis, are peculiar events 

of the annual reproductive cycle of Myotis nigricans (Chiroptera: Vespertilionidae). 

M. R. Beguelini 1 , C. C. Puga 2 , F. F. Martins 2 , S. R. Taboga 1 , E. Morielle-Versute 2 ; 1 Biology, 

UNESP - University of Estadual Paulista, São José do Rio Preto, Brazil, 2 Zoology and Botany, 

UNESP - University of Estadual Paulista, São José do Rio Preto, Brazil 

The reproductive data available for Myotis nigricans, an endemic Neotropical species of 

vespertilionid bat, are few and controversial; and indicate that its reproductive cycle is 

geographically variable, presenting an active pattern throughout the year in some regions and a 

hibernating pattern in others. Thus, this study aimed to evaluate the seasonal modifications in 

testicular morphology in a tropical environment. Twenty-eight mature specimens, collected in 

São Paulo State, Brazil (49W22'45" 20S49'11"), between September 2008 and August 2009, 

were submitted to morphometric and immunohistochemical (cell proliferation-PCNA; apoptosis- 

TUNEL; androgen receptor-AR) analysis. Morphological observations revealed that the testes 

present two periods of regression (TR) during the year: in late spring (November) and in early 

winter (June); and peaks of spermatogenic activity in early spring (September-October) and 

middle autumn (April). The TR in spring begins in October, with a sharply decrease in testicular 

epithelium and in the amount of PCNA+ cells; however, the amount of TUNEL+ and AR+ cells 

were basal in this period, indicating that the regression did not occur by apoptosis. In November 

the testes were totally regressed but, they already presented patterns of reactivation, as the 

increase in expression of AR and PCNA. In December was noted the greatest expression of 

PCNA and a great expression of AR, indicating an accelerated development, which can lead to 

errors that are eliminated by apoptosis, thus explaining the great amount of TUNEL+ cells. The 

recrudescence process extended from December to the production of the first spermatozoa only 

in March, with this period presenting a gradually decrease in the AR, PCNA and TUNEL 

expressions, culminating in the second period of TR in June. The TR in June was less 

accentuated than the first (November), presenting a more rapidly reactivation of the 

spermatogenesis (July). This reactivation was induced by the greatest AR expression and 

culminated in the greatest occurrence of apoptotic cells. All data demonstrated the presence of 

two periods of TR in the reproductive cycle of M. nigricans, a pattern never previously observed

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in bats; and that the apoptosis was not linked to the TR, but with the elimination of errors 

caused by the rapidly reactivation of the spermatogenesis. 

2309 

Reactive oxygen species cause reduced spore development and spore fitness in the 

budding yeast, Saccharomyces cerevisiae. 

M. M. Postema 1 , T. Stokes 1 , M. Scott 1 , S. W. Gorsich 1 ; 1 Biology, Central Michigan University, 

Mount Pleasant, MI 

The generation of spores in yeast is essential for sexual reproduction and survival of the 

organism. When diploid yeast cells undergo meiotic division it is important that four optimally fit 

spores are produced. During this specialized division the four resulting spores not only have a 

haploid copy of nuclear chromosomes, but also a complete complement of organelles, such as 

mitochondria. Mitochondria undergo temporally regulated fusion and fission events to assure 

that mitochondria are represented equally in each of the resulting spores. When this 

mitochondrial network is not maintained, due to mutations in mitochondrial fission genes (eg. 

dnm1/dnm1), it has been shown that fewer spores survive and the ones that do survive are 

more likely to have reduced respiratory fitness. We hypothesized that environmental factors 

could also influence spore development in wild-type diploid yeast. In the present study, we 

demonstrated that hydrogen peroxide phenocopied spore development phenotypes associated 

with mitochondrial fission mutants. Wild-type yeast exposed to hydrogen peroxide have 

mitochondrial morphology and distribution defects during spore development, reduced spore 

viability, and decreased respiratory competency just as seen in dnm1/dnm1 fission mutants. 

Next we demonstrated that reactive oxygen species (ROS) sensitive mutants have increased 

ROS accumulation during spore development. In addition spore development defects 

associated with these mutants and wild-type cells exposed to hydrogen peroxide were reduced 

when treated with various antioxidant strategies. These findings suggest that the ability of yeast 

to produce optimal numbers of fit spores is heavily influenced by their ability to protect 

themselves from exogenous or endogenous ROS. 

2310 

A Role of Xenopus Zygote Arrest 2 (Xzar2) in Translational Regulation of Key Cell Cycle 

mRNAs. 

J. M. Cook 1 , T. M. Yamamoto 1 , A. Charlesworth 1 ; 1 Department of Integrative Biology, University 

of Colorado Denver, Denver, CO 

Zygote arrest proteins, Zar1 and Zar2 (aka Zar1-like), have been implicated in the oocyte to 

embryo transition, zygotic genome activation, pre-implantation development and 

epidermalization. However, the mechanism of action of Zar proteins is unknown: transcriptional 

regulation, chromatin remodeling and RNA metabolism have all been suggested. Early Xenopus 

development is coordinated through the translational regulation of maternal mRNAs and is 

mediated by a variety of cis-elements in the 3’ untranslated region (3’UTR) of the mRNA. The 

recently described translational control sequence (TCS) is one such cis-element found in 

mRNAs, such as Wee1 and Pcm1. Here, we show that Xenopus Zar2 (Xzar2) binds the TCS of 

maternal mRNAs and can regulate translation in a progesterone responsive manner. 

In an electrophoretic mobility shift assay (EMSA), the C-terminal portion of XZar2 (CXzar2) 

bound a fluorescent labeled wild-type Wee1 3’UTR probe, resulting in a shifted protein-RNA 

complex. This band was super-shifted with a Zar2 antibody, verifying the presence of Zar2 

protein in the band. Complex formation was inhibited by the addition of unlabeled Wee1 3’UTR 

or Pcm1 3’UTR probe, but was not inhibited if the TCSs were mutated, indicating the TCS is 

required for RNA binding. In a dual luciferase reporter (DLR) assay, the N-terminal domain of

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Xzar2 fused to an MS2 tethering protein (NXzar2-MS2) was injected into oocytes and allowed to 

incubate with or without the addition of progesterone. NXzar2-MS2 repressed translation of the 

reporter mRNA in immature oocytes in a dose responsive manner. Furthermore, that repression 

was relieved during progesterone-induced meiotic maturation. Endogenous Zar2 levels were 

evaluated during maturation via Western blot, and it was found that levels drop dramatically 

during oocyte maturation. 

These data suggest that Xzar2 protein binds developmentally important mRNAs in a sequence 

specific manner and exerts translational control over those mRNAs differentially during meiotic 

maturation. We propose that one role of Xzar2 in early development may be to modulate the 

synthesis of maternal cell cycle regulatory proteins at the translational level in the maturing 

oocyte. 

2311 

Zygote Arrest Proteins Bind Maternal mRNAs. 

K. D. Silva 1 , T. Khat 1 , T. M. Yamamoto 1 , A. Charlesworth 1 ; 1 Department of Integrative Biology, 

University of Colorado Denver, Denver, CO 

Zygote arrest proteins, Zar1 and Zar2 (aka Zar1-like), are important in early development. Zar1 

has been implicated in the activation of the zygotic genome and progression past the two-cell 

stage. However, the mechanism of Zar protein action has yet to be characterized. The product 

of the maternal Wee 1 mRNA has also been identified as a regulator of early development. 

Xenopus laevis Wee 1 mRNA contains a newly characterized cis-element in its 3’untranslated 

region (UTR) called the Translation Control Sequence (TCS), which regulates mRNA translation 

during early development. The protein that binds to the TCS has yet to be identified. The 

objective of this study is to show that Zar proteins bind to Wee 1 mRNA. 

Zar1 and Zar2 proteins possess a highly conserved C-terminal region containing five pairs of 

invariable cysteine residues, four of which are thought to make up a putative atypical 

FYVE/plant homeodomain (PHD) zinc finger motif. Our data shows that Zar proteins use the 

conserved C-terminal region to bind the TCS of Wee1 maternal mRNA. To characterize binding 

to mRNA, Xenopus laevis Zar protein binding mutants, with point mutations at specified cysteine 

residues, were designed and used to perform electrophoretic motility shift assays (EMSA) with a 

Cy5 labeled Wee 1 3’ UTR probe. We show that cysteine to alanine mutation at any of the 

invariable cysteines disrupted Zar binding to Wee 1 mRNA. To test if zinc was a requirement for 

binding interactions, Zar protein was purified and an EMSA was performed in a zinc free buffer. 

This EMSA showed that without zinc, Zar proteins did not bind to Wee 1 mRNA. 

This data suggests that Zygote arrest proteins contain a new type of zinc finger motif that 

constitutes an mRNA binding domain, providing support to the hypothesis that Zar proteins play 

an important role in translational regulation of maternal mRNAs. 

2312 

Three-dimensional cell cultures of human urothelium by using a collagen gel scaffold. 

C. Wasén 1 , M. Levin 2,3 , D. Giglio 1,3 ; 1 Pharmacology, Neuroscience and Physiology, Göteborg, 

Sweden, 2 Wallenberg Laboratory, Sahlgrenska Center for Cardiovascular and Metabolic 

Research, Göteborg, Sweden, 3 The Sahlgrenska University hospital, Jubileumskliniken, 

Göteborg, Sweden 

In vivo, urothelial cells create a 3-6 cell thick epithelial layer in the urinary bladder. In vitro, 

urothelial cells are most often grown two-dimensionally (2D) forming a one-cell layer. Three 

dimensional cell culture (3D) is an alternative to classic 2D culture. In 3D, epithelial cells form in 

vivo-like structures. In the present study, we investigated if urothelial cells may be grown threedimensionally 

in a collagen gel scaffold. We used the UROtsa cell line, which is an immortalized

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normal human bladder urothelial cell line (Petzoldt et. al., 1995). UROtsa cells grown threedimensionally 

in a collagen I gel formed spherical multicellular formations after 7 days. The cells 

expressed occludin and laminin indicating presence of tight junctions and basal membrane, 

respectively. The present findings reveal a new and promising way to study urothelial function in 

vitro by growing urothelial cells three-dimensionally in a collagen gel scaffold. 

2313 

The 3Ds - development, differentiation and death, of yeast colony. 

L-C. Liou 1 , Q. Ren 1 , Z. Zhang 1 ; 1 Zoology and Physiology, University of Wyoming, Laramie, WY 

The budding yeast Saccharomyces cerevisiae has long been used as a model system to study 

all aspects of biology. Majority of the studies, however, are conducted in liquid culture. Yeast 

cells, when growing on solid medium, form organized colony structure. Cells inside the colony 

develop and differentiate into a mature structure under normal conditions. Colony cells may die 

when the colony encounters severe stress (internal, such as mutation or external, like drought). 

The objective of this study is to understand the mechanism of the development, differentiation 

and death of yeast colony. Using transmission electron microscopy (TEM), we showed that for a 

4 day-colony, the dividing cells are mostly located at the periphery and the bottom of the colony, 

where cells are in direct contact with the nutrient. Dividing cells are also observed at the top 

surface of the colony, where cells are far away from the nutrient. The non-dividing cells are 

mostly located at the middle portion of the colony. TEM analyses of mutants that are defective 

of colony growth and development, including chc1 and sro7/sro77 double deletion, revealed that 

cell death occurs mostly in the middle portion of the colony. Our results suggest the cells in the 

middle portion of the colony play an important, supporting role for the yeast colony development 

and differentiation. They likely cease to divide, or even die, to support the proliferation of other 

cells inside the colony. 

2314 

An immunoglobulin domain-containing protein, IGP, is involved in trafficking of FGFR1. 

S. Kim 1 , J. Liu 1 , G. Yagnik 1 , A. Stoner 1 , C. Stevens 1 , E. Cherkez 1 , J. kim 1 , S. Boyadjiev 1 ; 

1 UCDMC MIND Inst, Sacramento, CA 

Fibroblast growth factor receptors (FGFRs) are critical for cell growth, differentiation, and animal 

development. Mutations in this receptor family are associated with craniofacial defects and 

cancer. In this study, we characterize an immunoglobulin domain-containing protein (IGP) that 

influences stability and function of FGFR1. When IGP and FGFR1 are coexpressed, their levels 

are increased compared to individual expression. In addition, IGP enhances conversion of 

FGFR1 from ER forms to Golgi forms. Furthermore, IGP enhances phosphorylation of PLCgamma, 

but does not affect other FGFR1 signaling components such as AKT and ERK1/2. 

Interestingly, IGP predominantly exists as ER forms. These results indicate that IGP facilitates 

ER export of FGFR1 and activates the FGFR1-mediated PLC-gamma signaling pathway. 

