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

SUNDAY
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.)