Because aberrant activation of the FGFR signaling pathways is implicated in craniosynostosis 

(CS, premature fusion of cranial sutures), we have screened IGP for rare mutations from a 

cohort of nonsyndromic CS patients (431 individuals) who do not carry mutations in the 

previously known CS-causing genes. We have identified three rare missense mutations from 

this screening. Strikingly, one of these mutant IGPs further enhances phosphorylation of PLCgamma. 

In summary, IGP plays a critical role in ER export/maturation/signaling of FGFR1. 

Furthermore, our results further suggest that IGP mutations may be a risk factor for CS.

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2315 

Sonic Hedge Hog Pathway Expression in Normal and Megabladder Mice During 

Development. 

K. R. DeSouza 1 , M. Saha 1 , A. R. Carpenter 1 , K. M. McHugh 1 ; 1 Center for Molecular & Human 

Genetics, The Research Institute at Nationwide Children's Hospital, Columbus, OH 

In this study, we present the first comprehensive assessment of the expression pattern of the 

Sonic Hedge Hog (shh) signaling pathway in the bladders of normal mice at consecutive stages 

during development. Additionally, we compared the expression pattern of the shh signaling 

pathway in megabladder (mgb-/-) mice to wild type mice to determine if this critical signaling 

pathway is altered in the mutant bladders of these animals. 

We characterized the expression pattern of the shh pathway’s ligand (Shh), receptor (Ptch), and 

transcription factor (Gli) and the key smooth muscle regulatory gene, Myocardin (Myocd), in the 

bladders of male and female wild type and mgb-/- mutant mice at embryonic days 13.5, 14.5, 

15.5 and 16.5 (E13.5-E16.5), by in situ hybridization analysis. 

This study provides evidence that the shh pathway exhibits a canonical pattern of expression in 

wild type bladders, with each molecule displaying a specific spatial localization within the 

bladder. Shh expresses exclusively in the urothelium of the developing bladder. Ptch expresses 

in the proximal mesenchyme immediately adjacent to the urothelium, while Gli expresses in the 

distal mesenchyme, where smooth muscle cells eventually develop. Myocd, a key smooth 

muscle regulatory gene, expresses in the distal mesenchyme, where the presumptive smooth 

muscle develops in the bladder. Our study shows the shh pathway is functional in mgb-/- mutant 

bladders but shows a potential developmental delay with poorly restricted signal boundaries. In 

contrast, our results indicate that Myocd is minimally expressed in mgb-/- bladders when 

compared to controls. 

This study represents the first comprehensive analysis of the Shh signaling pathway during 

normal bladder development in mice. In addition, we show the shh pathway is functional in mgb- 

/- bladders but the key smooth muscle regulator, Myocd, is drastically reduced or absent. This 

finding suggests that the lack of detrusor smooth muscle development in mgb-/- bladders is not 

due absent short axis patterning but appears to result from a dramatic reduction in the 

expression of Myocd. 

2316 

Imbalance on cell proliferation and basal cell frequency of senile gerbil prostate: 

exposure effects of ethinylestradiol during the puberty. 

A. P. Perez 1 , M. F. Biancardi 1 , B. F. Gonçalves 1 , L. R. Falleiros Junior 2 , F. C. Santos 3 , S. R. 

Taboga 2 ; 1 Biology, Unicamp, Campinas, Brazil, 2 Biology, IBILCE/UNESP, São Joé do Rio Preto, 

Brazil, 3 Morphology, Federal University of Goias, Goiânia, Brazil 

Endocrine disruptor chemicals (EDCs) are exogenous compounds with the potential to disturb 

hormonal regulation. Male and female rodent exposed to EDCs during critical time points of 

development, such as prenatal and neonatal as well as puberty, affect the reproductive tract of 

both sexes. Some studies show that the prostate is a highly sensitive gland to the EDCs action 

that might increase the risk of emergence of prostatic pathologies with aging. Thus, this study 

examined the ethinylestradiol effects during puberal development on histological differentiation, 

cell proliferation and p63-positive cells frequency of senile gerbil male ventral prostate (VP) and 

female prostate. For this, male and female gerbil (Meriones unguiculatus) with 42 days old 

received by gavage 15µg/kg/day of 17α–ethinylestradiol (EE- component oral contraceptive) 

diluted in mineral oil during one week (Pub group) and control group (males and females) were 

remained untreated. When males and females of control and Pub groups completed 12 months 

of age, they were sacrificed and male VP and female prostate were retired, processed and

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histological sections for each experimental group were subjected to immunohistochemistry for 

proliferating (PCNA) and basal (p63) cells. Puberty exposure to ethinylestradiol promoted 

different alterations in the senile gerbil male VP and female prostate. The Pub group male VP 

presented a significant increase in percentage cell proliferation (25.65±3.19) compared to 

control group (10.57±1.37) mainly in regions with invasive lesions. While this increase was not 

observed in the Pub group female prostate. Regarding the prostatic basal cells frequency, was 

observed a significant increment in female Pub group (23.24±1.18) compared to control group 

(12.82±0.91), indicating difference in proportion of main cell populations in acinar epithelium. 

The Pub group male VP did not presented significant difference in this frequency. In conclusion 

the synthetic estrogen exposure during puberty causes aberrant proliferation in prostatic cells of 

senile male, while in female prostate was observed basal cells stratification, mainly in regions 

with epithelial hyperplasia. Although prostatic epithelium proliferation is regulated by androgen, 

the estrogen is also able to cause this event. Thus the synthetic estrogen exposure during 

prostatic morphogenesis can favor the emergence of invasive lesions mainly in senile male 

prostate. 

2317 

F3 ENU genetic screen – a quest for endothelial cells directional movement cues. 

S. Krispin 1 , J. Yu 2 , T. Mulligan 2 , A. Davis 2 , D. Castranova 2 , B. M. Weinstein 2 ; 1 Program in 

Genomics of Differentiation, National Institute of Child Health and Human Development, NIH 

Bethesda, MD, 2 National Institute of Child Health and Human Development, NIH Bethesda, MD 

Blood vessels are essential for tissue homeostasis within the complex bodies of vertebrates. 

Studies in developing vertebrates have uncovered many genes crucial for embryonic endothelial 

specification and for blood vessel differentiation and growth, but we still know little about what 

guides the patterning of developing blood vessels and determines the anatomical architecture of 

the vascular system. 

The metameric arrangement and relative anatomical simplicity of the zebrafish trunk primary 

angiogenic network makes it ideally suited to study the cues and mechanisms leading to the 

formation of vascular network. Of particular interest are the intersegmental vessels (ISVs), 

which emerge from the dorsal aorta (DA) and migrate dorsally along somites boundaries 

through angiogenesis. The cues that control this dorsal migration are still not understood. We 

have been using genetic screens to identify novel genes involved in endothelial cell (EC) 

directional movement. 

We used ENU (N-ethyl-N-nitrosourea) to induce random, single-base-pair mutations in the 

zebrafish genome, followed by a large-scale F3 genetic screen to uncover the homozygous 

vascular phenotypes of induced mutations. We facilitated the screening process by visualizing 

alterations in the vasculature primary network formation using a stable germline transgenic line, 

fli1-EGFP, which expresses enhanced green fluorescent protein in endothelial cells in vivo 

under the control of the fli1 promoter. We identified a new mutant, y210, in which trunk ISVs fail 

to migrate dorsally properly. We are in the process of mapping and positionally cloning the 

affected gene from the mutant, and then study its functional role in EC directional movements. 

Addressing this question will provide insight into one of the basic enigmas in 

developmental/vascular biology: what cues guide vessel positioning relative to other tissues and 

organs.

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2318 

Novel antibodies to track neurogenesis in planarians. 

K. G. Ross 1 , M. Taylor 1 , R. K. Munday 1 , A. Hubert 1 , R. M. Zayas 1 ; 1 Biology, San Diego State 

University, San Diego, CA 

Neural stem cells proliferate and integrate into the adult central nervous system (CNS) of 

diverse organisms, however there are gaps in our knowledge of the precise molecular 

mechanisms controlling these events. Planarian flatworms are amongst the simplest organisms 

to possess a CNS. Their nervous system is surprisingly complex at the molecular level, 

containing numerous diverse cell types, many of which demonstrate functional similarity with 

those found in vertebrates. Planarians maintain a population of adult pluripotent stem cells 

(neoblasts) that proliferate and differentiate in response to injury or during normal tissue 

homeostasis, enabling them to regenerate all of their tissues, including a complete nervous 

system. These features make planarians excellent models for in vivo studies of stem cell based 

CNS regeneration. However, many neurons in planarians have not been fully characterized and 

little is known about the spatial distribution of differentiating cells and temporal sequence of cell 

replacement during their regeneration. To improve our understanding of cell turnover and 

regeneration in the planarian CNS, we have performed a monoclonal antibody screen for 

neuronal markers in the planarian Schmidtea mediterranea. We have produced nine 

monoclonal antibodies thus far, each of which is specific to different planarian tissues, including 

discrete neuronal populations. We have characterized the staining patterns of these antibodies 

in intact and regenerating animals and are using them in combination with bromodeoxyuridine 

pulse-chase experiments to track neural differentiation in regenerating planarians. Our work has 

yielded new markers to help visualize CNS architecture and quantify differentiation of neuronal 

subpopulations. These analyses will help us understand how these animals maintain and 

reconstruct their nervous system and provide useful tools for phenotypic screening of gene 

knockdown experiments in planarians. This work was supported by CIRM Grant RN2-00940-1 

to RMZ. 

2319 

Imaging Epithelial Morphogenesis in the Developing Hair Placode. 

L. Ahtiainen 1 , S. Lefebvre 1 , P. Lindfors 1 , I. Thesleff 1 , M. L. Mikkola 1 ; 1 Institute of Biotechnology, 

University of Helsinki, Finland 

Development of different ectodermal appendages (hair follicle, mammary gland, tooth) shares 

similarities at the morphological and molecular level. All epidermal appendages begin as a 

thickening of the epithelium, a placode. The key signaling pathways regulating placode 

formation are rather well known, but the cellular mechanisms involved are still poorly 

understood. Two hypotheses have been proposed: local cellular proliferation or cell migration. 

Only very recently light microscopy imaging techniques have advanced to enable tracking of the 

fate of single cells in live tissue. This study aims at establishing and developing 3D and 4D live 

tissue imaging methodology for studying cellular processes regulating epithelial morphogenesis, 

in particular the role of the Ectodysplasin (Eda)/Eda receptor(Edar)/NF-κB signaling pathway in 

skin appendage development and disease. We have established laser scanning confocal 

imaging methodology that allows live visualization of E13.5-E14.5 mouse embryonic skin whole 

mount explants for up to 24h during the first wave of hair placode formation. As a tool we use 

K17-GFP embryonic mouse skin, where GFP expression driven by the K17 promoter marks 

emerging placode cells. We provide evidence indicating that epithelial cell migration is 

associated with hair placode formation and that inhibition of migration and actin remodeling 

suppresses placode formation. Imaging studies in the Fluorescent Cell Cycle Indicator (Fucci)

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mouse suggest that proliferation has a less prominent role in placode formation. Accordingly, 

inhibition of proliferation had no gross effect on timing or morphology of forming placodes. 

2320 

Multiparametric image analysis of lung branching morphogenesis. 

C. Schnatwinkel 1 , L. Niswander 1 ; 1 Pediatrics, Universtiy of Colorado, Denver/HHMI, Aurora, CO 

Morphogenesis remains a fascinating problem in biology and proper morphogenesis is required 

for the shape and function of all organs and tissues. However, the morphogenetic mechanisms 

by which the genome moulds developing tissue masses into specific shapes remain poorly 

understood. 

To provide the first insights into the cellular dynamics of lung branching, we developed a life 

imaging set up to study cellular dynamics during branching morphogenesis. This approach 

helped in many ways to understand the interpretation of various phenotypes we have observed 

in mutant lungs. In this study, we performed a systematic time-lapse analysis of two of the 

recently postulated branching subroutines domain branching and planar bifurcation of epithelial 

cells in both lung explant- and organotypic three-dimensional (3D) culture. Our analysis 

revealed morphological and molecular distinct stages during branching morphogenesis that 

occurred in normal cultured lungs. We have classified a single bifurcation event into four 

morphological and molecularly distinct stages. Throughout the stages, we noticed a proximal to 

distal increase in proliferation, as well as a consecutive reduction in bud size before undergoing 

another round of bifurcation. This phenomenon may be a potential reason why lungs in culture 

only have a limited branching capacity. The stages are the following: (1) The bud stage: the size 

of the initial epithelial bud is increased due to proliferation, which appears to occur throughout 

the epithelium. (2) The flattening stage: Initially the distal tip cells apply mechanical tension 

along the basal membrane, whereas the surrounding mesenchyme acts partially as a stabilizing 

scaffold. This step may be a checkpoint stage and is required to precisely position and 

determine the cleft region for the subsequent step. (3) The bifurcation stage: epithelial cells on 

the two sides of the bud move slightly outward and then towards the distal end of the lung 

explants. This movement is partially provided by proliferation along the epithelial axis, which 

leads to the extension of the monolayered epithelium. Cells in the cleft region seem not to 

respond to migrational cues and remain in place. (4) The outgrowing stage: The cells at the two 

sides of the bifurcating bud continue their directional growth. Previously lateral cells become tip 

cells and cleft cells stay behind and localize to what becomes the stalk. 

To address the role of distinct cellular behaviors, we applied pharmacological inhibitors. Lung 

explants treated with an inhibitor of cell proliferation and apoptosis, prevented bud growth but 

maintained the capability to branch. On the other hand, treatment of explants with inhibitors 

against MLCK to break force generating processes in the cell allowed the bud to continue 

growing but failed to bifurcate if the inhibitor was applied before the flattening stage. However, 

new formation of a bud from the stalk region (domain branch) was still present. In fact, blocking 

the ATPase activity of myosin by blebbistatin increased the number and location of domain 

branches. Among other findings, this suggests that proliferation is a key step in driving domain 

branch formation, whereas local changes in force generating mechanisms mold and divide the 

distal bud into two. 

Our finding defined a new set of parameters that can be applied to future studies with various 

types of mutant lungs as well as indicated that force generating mechanisms play an important 

role in the progression through the stages of branching morphogenesis.

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2321 

In vitro assessement of bioactivities of rmGDF-9 and rhBMP-15. 

L. Niu 1 , V. Hernandez 1 , J. Li 1 , V. Kalabokis 1 , G. Wu 1 , J. Humphrey 1 , R. Hao 1 ; 1 R&D Systems, 


Growth differentiation factor -9 (GDF-9) and bone morphogenetic protein -15 (BMP-15) are 

oocyte-derived growth factors that are well known to play important roles in regulating 

folliculogenesis and female fertility. However, recent publications implicating their biological 

functions in human cancers raised the questions of whether GDF-9 and BMP-15 exert diverse 

biological functions in non-ovarian tissues. We have purified recombinant mouse GDF-9 and 

human BMP-15 from Chinese Hamster Ovary cells and evaluated their bioactivities using an 

array of bioassays. As expected, both mouse GDF-9 and human BMP-15 bind to the 

recombinant human/mouse BMP-RII/Fc extracellular domain with Kd values of 1.4 nM and 2 nM 

respectively, in a functional ELISA. In addition, the two proteins activate Smad 2/3 in a mouse 

embryonic teratocarcinoma P19 cells. Furthermore, we discovered that the recombinant mouse 

GDF-9, similar to other members of the transforming growth factor -βfamily, is able to induce 

apoptosis of mink lung epithelia MV1Lu and prostate carcinoma DU145 cells. In contrast, the 

recombinant human BMP-15, like other BMP family members, is osteogenic, promoting 

differentiation of MC3T3-E1 cells to osteoblasts. We also found that human BMP-15 is very 

potent in supporting the survival and proliferation of NIH3T3 cells in nutrient deprived conditions. 

These in vitro differential functions of GDF-9 and BMP-15 provide the stepping stones for new 

research initiatives and also open a whole new avenue to explore their functions in cancer 

biology, in addition to the area of reproductive physiology. These studies in turn may eventually 

lead to an expanded role in their therapeutic potential. 

2322 

Reg1 and Reg3β expression in the pancreas of adult diabetic Goto-Kakizaki (GK) rats. 

J-C. Irminger 1 , M-H. Giroix 2 , S. Calderari 3 , J. Ehses 4 , J. Coulaud 2 , K. Rickenbach 1 , P. 

Serradas 3 , F. Homo-Delarche 2 ; 1 University of Geneva, Geneva, Switzerland, 2 University of 

Paris-Diderot, Paris, 3 University Pierre et Marie Curie, Paris, 4 University of British Columbia, 

Vancouver, BC, Canada 

Reg protein expression is associated with islet development, β-cell damage, diabetes and 

pancreatitis. We previously reported that islets of 4-month-old (4-mo) GK rats, a spontaneous 

model of type 2 diabetes, overexpress Reg1, 3α, 3β and 3γ vs age-matched Wistar control 

islets. Reg1 and 3β are involved in cell growth/survival control and inflammation, respectively. 

Diabetic GK islets also exhibit progressive inflammation reaction, consisting of high CCL2 

(MCP-1), CCL3 (MIP-1α), CXCL-1 (murine IL-8 analog) and IL-6 expression/release and mostly 

peri-islet macrophage infiltration. Importantly, Reg1 gene promoters contain IL-6-responsive 

elements. Here we analyzed in greater detail the pancreatic expression of Reg1 and Reg3β in 

diabetic GK rats. 

Isolated pancreatic islets and acinar tissue from male Wistar and GK rats were used for 

quantitative RT-PCR analysis. Islet IL-6, CCL2, CCL3 and CXCL1 release was measured by 

LuminexTM technology after a 48h islet culture on collagen. Pancreatic Reg 

immunohistochemistry (IHC) was performed on paraffin sections with a rabbit anti-human 

polyclonal antibody (Ab) and a mouse anti-rat monoclonal anti-Reg1 Ab. Macrophage infiltration 

was detected on cryostat sections using CD68 and MHC class II antibodies. Islet macrophage+ 

area was quantified and expressed as % of corresponding islet area. Statistical analyses used 

the Student’s t-test for unpaired data. 

The exocrine/endocrine ratio of Reg mRNA expression in 4-mo normoglycemic Wistar and 

diabetic GK rats was: 1) Reg1: 41.3±2.4 and 5.0±1.5, respectively (n=3 different

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isolations/group, p

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formation we are using the zebrafish gut as model system. The zebrafish gut tube forms from a 

solid rod of endodermal cells that differentiate and start to polarize and assemble tight junctions 

which give rise to multiple small lumens. These small lumens subsequently enlarge though the 

accumulation of fluid and eventually coalesce to form a single lumen. However, other cellular 

processes such as the remodeling of cell contacts must also be involved to facilitate 

coalescence into a single lumen. We are interested in these additional cellular processes that 

regulate the transition into a single lumen. Analysis of lumen formation in wild-type (WT) 

embryos revealed that the coalescence of multiple small lumens into one involves the formation 

of a double lumen intermediate. Interestingly, we found that smoothened (smo) mutants often 

fail to properly form a single lumen and instead display a double lumen phenotype. In smo 

mutants, cells are frequently found in between the two lumens and display mislocalization of 

adhesion proteins which suggests a defect in the remodeling of adhesive contacts. We analyzed 

the transcriptome of smo mutants and found that several genes associated with intracellular 

trafficking were down-regulated. In light of this finding, we generated transgenic zebrafish lines 

to determine the function of trafficking and recycling in the process of single lumen formation. 

Our analyses suggest that that intracellular membrane recycling is required for single lumen 

formation. Together, our data suggest that smo regulates a transitional step in single lumen 

formation that facilitates intercellular contact remodeling through recycling. 

Cancer Cell Biology II 

2325 

Rapamycin treatment increases mTORC2 activity and selectively activates the AKT1 

isoform in epidermis by inhibition of the mTORC1-dependent feedback loop to IRS-1. 

K. Sully 1 , O. A. Akinduro 1 , M. P. Philpott 1 , C. A. Harwood 1 , R. F. O'Shaughnessy 2 , C. Byrne 1 ; 

1 Cutaneous Research, Queen Mary University of London, London, United Kingdom, 

2 Immunobiology, UCL Institute of Child Health, London, United Kingdom 

Epidermal squamous cell carcinoma (SCC) is dramatically increased in patients undergoing 

immunosuppression following organ transplantation. The mammalian target of rapamycin 

complex 1 (mTORC1) inhibitor, rapamycin, when used as a post-transplantation 

immunosuppressive, significantly reduces SCCs, though the mechanism is not understood. 

Epidermal SCC is associated with changes in AKT signaling. AKT2, associated with less 

differentiated keratinocytes, is upregulated in SCC and in response to ultraviolet (UV) radiation, 

suggesting a tumour-promoting role. In contrast, AKT1, associated with differentiated 

keratinocytes, is downregulated in SCC and after UV radiation, suggesting a tumour-suppressor 

role. In some tumour types rapamycin activates AKT signaling via inhibition of mTORC1dependent 

negative feedback loops acting on the insulin receptor substrate-1 (IRS-1). It is 

unclear whether this feedback loop is active in epidermis. Upregulation of epidermal AKT 

signaling by rapamycin would have different effects on tumourigenesis depending on which AKT 

isoform is upregulated. The objective was to establish whether the mTORC1-dependent 

negative feedback loop to IRS-1 is active in epidermis and find which epidermal AKT isoform is 

affected. 

We show that rapamycin increases keratinocyte AKT phosphorylation at both S473 and T308, 

the two sites required for full activation, with a single treatment of rapamycin maintaining 

increased AKT phosphorylation for over 24 hours. Both acute and prolonged rapamycin 

treatment also increases epidermal mTORC2 activity, shown by upregulation of downstream 

targets. We demonstrate that the mTORC1-dependent negative feedback loop to IRS-1 is active 

in keratinocytes and responsible for rapamycin-induced mTORC2 activity and AKT 

phosphorylation. Rapamycin inhibits mTORC1-dependent phosphorylation of IRS-1 at

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Ser636/639 and increases IRS-1 tyrosine phosphorylation, protein stability and downstream 

signaling to AKT. Epidermal AKT1 isoform, but not AKT2, is selectively upregulated via 

rapamycin’s effects on epidermal mTORC2 and inhibition of the mTORC1-dependent feedback 

loop to IRS-1. 

These findings suggest a mechanism for rapamycin’s anti-tumour activity in skin. We propose 

that rapamycin’s selective activation of epidermal AKT1 promotes AKT1’s tumour-suppressing 

role in epidermis and protects against epidermal tumourigenesis. 

2326 

Gab2 regulates the migratory behaviors and E-cadherin expression via activation of the 

PI3K pathway in ovarian cancer cells. 

Y. Wang 1 , Q. Sheng 2 , M. A. Spillman 3 , K. Behbakht 3 , H. Gu 1 ; 1 Department of Pathology, 

University of Colorado Denver Anschutz Med Campus, Aurora, CO, 2 Department of Cancer 

Biology, Dana-Farber Cancer Institute, and Harvard Medical School, Boston, MA, 3 Department 

of Obstetrics and Gynecology, University of Colorado Denver Anschutz Med Campus, Aurora, 

CO 

Ovarian cancer, the most deadly gynecologic malignancy, is often diagnosed late and at the 

advanced stage when the cancer cells have already migrated and invaded into other tissues 

and organs. Better understanding of the mechanism of metastasis in ovarian cancer cells is 

essential to the design of effective therapy. In this study, we investigated the function of 

scaffolding adaptor protein Gab2 in ovarian cancer cells. Gab2 is found to be overexpressed in 

a subset of ovarian tumors and cancer cell lines. Gab2 expression mainly regulates the 

migratory behaviors of ovarian cancer cells. Overexpression of Gab2 promotes the migration 

and invasion, and down-regulates E-cadherin expression in ovarian cancer cells with low-Gab2 

expression. Conversely, knockdown of Gab2 expression inhibits the migration and invasion, and 

promotes E-cadherin expression in ovarian cancer cells with high-Gab2 expression. By 

expressing Gab2 wild type and Gab2 mutants that are defective in activation the PI3K and 

Shp2-Erk pathways, we find that Gab2 inhibits E-cadherin expression and enhances the 

expression of Zeb1, a transcription factor involved in epithelial-to-mesenchymal transition 

(EMT), and cell migration and invasion through the activation of the PI3K pathway. Knockdown 

of Zeb1 expression blocks Gab2-induced suppression of E-cadherin expression and increase in 

cell invasion. LY294002 and GDC-0941, inhibitors of PI3K, or Rapamycin, an inhibitor of PI3K 

downstream target mTOR, can reverse the effects of Gab2 on migration and invasion. Overall, 

our studies reveal that Gab2 overexpression, via activation of the PI3K-Zeb1 pathway, promotes 

characteristics of EMT in ovarian cancer cells. Ovarian cancer cells with high Gab2 expression 

are potentially more metastatic in vivo and drugs that target the PI3K pathway may be used to 

treat a subset of ovarian cancer with high Gab2 expression. 

2327 

LRIG2 promotes PDGF induced experimental glioma. 

C. Holmlund 1 , T. Karlsson 1 , M. Faraz 1 , O. Casar Borota 2,3 , J. Miller 4 , B. Wang 1 , C. Sweeney 4 , R. 

Henriksson 1 , H. Hedman 1 ; 1 Radiation Sciences, Oncology, Umeå, Sweden, 2 Medical 

Biosciences, Pathology, Umeå, Sweden, 3 Laboratory Medicine, Pathology/Cytology, Umeå, 

Sweden, 4 University of California, UC Davis Medical Center, Sacramento, CA 

Leucine-rich and immunoglobulin-like domains protein 2 (LRIG2) is a transmembrane protein, 

whose expression is associated with poor survival of oligodendroglioma patients. This is in 

contrast with findings for the homologous protein LRIG1, a negative regulator of growth factor 

signaling, whose expression is associated with good survival in several different malignancies. 

Here, we address the role of LRIG2 in an experimental glioma model and its possible regulation

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of platelet-derived growth factor (PDGF) receptor (PDGFR). Gliomas were induced in Ntv-a 

mice by intracranial injection of PDGFB-expressing RCAS viruses. All injected Lrig2-wild-type 

mice developed oligodendroglioma-like brain tumors of grade II/III (82%) or glioblastoma-like 

tumors of grade IV (18%), whereas Lrig2-deficient mice only developed grade II/III tumors (77%) 

or no detectable tumors (23%). In transfected cells, LRIG2 and PDGFRα co-localized in 

intracellular vesicle-like structures. Intriguingly, LRIG2, in contrast to LRIG1, did not negatively 

regulate the protein levels of PDGFRα or PDGFRβ. Instead, LRIG2 up-regulated the protein 

levels of PDGFRα. In summary, we show that LRIG2 promoted the genesis and/or progression 

of PDGF-induced oligodendroglioma and positively regulated PDGFRα protein levels. This 

further supports the notion that LRIG proteins play important roles in cancer biology and 

suggests that LRIG1 and LRIG2 may have opposing functions in the regulation of PDGF 

signaling. 

2328 

The Study of Androgen Effects on LPA-induced VEGF-C Expression in Prostate Cancer 

Cell Lines. 

Y-C. Lin 1 , H. Lee 2 ; 1 Institute of Zoology, National Taiwan University, Taipei, Taiwan, 

2 Department of Life Science, National Taiwan University, Taipei, Taiwan 

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that binds to a family of G proteincoupled 

receptors (GPCRs), termed LPA1~LPA5. It has been reported that vascular endothelial 

growth factor-C (VEGF-C) was related to the lymph node metastasis in human prostate 

cancer. Our previous study has indicated that LPA induces the expression of VEGF-C in human 

prostate cancer PC-3 cell line. Moreover, a well-known effector, androgen, modulating prostate 

cancer cell progression was also shown to negatively regulate VEGF-C expression in prostate 

cancer. Therefore, we attempt to investigate whether LPA-induced VEGF-C expression is 

affected by androgen in different prostate cancer cells. By real-time PCR analysis, we proved 

that LPA could trigger VEGF-C expression in dosage and time-dependent manners in different 

prostate cancer cell lines. In addition, our results further showed that androgen agonist 

decreased LPA-induced VEGF-C expression. On the contrary, androgen antagonist enhanced 

LPA-induced VEGF-C expression. In summary, our results demonstrated that androgen was 

involved in LPA-induced VEGF-C expression in human prostate cancer cells. 

2329 

Mitochondrial Genome Instability and ROS Enhance Intestinal Tumorigenesis in 

APCMin/+ mice. 

D. Woo 1 , P. Green 2 , J. Santos 2 , A. D’Souza 3 , Z. Walther 3 , D. Martin 4 , B. Christian 3 , N. Chandel 5 , 

G. Shadel 3 ; 1 Gyeongsang National University, Jinju, Korea, 2 University of Medicine and 

Dentistry, New Jersey-New Jersey Medical School, Newark, NJ, 3 Yale University School of 

Medicine, New Haven, CT, 4 Emory University School of Medicine, Atlanta, GA, 5 Northwestern 

Medical School, Chicago, IL 

Alterations in mitochondrial oxidative phosphorylation have long been documented in tumors. 

Other types of mitochondrial dysfunction, including altered reactive oxygen species (ROS) 

production and apoptosis, can also contribute to tumorigenesis and cancer phenotypes. 

Furthermore, mitochondria contain mtDNA, mutation and altered amounts of which have been 

observed in cancer cells. However, how mtDNA instability per se contributes to cancer remains 

largely undetermined. Tfam is a mitochondrial transcription factor and mtDNA-packaging protein 

required for expression and maintenance of mtDNA. Tfam heterozygous knock-out (Tfam +/- ) 

mice exhibit mild mtDNA depletion, but have no overt phenotypes. Here, we show that Tfam +/- 

mouse cells and tissues not only have less mtDNA, but also increased oxidative mtDNA

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damage. Crossing Tfam +/- mice to the APC Min/+ mouse cancer model revealed that mtDNA 

instability increases tumor number and growth in the small intestine. This was not due to 

enhancement of Wnt/ -catenin signaling, but rather appears to involve a propensity for 

increased mitochondrial ROS production. Direct involvement of mitochondrial ROS in intestinal 

tumorigenesis was demonstrated by crossing APC Min/+ mice to those that have catalase targeted 

to mitochondria (MCAT), which resulted in a significant reduction in tumorigenesis in the colon. 

Altogether, these results show that mitochondrial genome instability and ROS can enhance 

tumorigenesis, and elucidate Tfam +/- mice as relevant model to address the role of mtDNA 

instability in disease states where mitochondrial dysfunction is implicated, such as cancer, 

neurodegeneration and aging. 

2330 

Upregulation of Plk1 in castration-resistant prostate cancer cells. 

A. Deeraksa 1 , J. Pan 1 , Y. Sha 1 , X. Liu 1 , A. C. Ferrari 2 , N. T. Eissa 1 , S-H. Lin 3 , L-Y. Yu-Lee 1 ; 

1 Medicine, Baylor College of Medicine, Houston, TX, 2 Medicine, New York University School of 

Medicine, New York, NY, 3 Molecular Pathology, The University of Texas M. D. Anderson 

Cancer Center, Houston, TX 

Castration-resistant progression of prostate cancer (PCa) is a challenge in PCa treatment. A 

recent study found that PCa cells cultured in charcoal-stripped media (androgen-independent AI 

condition) have undergone a genetic reprogramming that selectively upregulated the expression 

of M-phase cell cycle genes. We found that the mitotic kinase Plk1 is upregulated during the 

reprogramming process in AI PCa cells. We employed a pair of PCa cells, LNCaP (androgendependent 

PCa cells derived from lymph node metastasis) and LNCaP-AI cells (AI PCa cells 

derived from LNCaP), to determine the role of Plk1 in castration-resistant progression of PCa. 

We found that LNCaP-AI cells express 3 – 5 fold higher levels of Plk1 relative to LNCaP cells. 

Cell cycle proteins along the Plk1-regulated mitotic pathway, including Cdc25C (mitotic entry), 

Sgo2 (chromosome cohesion), PICH (chromosome architecture), BubR1 (spindle checkpoint), 

and NudC (cytokinesis) are all elevated in LNCaP-AI cells. Both LNCaP and LNCaP-AI cells 

were highly sensitive to a 5-day treatment with a small molecule inhibitor of Plk1, BI2536, with 

IC50 of 0.6 nM and 0.2 nM, respectively, compared to many other cell types with IC50 between 

10 – 50 nM. Under charcoal-stripped culture conditions, LNCaP did not grow or respond to 

BI2536 treatment. In contrast, LNCaP-AI cells remained highly sensitive to BI2536 inhibition, 

suggesting that the upregulated Plk1 pathway is inhibited by BI2536 in LNCaP-AI cells. 

We further examined whether growth inhibition by BI2536 leads to cell death. In full medium 

culture conditions, LNCaP cells responded to BI2536 with caspase 3 cleavage and membrane 

blebbing, characteristics of apoptosis. In contrast, LNCaP-AI cells did not show caspase 3 

cleavage but exhibited PARP-1 cleavage, a common end point for cell death. 

Immunofluorescence microscopy showed unusual nuclear morphologies in BI2536-treated cells. 

LNCaP cells contained a single enlarged/lobular nucleus, a phenomenon previously observed 

with Plk1 inhibition in other cell types. In contrast, LNCaP-AI cells formed giant cells that 

contained up to 30 small and large independent nuclei within a single cell unit, likely due to 

nuclear fragmentation and cytokinesis failure. Live cell imaging indicate that these giant cells 

underwent membrane disintegration and cell death characteristic of necroptosis. These results 

suggest that LNCaP-AI cells utilize a caspase 3-independent cell death modality in response to 

BI2536 inhibition. These observations suggest that Plk1 has a novel role in cell growth/death 

regulation in LNCaP-AI cells and Plk1 may be targeted for therapy of castration-resistant PCa.

2331 

HPV E6 oncoproteins from high and low risk types associate with the proteasome 

independently of E6AP. 

L. M. Banks 1 , V. Tomaic 1 ; 1 ICGEB, Trieste, Italy 

TUESDAY 

Human papillomavirus (HPV) E6 oncoproteins associate with components of the ubiquitin 

proteasome pathway, including the cellular ubiqutin ligases E6AP, EDD and HERC2. E6AP was 

shown to play a major role in E6’s degradatory activities, while more recent studies suggest that 

its presence is also critical for E6 protein stability. In addition, it has been suggested that E6’s 

association with EDD could have a profound effect upon the ability of E6 to target various 

substrates for proteolytic degradation and thereby directly influence the development of HPVinduced 

malignancy. To further investigate E6 function, we performed a mass spectrometric 

analysis on HPV-18 E6 and E6AP protein complexes. Besides identifying several known binding 

partners of these two proteins, we also identified several proteasome regulatory subunits as 

being associated with E6 and E6AP. We also show that both HPV-18 E6 and E6AP, 

independently of each other, interact with proteasome regulatory subunits in vitro. HPV-18 E6 

preferentially interacts with S2 and S4 subunits, while E6AP interacts with S2, S4, S5a, S7 and 

S8 subunits, an interaction that is independent of E6AP’s catalytic activity. Furthermore, HPV-16 

E6 and HPV-11 E6 also preferentially interact with the S2 and S4 subunits, although the HPV- 

16 E6 interaction is significantly stronger than that with HPV-11 E6. In presence of endogenous 

E6AP HPV-18 E6 also binds strongly to the subunit S5a, while binding to S2 and S4 subunits is 

reduced. These results demonstrate that E6AP can recruit E6 to the S5a subunit. Ongoing 

studies will now aim to elucidate these aspects further and clarify how E6AP mediates E6s’ 

association with the proteasome. 

2332 

Cyclin B1 and B2 have oncogenic properties but drive tumorigenesis through distinct 

mechanisms. 

H-J. Nam 1 , J. van Deursen 1,2 ; 1 Pediaric and Adolescent Medicine, Mayo Clinic, Rochester, 

Rochester, MN, 2 Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Rochester, MN 

High cyclin B1 and B2 levels are observed in a large proportion of human tumors and correlate 

with poor clinical outcome. However, the molecular and cellular defects caused by increased 

expression of these mitotic cyclins and their roles in tumorigenesis are unknown. To address 

these questions, we generated transgenic mice that overexpress cyclin B1 or B2. These mice 

show high aneuploidy rates, with cyclin B1 overexpression inducing chromatin bridges and 

cyclin B2 overexpression lagging chromosomes. Furthermore, cyclin B1 overexpression leads 

to persistent inhibition of separase, while cyclin B2 overexpression does not. Conversely, cyclin 

B2 overexpression induces supernumerary centrosomes, whereas cyclin B1 does not. These 

results demonstrate that both cyclin B1 and B2 induce chromosome missegregation, although 

through distinct mechanisms. Furthermore, preliminary results indicate that both cyclin B1 and 

B2 transgenic mice are prone to spontaneous tumors, although the tumor spectra of the 

transgenes are remarkably different. In addition, it seems that cyclin B2 overexpression, but not 

cyclin B1 overexpression, predisposes mice to DMBA-induced cancers. Taken together, the 

data suggest that both cyclin B1 and cyclin B2 drive chromosomal instability and tumorigenesis, 

but through markedly different mechanisms and in distinct tissues.

TUESDAY 

2333 

Reactive oxygen species regulates CDK2 activity differently in normal and cancer cells to 

induce senescence and proliferation. 

C. Hwang 1 , S-M. Lee 1 , K-S. Kwon 1 ; 1 Aging Research Center, KRIBB, Daejeon, Korea 

Reactive oxygen species (ROS) were previously shown to trigger cellular senescence through 

several pathways such as Ras, p38 MAPK , p66shc, Rb, p53, and p21 Cip1 . However, the detailed 

mechanisms remain to be elucidated. Previously we demonstrated that p21 Cip1 was degraded at 

an early phase after low dose H2O2 treatment in a variety of cell types. Here, we observed that 

low dose of H2O2 induced cellular senescence in HDFs and IMR90 cells but induced cellular 

proliferation in cancer cells, even though p21 Cip1 was degraded in both cases. Interestingly, 

normal cells showed sustained inhibition of CDK2 activity upon H2O2 stimulation, which is a 

likely mechanism in cell cycle arrest and subsequent senescence. However, H2O2 stimulation 

resulted in temporary CDK2 activation in cancer cells, which might cause H2O2-induced 

proliferation. Still unclear are how ROS control CDK2 activity differently in the normal and 

cancer cells. 

Key words: H2O2, CDK2, p21 Cip1 , senescence, proliferation, cell fate 

2334 

The nuclear translocation of hTERT is mediated by the classical importin alpha/betadependent 

import pathway. 

S. Jeong 1 , J. Lee 1 , I. Chung 1 ; 1 Department of Biology and Integrated Omics for Biomedical 

Science, WCU Program of Graduate School, Yonsei university, Seoul, Korea 

The maintenance of eukaryotic telomeres requires telomerase, which is minimally comprised of 

a telomerase reverse transcriptase (TERT) and an associated RNA component (TERC). 

Telomerase activity is tightly regulated by expression of hTERT at both the transcriptional and 

posttranslational levels. In our previous study, we identified the region of bipartite nuclear 

localization signal (NLS) of hTERT. However, the molecular mechanism of nuclear translocation 

of hTERT remains unclear. To investigate clear nuclear translocation mechanism, we minimized 

the NLS region of hTERT, and then this NLS region was conjugated to Glutathionine Stransferase 

(GST). Clearly, GST protein was diffused at both cytoplasm and nucleus. In contrast 

to GST protein, GST-conjugated wild type NLS protein was predominantly localized in the 

nucleus and GST-conjugated mutant (7A+S227A) NLS protein was not. To prove the molecular 

mechanism of nuclear translocation, we co-immunoprecipitated GST-conjugated wild-type NLS 

or endogenous hTERT with importin α/β1. And we also measured telomerase activity after 

importin alpha, beta and Ran overexpression by TRAP assay. 

We next investigated cells selectively depleted of importin alpha and beta1 by transfection with 

siRNAs. We observed the effect of importin siRNAs on endogenous hTERT by 

immunofluoresence analysis, subcellular fractionation assay and TRAP assay. These findings 

demonstrate that the nuclear translocation of hTERT is mediated by the classical importin α/βdependent 

import pathway. 

2335 

P300-mediated Acetylation of TRF2 is required for Telomere Maintenance. 

Y. Her 1 , I. Chung 1 ; 1 Department of Biology and Integrated Omics for Biomedical Science, WCU 

Program of Graduate School, Yonsei University, Seoul, Korea 

Human telomeres are protected from DNA damage by a nucleoprotein complex that includes 

the repeat-binding factor TRF2. The Abundance of TRF2 protein at telomeres is tightly

TUESDAY 

regulated by post-translational modifications, protein-protein interactions and protein 

stabilization. Here we report that TRF2 is physically interacts with p300 and its lysine 293 is 

acetylated by p300 in vitro and in vivo. Whereas overexpression of p300 increases the level of 

telomere-bound TRF2, depletion of endogenous p300 expression decreases the level of 

telomere-bound TRF2. We show that loss of TRF2 from telomeres results in ubiquitination and 

degradation of TRF2 by the proteasome. In addition, we found that p300-mediated TRF2 

stabilization is dependent of p300 catalytic activity, using either the inactive form of p300 or the 

acetylation mutant TRF2-K293R. Depletion of p300 in cancer cells induced DNA-damage 

response foci at internal genome as well as telomeres as evidenced by telomere dysfunctioninduced 

foci, indistinguishable from TRF2-K293R stably expressing cell lines. Some of the 

metaphase chromosomes showed no telomeric signal at chromatid ends, suggesting an 

aberrant telomere structure. Furthermore, we found that stable overexpression of K293R 

induces growth arrest in human cancer cells, suggesting that K283R may control cell 

proliferation. Taken together, these results suggest p300-mediated acetylation of TRF2 is 

required for the efficient telomere binding of TRF2 as well as telomere protection and represents 

a new pathway for telomere maintenance by modulating the level of TRF2 at telomeres. 

2336 

Human chromosomal passenger Borealin co-exists with active telomerase and regulates 

telomerase activity. 

P. Khadka 1 , J. Lee 1 , S. Beak 1 , I. Chung 1 ; 1 Department of Biology and Integrated Omics for 

biomedical science, WCU program of Graduate school, Yonsei University, Seoul, Korea 

From different studies it has shown that the conventional DNA metabolism machineries directly 

or indirectly regulate telomerase activity and telomere, still it is not clearly known how DNA 

replication proteins regulate telomerase. Here, we report the role of borealin, member of 

chromosomal passenger, which binds with hTERT and regulates telomerase activity. Borealin is 

a member of the chromosomal passenger complex that plays important role as a regulator of 

DNA replication, chromosome alignment, and spindle assembly checkpoint. Here, we show that 

borealin co-exists with active telomerase in vivo via telomeric DNA in telomerase positive cells 

and telomerase-immortalized human somatic cells but not in human ALT cells. In the present 

study, we demonstrate that hTERT interacts with borealin both in vitro and in vivo. Here, we 

show that the association of borealin with hTERT is independent of RNA but requires DNA and 

also we show that N-terminal of hTERT binds to borealin. Furthermore, we show that borealin 

depletion by RNA interference reduces the telomerase activity inducing the cell growth arrest in 

MCF7 cells. Borealin has until now been described as a component of chromosomal passenger, 

mainly involved in DNA replication, here, we confirmed that about 30%-40% of borealin can bind 

to telomeric end. This is, to our knowledge, the first evidence that Borealin coexist with active 

telomerase and regulates telomerase activity, revealing a not yet recognized link of this CPC 

protein to telomere. 

2337 

PIAS1 and RNF4 regulate the homeostasis of SUMO-conjugated TRF2. 

J. Her 1 , I. Chung 1 ; 1 Department of Biology and Integrated Omics for biomedical science, WCU 

program of Graduate school, Yonsei University, Seoul, Korea 

Eukaryotic chromosome end is composed of DNA and proteins, called telomere and forms T 

loop to protect chromosome ends. TRF2 is a major component of sheltrin complex and function 

to prevent loss of genetic materials and shelter chromosome end to avoid DNA damage 

machinery. Without TRF2, DNA damage signal occurs and cells go to apoptosis. To avoid it, 

TRF2 mostly binds to telomere and is very stable. So we think that there should be complex

TUESDAY 

mechanism to degrade. Sheltrin complex proteins are regulated by several post translational 

modifications. Among these modifications, SUMOylation is one of essential modifications in ALT 

cells. It is known that TRF1 and TRF2 are sumoylated by MMS21 and their SUMOylation is 

required for APB formation in ALT cells but it is not clear what the function of TRF1 and TRF2 

SUMOylation is in telomerase positive cancer cells. Here, we found that PIAS1 is another 

SUMO E3 ligase for TRF2 and SUMOylated TRF2 is regulated by proteosomal degradation. 

SUMO-specific E3 ubiquitin ligase, RNF4, is also involved in SUMOylated TRF2 degradtion and 

control the levels of SUMOylated TRF2. 

2338 

Disruption of CTCF at the miR-125b1 locus in gynecological cancers. 

E. Soto-Reyes 1 , R. Gonzalez 1 , F. Cisneros-Soberanis 1 , C. Castro 2 , D. Cantú 1 , F. Recillas- 

Targa 3 , L. A. Herrera 4,5 ; 1 Dirección de Investigación, Instituto Nacional de Cancerología, 

Mexico, Mexico, 2 Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas 

UNAM, Mexico, 3 Biología Molecular, Instituto de Fisiología Celular UNAM, 4 Instituto Nacional de 

Cancerología, Mexico, 5 Instituto de Investigaciones Biomédicas UNAM, Mexico 

In cancer cells, transcriptional gene silencing has been associated with genetic and epigenetic 

defects. The disruption of DNA methylation patterns and covalent histone marks has been 

associated with cancer development. Until recently, microRNA (miRNA) gene silencing was not 

well understood. In particular, miR-125b1 has been suggested to be an miRNA with tumor 

suppressor activity, and it has been shown to be deregulated in various human cancers. Herein, 

we characterized the CCCTC-binding factor (CTCF) at the miR-125b1 locus in normal and 

breast cancer cells. The disruption of CTCF in breast cancer cells correlated with the 

incorporation of repressive histone marks such H3K9me3 and H3K27me3 as well as with 

aberrant DNA methylation patterns. To determine the effect of DNA methylation at the CpG 

island of miR-125b1 on the expression of this gene, we performed a qRT-PCR assay. We 

observed a significant reduction on the expression of miR-125b1 in cancer cells in comparison 

with controls, suggesting that DNA methylation at the CpG island might reduce miR-125b1 

expression. These effects were observed in other gynecological cancers, including ovarian and 

cervical tumors. Our data strongly suggest that the loss of CTCF may contribute to the 

destabilization of chromatin permissive for transcription and the establishment of an aberrant 

repressive chromatin configuration, including DNA hypermethylation, that induce the epigenetic 

silencing of the miR-125b1 locus. 

This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT: 83959) 

and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica of the 

Universidad Nacional Autónoma de México (PAPIIT, IN213311). 

2339 

Cancer cell proliferation regulated by DOT1L histone methyltransferase. 

W. KIM 1 , R. KIM 1 , G. PARK 1 , J. KIM 1 ; 1 Kyung Hee University, Seoul, Korea 

DOT1L, a histone methyltransferase for histone H3 K79, participates in the regulation of 

transcription, development, erythropoiesis, differentiation and proliferation of normal cells. With 

respect to cancer, DOT1L-mediated methylation contributes to development of MLL-rearranged 

leukemias. However, the role of DOT1L in cancer cell proliferation has not been fully elucidated. 

DOT1L siRNA-transfected A549 and NCI-H1299 lung cancer cells displayed a non-proliferating 

multinucleated phenotype. DOT1L-deficient cells also showed abnormal mitotic spindle 

formation and centrosome number, suggesting that DOT1L deficiency leads to chromosomal 

missegregation. This chromosomal instability led to cell cycle arrest at the G1 phase and

TUESDAY 

induced senescence determined by enhanced activity of senescence-associated (galactosidase 

activity. Meanwhile, overexpression of siRNA-resistant DOT1L, not a catalytically 

inactive mutant, substantially restores DOT1L siRNA-induced phenotypes. Overall, these data 

imply that downregulation of DOT1L-mediated histone H3 Lys79 methylation inhibits 

proliferation of cancer cells. Therefore, the inhibition of DOT1L activity might act as a barrier to 

aggressive growth of cancer cells. 

2340 

Class 3 semaphorins and their receptor complexes- neuropilin/plexins regulated by E2F1 

and SOX4 in pancreatic ductal adenocarcinoma. 

H-Y. Huang 1,2 , Y-Y. Cheng 1 , P-H. Huang 1,2 ; 1 Department of Pathology, National Taiwan 

University Hospital, Taipei, Taiwan, 2 Institute of Pathology, National Taiwan University, College 

of Medicine, Taipei, Taiwan 

Ligand/receptor co-expression can allow cells to respond to growth and survival demand 

through signal transduction in a certain circumstance, like Eph/Ephrin co- expression modulates 

axon guidance in the developing central nervous system 

Individual members of class 3 semaphorins (SEMA3), secreted cues for axonal growth cone 

guidance molecules, are frequently over-expressed with their receptor complexes 

plexins/neuropilins (PLXNs/NRPs) in tumors such as breast cancer, hepatocellular carcinoma, 

pancreatic cancer, etc. We thoroughly examined expression of SEMA3 and their receptors in 

pancreatic ductal adenocarcinoma (PDAC) and found that, unlike other epithelial malignancies 

that often over-express one or few specific members of SEMA3/PLXN family, combined 

expression of more than 5 members of SEMA3/Plexin family are noted in most human PDAC 

cases. Analysis of promoter regions of SEMA3, plexins and NRPs revealed that putative binding 

sites of E2F1 and SOX4 are almost always present in all SEMA3 and plexin promoters. With 

chromatin-immunoprecipitation, luciferase promoter activity assay, and electrophoresis mobility 

shift assay, E2F1 and SOX4 were demonstrated to directly bind the consensus sites at the 

promoter of each SEMA3 and plexin gene we examined to enhance the transcriptional activity. 

Statistical analysis further showed correlation between SOX4 expression and poor survival in 

human PDAC. Conversely, RNAi-knockdown of SOX4 resulted in concomitant decrease in 

expression level of SEMA3/plexin family members and accentuated tumor growth by affecting 

cell proliferation. 

2341 

microRNAs regulating matrix metalloproteinase 2 activities. 

S-Y. Choi 1 , J. Kim 1 , B. Shin 1 ; 1 Chonnam National University Medical School, Gwangju, Korea 

MMP-2 plays an important role in tumor cell invasion and metastasis. Because microRNAs 

(miRNAs) have recently been reported to be implicated in these processes, we set out to 

determine a role for miRNAs regulating tumor cell invasion and metastasis. We screened 215 

human miRNAs for upregulating MMP2 activities in human fibrosarcoma HT1080 cells. 

Transfection of HT1080 cells with miRNAs followed by zelatin zymography identified 14 miRNAs 

raising MMP2 activities. Of the 14 miRNAs, further analyses with Western blotting, luciferase 

assay and Matrigel invasion assay pointed to miR-105 and miR-128 as final candidates. We will 

perform in vivo pulmonary metastasis assay in mice under gain-of-function and loss-of-function 

contexts to validate the effect of miR-105 and miR-128 on tumor cell invasion and metastasis.

TUESDAY 

2342 

Profiling of linker histone variants in ovarian cancer. 

M. Medrzycki 1 , Y. Zhang 1 , J. McDonald 1 , Y. Fan 1 ; 1 Georgia Institute of Technology, Atlanta, GA 

H1 linker histones play a key role in facilitating higher order chromatin folding. There are 11 H1 

variants in mammals that are differentially regulated during development and cellular 

differentiation. Emerging evidence suggests that H1 and its specific H1 variants are important 

epigenetic factors in modulating chromatin function and gene expression. Ovarian cancer is a 

devastating disease, ranking the fifth leading cause of all women cancer death due to its poor 

prognosis and difficulty in early diagnosis. Although epigenetic alterations in ovarian cancers are 

being appreciated in general, the role of H1 in ovarian cancers has not been explored. Here, 

using quantitative RT-PCR assays, we systematically examined the expression levels of 7 H1 

genes in 33 human epithelial ovarian tumors. We found that the expression of H1.3 is markedly 

increased, whereas the expression levels of H10, H1.1, H1.4 and H1x are significantly reduced 

in the malignant adenocarcinomas compared to benign adenomas. Strikingly, ovarian 

adenocarcinomas and adenomas exhibit characteristic expression patterns, and expression 

profiling of these 7 H1 genes in tumor samples can discriminate adenocarcinomas vs. 

adenomas. We identified 6 H1 genes as the minimum gene set required for correct 

discrimination of ovarian adenomas and adenocarcinomas with high accuracy. These findings 

indicate that the expression of H1 variants is exquisitely regulated and may serve as potential 

epigenetic biomarkers for ovarian cancer. 

2343 

Investigating the contribution of centrosome amplification in tumorigenesis. 

B. D. Vitre 1 , A. J. Holland 1 , Y. Wang 1 , D. W. Cleveland 1,2 ; 1 Ludwig Institute for Cancer Research, 

La Jolla, CA, 2 Department of Cellular and Molecular Medicine, University of California San 

Diego, La Jolla, CA 

As the major microtubule organizing centers, centrosomes play a central role in facilitating the 

formation of a bipolar mitotic spindle. Defects in centrosome biogenesis can induce an abnormal 

centrosome number and may lead to chromosome missegregation and subsequent aneuploidy. 

Although aneuploidy is an extremely common feature of tumor cells, its status as a cause or a 

consequence of cancer is highly controversial. In vertebrates and invertebrates, the conserved 

protein kinase Polo-like kinase 4 (Plk4) plays a key role in initiating centriole duplication and 

overexpression of Plk4 promotes the formation of extra centrosomes. Here we will describe the 

construction of two mouse models in which centrosome amplification can be induced through 

conditional overexpression of Plk4. We have made use of both a doxycycline-inducible promoter 

and the Cre-LoxP system to allow reversible and non-reversible expression of Plk4 in a tissue 

specific manner. These mice will be used to study the contribution of centrosome amplification 

in tumorigenesis. 

2344 

LEKTI re-expression up-regulates p53 and p21 in OSC-19, a metastatic squamous cell 

carcinoma. 

A. Iarrobino 1,2 , T. Shellenberger 3 , S. Klemann 2,3 ; 1 School of Medicine, University of Central 

Florida, Orlando, FL, 2 Rollins College, Winter Park, FL, 3 MD Anderson Cancer Center - Orlando, 

Orlando, FL 

Lymphoepithelial Kazal Type Inhibitor (LEKTI) is a 15-domain serine proteinase inhibitor with 

constitutive expression in squamous mucosa and with reduced or absent expression in the 

majority of head and neck squamous cell carcinomas (HNSCC) and cell lines. The loss of

TUESDAY 

expression may alter dynamics in the extracellular matrix of the tumor environment to favor local 

invasion and metastasis. Re-expression of LEKTI in HNSCC lines alters cell behavior in vitro 

and reduces local invasion in vivo. p53 is a well-characterized tumor suppressor gene with lost 

expression in many cancers, including HNSCC. In the context of examining LEKTI and matrix 

metalloproteinase expression in the HNSCC line OSC-19, we found the expression of p53 was 

restored upon stable transfection of OSC-19 with LEKTI to induce re-expression. Using OSC-19 

parental, vector, and clones re-expressing LEKTI (LEKTI-17 and LEKTI-25), semi-quantitative 

and quantitative analysis demonstrate restored expression of p53 in OSC-19 cells after 

transfection with LEKTI and cultivation both in vitro and in vivo (murine flank and orthotopic 

tongue tumor models). With re-expression of LEKTI and the subsequent restoration of p53, the 

expression of p21 was in turn up regulated. Differential expression of LEKTI was noted for 

parental cells cultivated in vitro and in vivo. Whereas LEKTI expression did not take place in 

vitro, its expression took place in both tumor types. We found p53 and p21 expression in 

xenografts derived from parental cells and both OSC-19 clones transfected to express LEKTI. 

Whenever LEKTI was expressed, re-expression of p53 and p21 was observed. Lastly, in vitro 

treatment of parental and vector cell lines with conditioned medium containing secretory factors 

of LEKTI-25 cells resulted in the re-expression of p53 and p21. Our results demonstrate the 

integrity of signaling pathways through which extracellular factors act and highlight the 

importance of the tumor microenvironment in regulating downstream mediators involved in the 

biologic behavior of OSC-19. [The James and Esther King New Investigator Award of the 

Florida Biomedical Research Program (TS) and the Critchfield Fund of Rollins College (SK) 

funded this work.] 

Normal and Diseased Organs and Therapeutics 

2345 

Therapeutic Multiple Exon-skipping Using Cell-penetrating Morpholinos for Dystrophic 

Dogs. 

T. Yokota 1 , T. Nagata 2 , A. Nakamura 3 , N. Urasawa 4 , T. Saito 2 , R. Kole 5 , P. Sazani 5 , T. 

Partridge 6 , E. Hoffman 6 , S. Takeda 2 ; 1 University of Alberta, Edmonton, AB, Canada, 2 National 

Center of Neurology and Psychiatry, Japan, 3 Shinshu University, Japan, 4 Nagano Red Cross 

Hospital, Japan, 5 AVI Biopharma, 6 Children's National Medical Center, Washington, DC 

Duchenne muscular dystrophy (DMD), the most common and fatal X-linked myopathy, and its 

milder form, Becker muscular dystrophy (BMD), are caused by mutations in the dystrophin 

(DMD) gene. Antisense-mediated exon skipping therapy is currently one of the most promising 

molecular therapies for DMD. The exon skipping leads to the production of internally deleted inframe 

mRNA transcripts but the truncated quasi-dystrophin retains some functions like BMD. 

Previously we demonstrated the first successful exon-skipping treatment in body-wide skeletal 

muscles in Canine X-linked muscular dystrophy (CXMD) using a cocktail of phosphorodiamidate 

morpholino oligomers (PMOs, morpholinos) targeting exon 6 and exon 8 of dystrophin mRNA. 

However, unmodified (bare) morpholino injections led to inefficient delivery to the heart, and 

dystrophin induction was barely detectable in the cardiac muscle. Here, we sought to recover 

the dystrophin expression in cardiac muscles in dystrophic dogs using morpholinos conjugated 

with negatively charged arginine-rich cell-penetrating peptides (PPMOs). We demonstrated that 

the delivery moieties significantly improved dystrophin production in both skeletal and cardiac 

muscles. Intravenous injections with PPMOs restored dystrophin expression in cardiac muscles 

accompanied by ameliorated histology. No obvious toxicity was detected by blood tests and 

histology. Our results show the potential of PMO conjugates as therapeutic agents for DMD.

TUESDAY 

2346 

The Regenerative Response of Zebrafish Hearts to Long-Term Induced Exercise Stress. 

B. Schoffstall 1 , P. deVerteuil 1 , M. Jean 1 , N. Lopez 1 , J. Tapia 1 ; 1 Biology, Barry University, Miami 

Shores, FL 

While adult human cardiomyocytes are capable of low level cell division, this hyperplastic 

response is not sufficient to repair damaged cardiac tissue. Danio rerio (zebrafish) 

cardiomyocytes have recently been shown to be capable of proliferating at high levels to 

completely repair hearts damaged by apex amputation or cryoinjury; thus, these fish have 

become a popular model to study heart regeneration. We hypothesized that exposure of 

zebrafish to long-term cardiac overload stress would elicit a similar response without invasive 

surgery, to provide us with a simple cardiac regeneration research model. In addition, this model 

may have useful applications to human stress-induced cardiac responses, which can lead to 

pathological cardiac hypertrophy and ultimately death. We stressed zebrafish cardiac systems 

using a 10-week forced swimming exercise regimen, then compared fully remodeled hearts to 

non-exercised controls for physiological function and histological evidence of cell proliferation. 

We sought to determine if the zebrafish heart response to cardiac overload stress results in 

heart enlargement, and whether hypertrophy or hyperplasia is involved. We also used a 

zebrafish full-genome expression microarray to screen for genes that may be key initiators of a 

regenerative response. Adult zebrafish were forced to swim twice daily against a strong water 

current. After a period of 10 weeks, individual fish were measured for overall size; hearts were 

filmed to determine heart rate, ventricular surface area, and percent shortening fraction; and 

hearts were extracted for histological tissue sectioning, cDNA preparation, and analysis of total 

protein. Our results indicate that fully remodeled exercised zebrafish heart ventricles are ~32% 

larger than non-exercised (p

TUESDAY 

1) Malformation of great toes present at birth. 2) Progressive heterotropic ossification which 

starts at the first decade of life. Ossification is manifested by recurrent and painful swelling flare 

ups of soft tissues that involve fascias, ligaments, aponeuroses, tendons and skeletal muscles. 

A Mexican family with one member affected of FOP was studied. The patient with the illness 

was a 19 years old female, who started with this disease at the age of 8 years old; she 

developed spontaneous and painful swelling of the right scapular area accompanied of 

functional limitation for movement, among clinical laboratory abnormalities it was found alkaline 

phosphatase activity increased. By radiography there was abnormal calcification and "pseudoexostosis 

dependent of ligament ossification at the site of attachment to the long bones. 

Mutation analysis was carried out using genomic DNA from peripheral blood lymphocytes; 

samples were assessed by PCR amplification using exon flanking primers (Shore et al, Nature 

Genetics 38; 525), PCR products were digested with respective restriction enzymes 1h at 37°C. 

Main results were obtained with the exon 4 flanking primers and Cac81 restriction enzyme, this 

gave a 253 bp product that carries the ACVR1 617→A mutation which causes aminoacid 

substitution of arginine by histidine in codon 206. ACVR1 gene encodes a type I bone 

morphogenic protein (BMP) transmembrane receptor, and mutation of this gene results in 

dysregulation of BMP signaling. This rare case is a novo mutation since her relatives were 

clinically healty and molecular analysis did not show ACVR1 617→A mutation. 

2348 

TGF-β and BAFF derived from CD4+CD25+Foxp3+ T cells mediate mouse IgA isotype 

switching. 

G-Y. Seo 1 , P-H. Kim 1 ; 1 Department of Molecular Bioscience, Kangwon National University, 

Chuncheon, Korea 

TGF-β is generally accepted as the physiological IgA isotype switch factor. Nevertheless, it is 

virtually unknown what kinds of cells in mucus-associated lymphoid tissue (MALT) mainly 

provide this cytokine to B cells. Regulatory T cells (Tregs) has immune suppressive activity by 

secretion of inhibitory cytokine such as TGF-β and IL-10. Thus, it is plausible that Tregs may be 

involved in IgA class switching recombination (CSR) in MALT. We explored, in the present 

study, the possibility that CD4 + CD25 + T cells facilitate IgA CSR in mouse B cells. 

CD4 + CD25 + Foxp3 + T cells were more stimulatory in IgA production by the cocultured splenic B 

cells than CD4 + CD25 - Foxp3 - T cells, and this effect was markedly abrogated by anti-TGFβAb. 

This was paralleled by an increase of germ line transcriptα (GLTα), an indicative of IgA CSR. In 

contrast, CD4 + CD25 - Foxp3 - T cells were more potent in induction of GLTγ1 and GLTε by the 

cocultured splenic B cells. Consistent to these results, phenotypic analyses revealed that TGF-β 

and IL-4 were predominantly expressed by CD4 + CD25 + Foxp3 + T cells and CD4 + CD25 - Foxp3 - T 

cells, respectively. Further, we found that CD4 + CD25 + T cells strongly express BAFF leading to 

AID expression in B cells. Taken together, our results suggest that CD4 + CD25 + T cells may 

have an important effect on IgA isotype commitment through TGF-β and BAFF in MALT.

2349 

Anti-platelet activity of yuzu and its components is mediated by inhibition of TXA2 

production. 

H. Yu 1,2 , S. Park 3 , I. Chung 4 , Y-S. Jung 1,5 ; 1 College of Pharmacy, Ajou University School, 

Suwon, Korea, 2 Brain Korea 21 for Medical Sciences, Ajou University, Suwon, Korea, 

3 Molecular Biotechnology, Konkuk University, Seoul, Korea, 4 Applied Life Science, Konkuk 

University, Seoul, Korea, 5 Brain Korea 21 for Molecular Science and Technology, Ajou 

University, Suwon, Korea 

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The platelet activation and subsequent platelet aggregation play an essential role in the 

pathogenesis of atherothrombotic disease such as cardiovascular, cerebrovascular, and 

peripheral vascular diseases. Food can be one of the most important factors that influence risks 

for atherothrombotic diseases. Yuzu included in citrus is one candidate that may benefit the 

cardiovascular system. In this study, we investigated whether the methanolic extract of yuzu 

(yuzu ME) and its components (hesperidin and naringin) have anti-platelet activities. Yuzu ME 

and hesperidin inhibited collagen-, arachidonic acid (AA)-, ADP- and thrombin-induced rat 

platelet aggregation in vitro and ex vivo. Naringin also inhibited platelet aggregation induced by 

collagen, AA, or thrombin, but not aggregation induced by ADP. The oral administration of yuzu 

ME or hesperidin prolonged mouse tail vein bleeding time in a dose-dependent manner in vivo. 

In addition, yuzu ME including hesperidin and naringin inhibited collagen and thrombin-induced 

platelet aggregation by interfering with TXA2 production as established by the measurement of 

TXB2. These results suggest that yuzu ME and its component such as hesperidin and naringin 

have anti-platelet activity, and that intake of yuzu, which includes various flavonoids such as 

hesperidin, may be beneficial for individuals at high risk of cardiovascular diseases. This 

research was supported by the Technology Development Program (#308013-3) for ′Food′ 

Ministry for Food, Agriculture, Forestry and Fisheries, and the Cooperative Research Program 

for Agriculture Science & Technology Development (Project No. PJ006986) Rural Development 

Administration, Republic of Korea. 

2350 

Dehydroepiandrosterone inhibits the activation and dysfunction of endothelial cells 

induced by high glucose concentration. 

E. Huerta García 1 , J. L. Ventura Gallegos 2,3 , M. E. Crescencio Victoriano 1 , A. Montiel Dávalos 1,4 , 

G. Tinoco Jaramillo 5 , R. López-Marure 6 ; 1 Biología Celular, Instituto Nacional de Cardiología 

“Ignacio Chávez”, México, 2 Departamento de Bioquímica, Instituto Nacional de Ciencias 

Médicas y Nutrición "Salvador Zubirán", México, 3 Departamento de Medicina Genómica y 

Toxicología Ambiental, Instituto de Investigaciones Biomédicas, UNAM, Mexico, 4 Subdirección 

de Investigación Básica, Instituto Nacional de Cancerología, México, 5 Jefatura de Ginecología, 

Hospital General de Zona 2A “Troncoso”, IMSS, México, 6 Biología Celular, Instituto Nacional de 

Cardiología “Ignacio Chávez”, México City, Mexico 

Dehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes; 

however, its mechanisms of action are unknown. Here, we focus on the effect of DHEA on the 

activation of endothelial cells induced by a high concentration of glucose. The adhesion on 

U937 cells, the expression of adhesion molecules, the production of ROS and NO, and the 

translocation of NF-[kappa]B were evaluated in human umbilical vein endothelial cells (HUVEC) 

treated with high concentrations of glucose, DHEA, and both. High concentrations of glucose (> 

20 mM) induced an increase in adhesion, an increase in mainly E-selectin and PECAM-1 

expression, ROS and NO production, translocation of NF-[kappa]B, and degradation of its 

inhibitor I[kappa]B-[alpha]. DHEA abolished adhesion and the increase of E-selectin, ICAM-1, 

VCAM-1, and PECAM-1 induced by glucose. In addition, DHEA completely blocked oxidative

TUESDAY 

stress and decreased translocation of NF-[kappa]B and the degradation of I[kappa]B-[alpha] 

induced by glucose. These results suggest that DHEA protects against the activation of 

endothelial cells induced by high concentrations of glucose, indicating that DHEA could be 

useful in the treatment of hyperglycemia and diabetes. 

2351 

Activin A stimulates mouse antigen presenting cells to express APRIL and BAFF. 

G. Seo 1 , P-H. Kim 1 ; 1 Department of Molecular Bioscience, Kangwon National University, 

Chuncheon, Korea 

The TNF family ligands APRIL and BAFF are primarily expressed by macrophages and 

dendritic cells and stimulate the differentiation and survival of B cells and their Ig production. In 

this study, we investigated the role of activin A in APRIL and BAFF expression by mouse 

antigen presenting cells and the signaling mechanism involved. Activin A markedly enhanced 

APRIL expression in mouse macrophages at both the transcriptional and protein levels. 

Overexpression of DN-Smad3 and SB431542 abrogated activin-induced APRIL transcription. 

Furthermore, activin A induced Smad3 phosphorylation. These results indicate that activin A 

enhances APRIL expression through both ALK4 and Smad3. In a subsequent analysis of activin 

A signaling, it was found that PD98059, an ERK inhibitor, eliminated activin A-induced APRIL 

expression. On the other hand, overexpression of CREB, a molecule downstream of ERK, 

augmented activin A-induced APRIL expression, and this effect could be abolished by 

PD98059. This finding that activin A induces ERK and CREB phosphorylation suggests that 

ERK and CREB act as intermediates in APRIL expression. Therefore, these results 

demonstrate that activin A can enhance APRIL expression through two different pathways, 

Smad3 and ERK/CREB. On the other hand, activin A also enhanced BAFF expression in mouse 

macrophages and dendritic cells at both the transcriptional and protein levels. Overexpression 

of DN-Smad3 and SB431542 abrogated activin-induced APRIL transcription. These results 

demonstrate that activin A can enhance BAFF expression through ALK4-Smad3 pathway. 

Taken together, these results suggest that activin A can modulate mouse APCs to express 

APRIL and BAFF. 

2352 

High-fat diet during sexual maturation influences the epithelium cell turnover kinetics in 

the adult rat ventral prostate. 

E. Z. Pytlowanciv 1 , D. L. Ribeiro 2 , R. M. Góes 3 ; 1 Biology Institute, State University of Campinas 

(Unicamp), Campinas, Brazil, 2 Histology Sector, Institute of Biomedical Sciences, Federal 

University of Uberlandia (UFU), Uberlândia, Brazil, 3 Department of Biology, University of 

Estadual Paulista (Unesp), São José do Rio Preto, Brazil 

The obesity epidemic has been recognized as one of the major global health problems and is 

associated to several diseases including reproductive problems. Various consequences of 

obesity in the male genital tract have been related to changes in sex steroid hormones. 

Although this could be applied to prostate, the influence of obesity and high-fat ingestion to the 

prostate histophysiology is unknown. The aim of this study was to evaluate if a high-fat diet (HF) 

interferes on rat ventral prostate pubertal maturation and harm the histology of adult gland. Male 

Wistar rats (4w old) were fed for 3, 6 or 9 weeks (HF3, 6 and 9) with high-fat diet (20%fat, 

4.9kcal/g) whereas control animals (C3, 6 and 9) received balanced chow (4%fat, 3.2Kcal/g). 

Ventral prostate was analyzed using stereological, immunocytochemical and western blotting 

methods. The relative proportion of tissue gland components were maintained in HF, except for 

an 1.1-fold increase in acinar epithelium volume at HF6. Morphological analyses indicated that 

acinar epithelium was significantly higher in HF groups, more pleated and exhibited mild diffuse

TUESDAY 

epithelial hyperplasia. The high cellularity observed in acinar epithelium of all HF-treated groups 

is evidenced by a significant rise in cell densities. HF during sexual maturation promoted a 4fold 

increment in cell proliferation as well as a 4-fold reduction in apoptosis rate and also 

elevated circulating testosterone serum levels in HF9 compared to C9 group. 

Immunocytochemistry indicated an increase in cells expressing AR under all HF conditions. 

However, the western blotting analyses surprisingly pointed to a tendency of 110kDa-AR band 

down expression and 45kDa band over expression in prostate crude protein extracts. This may 

be due to primary polyclonal antibody non-specific cross reaction and the small molecular 

weight bands found on prostate extracts possibly correspond to AR isoforms. Interestingly, there 

was an inverse correlation between 110kDa AR and smaller band expression in HF3 and HF9, 

which may suggest a fat dietary interference on differential expression of AR isoforms. The 

circulating androgen elevation, could explain the cell proliferation increment, apoptosis reduction 

and high prostate growth observed during HF treatment, since the androgens are the main 

factors responsible for maintaining the homeostasis of the prostate, through the activation of its 

receptor (AR). Finally, these experiments indicate that HF diet-treatment during puberty 

influences prostatic maturation causing hiperplastic alterations and higher gland volume at 

adulthood. In addition, the quantity of satured fat in diet interferes in the cell epithelium turnover 

kinetics in the adult prostate. 

2353 

Effect of peripheral serotonin on glucose uptake in murine brown adipocyte. 

H. Watanabe 1 , K. Saito 1 , R. Saito 1 , T. Nakano 1 , N. Okada 1 , K. Sumiyoshi 1 , S. Ohwada 1 , K. 

Watanabe 1 , H. Aso 1 ; 1 Tohoku University, Miyagiken Sendaishi, Japan 

Serotonin is a neurotransmitter synthesized in the raphe nuclei of the brain stem and involved in 

the central control of food intake, sleep and mood. Serotonin is also a peripheral hormone 

produced by enterochromaffin cells in the intestine and involved in vasoconstriction, 

haemostasis and immune system. Serotonin is synthesized by two distinct tryptophan 

hydroxylase (TPH) rate-limiting enzyme in brain (TPH2) and in peripheral (TPH1). As serotonin 

is inability to cross the blood-brain barrier, there are two serotonin systems in brain and 

periphery with independent functions. We revealed that serotonin increased plasma glucose 

concentration through inhibiting glucose uptake from blood to tissue. However, the functions of 

serotonin in periphery have not yet been fully elucidated. 

The major thermogenic center is the brown adipose tissue (BAT), which is composed of 

multilocular lipid droplets and a large number of mitochondria. Thus, BAT is known as an 

important tissue in the regulation of body weight and energy metabolism. In this study, we have 

established a clonal murine brown preadipocyte (MBP) line and investigated the function of 

serotonin on glucose metabolism in MBP cells. After fasted mice were intraperitoneally injected 

with 1 mg serotonin, the glucose uptake in BAT was elevated. Next, we tried to reveal the effect 

of serotonin on glucose uptake in BAT using established MBP cells. MBP cells showed the 

characteristic of BAT followed induction of differentiation, such as multilocular lipid droplets and 

the expressions of BAT related genes including uncoupling protein 1 (UCP-1). Additionally, the 

stimulation of serotonin induced the increase of glucose uptake in MBP cells in a dose 

dependent manner. To determine what kind of serotonin receptors were related to the glucose 

uptake, we confirmed the expression of several serotonin receptors and measured the glucose 

uptake in MBP cells using three kinds of serotonin receptor antagonist: SB-204070 (5HT4), Ro- 

04-6790 (5HT6) and methysergide (5HT1, 5HT2 and 5HT7). The pretreatment of serotonin 

antagonist SB-204070 inhibited the glucose uptake in MBP cells after serotonin treatment. 

These data indicate that serotonin regulates a glucose metabolism in BAT through serotonin 4 

receptor.

TUESDAY 

2354 

Topical application of capsaicin induces adipokine secretion in visceral adipose tissues. 

G-R. Lee 1 , D-J. Yoon 2 , I-S. Han 2 ; 1 Medical Sciences, University of Ulsan, Ulsan, Korea, 

2 Biological Sciences, University of Ulsan, Ulsan, Korea 

We attempted to determine whether topical application of capsaicin can protect fat accumulation 

in visceral adipose tissues. We first observed that the topical application of 0.075% capsaicin to 

male obese mice fed a high-fat diet significantly reduced weight gain and visceral fat compared 

to those of untreated obese control mice (p < 0.001). Fat cells were markedly reduced in 

mesenteric and epididymal adipose tissues of the mice treated with capsaicin cream. Capsaicin 

treatment lowered serum levels of fasting glucose, total cholesterol, and triglycerides. 

Immunoblot analysis and RT-PCR revealed increased adiponectin expression in capsaicintreated 

mice. In addition, application of capsaicin to obese mice downregulated mRNA levels of 

TNF-ƒÑ and IL-6 but upregulated other adipokines including PPARs,visfatin, and adipsin. These 

results indicate that topical application of capsaicin to obese mice limited fat accumulation in 

adipose tissues and may contribute to insulin sensitivity and anti-inflammation. 

2355 

Interleukin-6 / Stat3 axis mediates S100a9 expression in the colonic epithelial cells in a 

mouse model of ulcerative colitis. 

J-K. Lee 1 , M-J. Lee 1 , J. Choi 1 , C-H. Cho 1 , S-K. Ye 1 ; 1 Department of Pharmacology, Seoul 

National University College of Medicine, Seoul, Korea 

In the intestinal epithelium lining, the mucosal surface is a key player in homeostasis. However, 

when the balance is disturbed, it might lead to intestinal inflammation, including ulcerative colitis 

(UC). Interleukin-6 (IL-6) and the signal transducer and activator of transcription 3 (STAT3) 

phosphorylation have been reported to be highly expressed in the colonic tissues in UC. Also, 

S100A9, as a fecal biomarker, is correlated with the severity of UC. However, the expression 

mechanism of S100A9 in colonic epithelial cells (CECs) remains elusive. Here, a mouse model 

of UC was developed to investigate the linkage of IL-6 and S100a9 in CECs. We injected 

sgp130Fc or siSTAT3/CH-NP into dextran sodium sulfate (DSS)–treated mice and observed a 

markedly decreased S100a9 expression in CECs compared to those treated with PBS or 

siNegative/CH-NP. Furthermore, the administration of JAK2 or STAT3 inhibitors post IL-6 

stimulation into Caco-2 indicated that IL-6-mediated S100A9 expression occurs through STAT3 

activation. Also, we found that STAT3 binds directly to the S100A9 promoter and enhances its 

activity. We provide insights on the role of S100a9, which may recruit granulocytes into inflamed 

colon tissues. Thus, we conclude that elevated S100a9 expression mediated by IL-6/STAT3 in 

CECs plays an important role in the manifestation of UC. 

2356 

Immune cell monitoring using a handheld cell counter. 

A. Cappione 1 , N. Thirumalapura 2 , E. Crossley 2 , D. Hoover 1 ; 1 EMD Millipore, Danvers, MA, 

2 University of Texas Medical Branch, Galveston, TX 

Biological samples, such as primary isolates or cultured cells, are often comprised of a phenotypically heterogeneous 

population. The ability to discriminate subsets and determine their frequencies (and concentrations) is critical to many aspects 

of research. Cellular complexity is typically resolved by flow cytometry using panels of protein-specific fluorescent antibodies. 

Notably, many cell states are also uniquely distinguishable on the basis of size alone. 

Cell counting devices, operating via the principle of Coulter-based particle detection, permit the determination cell diameter 

measurements at the submicron level, as well as providing accurate concentrations. We describe three examples where a 

handheld cell counter provided qualitative assessment of individual population frequencies in complex cell mixtures. These

TUESDAY 

include: (1) Determination of lymphocyte and monocyte concentrations and their relative frequency 

in PBMC isolates, (2) Assessment of T-cell activation as a function of cell size shift in PBMC cultures, and (3) 

Identification of changes in splenocyte population dynamics in a murine model of Human Monocytotropic Ehrlichiosis (HME). 

Following cell harvest, single cell suspensions were prepared prior to acquisition. All samples 

were also stained with cell-specific fluorescent antibodies and analyzed by flow cytometry. In 

PBMC, the cell counter identified the lymphocyte and monocyte fractions as distinctly sized 

peaks (7.2 and 10.0µm, respectively). Across the 9 samples tested, Counter-derived frequency 

and concentration values for each subset were consistent with flow cytometry. In PBMC cultures 

co-stimulated with anti-CD3/CD28 antibodies, the counter confirmed the presence of a larger 

activated T-cell fraction (7.3 and 12.4 µm, respectively); the increase in cell size was coincident 

with expression of CD25. In the murine HME model, a change in the relative frequency of 

splenic erythroid (4.1µm) to lymphocyte (6.2µm) fractions was shown to be due to an increase 

in the Ter119+ erythroid fraction. 

The availability of simplified methods for monitoring changes in culture dynamics is very useful 

for accelerating research in the areas of diagnostics and drug design. While flow cytometry 

provides a more sophisticated and quantitative cell analysis platform, our findings indicate that 

cell counting devices can fill this role providing in-line insight into cellular responses. 

2357 

Effect of IL-12 on Negative Selection in the TNC microenvironment. 

S-A. Miller 1 , S. C. Henley 1 , F. R. Davis 1 , R. H. Lewis 1 , G. C. Bernard 1 , S. Samuels 1 , M. T. 

Martinez 1 ; 1 Biology, Tuskegee University, Tuskegee, AL 

Thymic nurse cells (TNCs) are cortical epithelial cells of the thymus that appear to be significant 

participants in the negative selection of thymocytes. TNCs express both class I and class II 

major histocompatibility (MHC) proteins on their cell surfaces. TNCs internalize thymocytes that 

are CD4 + CD8 + TCR lo (triple positive) into cytoplasmic vacuoles. These thymocytes are at the 

developmental stage where they undergo MHC restriction. In addition to thymocytes, TNC 

vacuoles also contain macrophages that interact intimately with the thymocyte subset. Greater 

than 95% of the TNC-interactive thymocyte population has been reported to undergo apoptosis 

within TNC vacuoles suggesting a strong role for TNCs in the negative selection of thymocytes. 

Triple positive thymocytes are reported to be non-responsive to a variety of cytokines including 

IL-6, IL-7, IL-10, IL-15 and IFN-γ. However, these thymocytes were found to be responsive to 

IL-12. Although IL-12 promotes pro-inflammatory responses in the periphery it was shown to 

significantly influence the deletion of the triple positive thymocyte subset in the thymus. We 

therefore hypothesized that within the TNC microenvironment IL-12 can safely facilitate negative 

selection. We used cOVA-TCR transgenic (Tg), D011.10 mice that recognize the cOVA 323-339 

peptide to analyze negative selection within the TNC microenvironment. The Tg mice were 

injected with different combinations of anti-IL-12 antibody or rat IgG2a antibody and/or cOVA 

peptide over a four day period. TNCs and thymocyte populations were harvested and analyzed 

for apoptotis using Annexin V with flow cytometry and TUNEL with fluorescent microscopy. 

Unexpectedly, there were no observable differences in thymocyte apoptosis within the TNCs of 

mice injected with rat-IgG2a, anti-IL-12, or those that were not injected. These data suggest that 

IL-12 does not play a role in negative selection within the TNC microenvironment.

TUESDAY 

2358 

Overexpression of human alpha-1 antitrypsin (AAT) in PiZZ liver reduces the 

polymerization and facilitates secretion in vitro and in vivo. 

K. Xiao 1,2 , L. Wang 1 , Y. Lu 3 , R. Oshins 1 , R. L. Bridges 1 , E. J. McAndrew 1 , A. Huegel 1 , S. Song 3 , 

A. D. Fu 4 , C. Liu 4 , F. Rouhani 1 , M. L. Brantly 1 ; 1 Department of Medicine, University of Florida, 

Gainesville, FL, 2 Genetics & Genomics Program, University of Florida, Gainesville, FL, 

3 Departments of Pharmaceutics, University of Florida, Gainsville, FL, 4 Dept of Pathology, 

University of Florida, Gainsville, FL 

The Alpha-1 Antitrypsin Deficiency (AATD) is one of the most prevalent inherited lung diseases 

next to the cystic fibrosis and also most common metabolic-genetic indication for pediatric liver 

transplantation. However, in heterozygote PiMZ patient, the liver damage is mild. To evaluate 

the effect of human wild (M) type AAT (MAAT) as a possible chaperone on the trafficking of 

secretion-incompetent PiZZ AAT protein (ZAAT), we established a PiZZ cell model from 

pediatric patient. Over-expression of human MAAT in this cell model prevents process of 

polymerization of ZAAT in the cytoplasma. The result was confirmed by AAT Western blot, 

immunostaining and electron microscope (EM). We further test this treatment on PiZ transgenic 

mouse through portal vein injection of 1 X 10 11 vg of AAV8 that expressing MAAT. The 

characteristic accumulation of ZAAT as polymer in hepatocyte has decrease sharply by polymer 

specific Ab staining and ELISA. Furthermore, the secretion of ZAAT in the serum increases 

about 5-fold after 12 weeks of treatment. The secretion of ZAAT is dosage dependent on the 

MAAT secretion level. Secretion of one molecular of MAAT brings out approximately one 

molecular of ZAAT. The purified MAAT can also successfully block the formation of polymer of 

ZAAT in vitro which explains the mechanism of this treatment. The average serum SGOT level, 

which reflects liver function, decreased about 21% after the treatment. Our result may be further 

tested on clinical trial in patients with AAT liver diseases. 

2359 

Identification of Serum miRNAs as Potential Biomarkers for Acute Myocardial Infarction 

P-H. Chu 1 , H-C. Chen 2 , S-J. Chen 2 ; 1 Chang Gung Memorial Hospital, Taipei, Taiwan, 2 Chang 

Gung Univeristy, Gueishan, Taoyuan 

It is important to find specific biomarkers to understand the pathophysiology and identify 

patients with ST-segment elevation myocardial infarction (STEMI). Recent studies have 

revealed the role of microRNAs (miRNAs) in a variety of biological and pathological processes 

including acute myocardial infarction (AMI). We hypothesized that circulating serum miRNAs 

may be useful in the diagnosis of STEMI. The aim of this study was to establish the ability to 

detect the miRNAs, and then to elucidate the relationship between miRNAs and STEMI. We 

initially profiled the differential expressions of 270 serum miRNAs, and identified potential serum 

microRNAs from 8 STEMI patients and matched healthy controls. We then used the candidate 

miRNAs to evaluate 62 patients, including 31 STEMI and 31 normal patients. The t-test and 

receiver operating characteristic (ROC) curves were established to discriminate AMI patients 

from the normal patients. P values less than 0.05 were defined as statistically significant. We 

initially identified 12 up-regulated miRNAs and 13 down-regulated miRNAs showing at least a 

two-fold change. Serum miR-486-3p, miR-150*, miR-126, miR-26a and miR-191 were validated. 

ROC analysis using the expression ratio of miR-486-3p and miR-191 showed an area under the 

curve of 0.863. Our findings implicate that serum miRNAs may be used as potential diagnostic 

biomarkers for STEMI.

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2360 

Therapeutic Potential of TGF-β2 -Treated Mesenchymal Stem Cells from Wharton′s Jelly 

in a Rat Myocardial Infarction Model. 

H-S. Wang 1 , P-C. Chu 1 ; 1 Anatomy, Natl Yang-Ming University, Taipei, Taiwan 

Myocardial infarction (MI) is one of the leading causes of morbidity and mortality worldwide. The 

development of stem cell therapy for treating MI patients is a contemporary challenge in the field 

of cardiovascular medicine. In this study, left anterior descending artery ligation rats were used 

as a myocardial infarction model. The induction of left anterior descending artery ligation was 

confirmed by electrocardiogram, echocardiography, and the presence of c-troponin I in the 

serum. To investigate the improved myocardial function after transplantation of undifferentiated 

and TGF-β2-treated cells, electrocardiogram, echocardiogram, Masson′s Trichrome staining, 

and immunohistochemical staining of heart tissues at different time points were assessed in the 

infracted rat hearts. Our results indicated that transplantation of undifferentiated or TGF-β2treated 

human umbilical cord mesenchymal stem cells from Wharton′s jelly both improve left 

ventricular systolic function, reduce the fibrotic area of heart, and increase the survival rate of MI 

rat model. These results suggested that deliver stem cells from human umbilical cord 

mesenchymal stem cells to sites of injury are able to ameliorate the dysfunctional heart failure.

SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

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