cytoskeleton - Institut d'études scientifiques de Cargèse (IESC)

2010
22 Février
26 Février
CYTOSKELETON:
CONTRACTILITY
AND MOTILITY
Laurent BLANCHOIN
institut de Recherches en
Technologie et Sciences pour le Vivant -
iRTSV
CEA Grenoble
17, rue des Martyrs 38054 Grenoble
cedex 9
33(0)438784978
laurent.blanchoin@cea.fr
Direction scientifique :
Giovanna Chimini
Contact :
Dominique Donzella
tél : 04 95 26 80 40
www.iesc.univ-corse.fr

FEBS Advanced Lecture Course on
« Cytoskeleton, Contractility and
Motility »
Pierre Gilles de Gennes Winter School 2010
22 - 26 February 2010
Monday 22th February
Institut d'Etudes Scientifiques de Cargèse
Corsica, France
08h30 09h00
09h00 10h00
10h00 11h00
11h00 11h30
11h30 12h30
12h30 14h00
14h00 16h00
16h00 16h30
16h30 17h30
17h30 18h30
19h00
09h00 10h00
10h00 11h00
11h00 11h30
11h30 12h30
12h30 14h00
14h00 16h00
16h00 16h30
16h30 17h30
17h30 18h30
19h00
Participants welcome
Phong Tran University of Pennsylvania, USA, and Institut Curie, Paris, France
Cytoskeleton , Ce Polarity and Ce Shap
Michael Sixt Max Planck Institute, Martinsried, Germany
Force generation and force transduction in crawling ce s
Co ee break
David Kovar The University of Chicago, USA
Biochemistry of actin and actin binding proteins
Lunch
Oral presentations participants
Co e break
Laurent Blanchoin Institute of life sciences research and technologies iRTSV , Grenoble, France
Actin Dynamics
Jonathan Howard Max Planck Institute, Dresden, Germany
Motor proteins as nanomachines
Dinner
Tuesday 23th February
Enrique M. de la Cruz Molecular Biophysics & Biochemistry Department, Yale University, USA
Methods of equilibrium and transient kinetic analysis
Matthias Rief Technische Universität, München, Germany
How forces can be used to influence and investigate biomolecular kinetics in a complex energy landscape part 1
Co ee break
Gijsje Koenderink AMOLF, the Netherlands
Mechanical properties of active cytoskeletal networks part 1
Lunch
Poster presentations + Practical demonstrations
Co ee break
Pekka Lappailainen Institute of Biotechnology, Helsinki, Finland
Cytoskeleton membrane interactions
Marko Kaksonen EMBL, Heidelberg, Germany
Actin cytoskeleton in membrane trafic
Dinner

Oral Présentations
Names in alphabetical order
5

Timo BETZ : Timo.Betz@curie.fr
Laboratoire P.C.C. UMR 168, Paris, France
ATP-dependent mechanics of red blood cells
Msham BRITTO : mishanv@gmail.com
Dublin City University, Ireland
Moesin is a pivotal cytoskeletal modulator in vascular cells during
mechanotransduction, globally regulated by microRNAs
Sebastian FUERTHAUER : bastian@pks.mpg.de
Max Planck Institut für Physik komplexer Systeme, Dresden, Germany
Antisymmetric stress and the role of angular momentum conservation in complex
fluids
Florian HUBER : florian.huber@uni-leipzig.de
University of Leipzig, Germany
Linker Induced Actin Network Formation under Cell-Sized Confinement
Svenja-Marei KALISCH : s.kalisch@amolf.nl
FOM-Institute AMOLF Amsterdam, Netherlands
Against the wall: Microtubule dynamics is regulated by force and end-binding
proteins
Jimmy Le DIGABEL : j
jimmy.ledigabel@univ-paris-diderot.fr
Matière et Systèmes Complexes, Benoit Ladoux, Paris, France
Active substrates to study mechanotransduction
Chiu Fan LEE : cflee@pks.mpg.de
Max-Planck-Institute, Dresden, Germany
Protein amyloid self-assembly
Olga MARKOVA : markova@ibdm.univ-mrs.fr
IBDML – UMR 6216, Institute of Developmental Biology Marseilles
France
Hydrodynamic description of tissue elongation during Drosophila embryo
morphogenesis: the role of active anisotropic cytoskeletal stresses
Helen MATTHEWS : h.matthews@ucl.ac.uk
MRC Laboratory for Molecular Cell Biology, London; UK
Amsha PROAG : amsha.proag@univ-paris-diderot.fr
Laboratoire Matière et systèmes complexes UMR 7057, Paris, France
Microstructured chambers to study the sensitivity of cells to the geometry and
stiffness of their environment
6

Michal REICHMAN-FRIED : mreichm@uni-muenster.de
Institute of Cell Biology, ZMBE, Munster, Germany
Migration of primordial germ cell in zebrafish embryo
Nessy TANIA : ntania@math.ubc.ca
Department of Mathematics The University of British Columbia
Vancouver, Canada
Stanilas VINOPAL : vinopal@img.cas.cz
Institute of Molecular Genetics ASCR, Prague, Czech Republic
Localization of gamma-tubulin in nucleolus and its dynamics
7

Posters
Names in alphabetical order
9

Julia ARENS : julia.arens@mpi-dortmund.mpg.de
Max-Planck Institute of Molecular Physiology, Dortmund, Germany
A HIGH-CONTENT SCREEN TO IDENTIFY MICROTUBULE
REGULATORSINVOLVEDIN NEURITE INITIATION
During neurite initiation, microtubules are reorganized from a disordered array to form
thick bundles, which fill the neurite shaft. Microtubule associated proteins (MAPs) are
important regulators of this transition. Neuronal cells express MAPs, which modulate
various aspects of microtubule behavior, such as their dynamic stability, their physical
rigidity and force-generating interactions with each other as well as other cellular
compartments.
To evaluate their role in neurite initiation, we knocked-down all known MAPs in a stem
cell-like model system via mixtures of siRNAs, and quantified resulting changes in
neuronal morphology via automated fluorescence microscopy and image analysis. In
this screen, we identified 40 candidate MAPs, which show significant morphological
changes compared to control cells. Such changes include increases and decreases in
neurite length as well as enlarged neurite diameter. Of these primary candidates, 24
were confirmed in an independent screen using individual siRNAs. The confirmed
candidates include well known MAPs which were expected to play important roles in
neurite initiation, and new candidate regulators, for which no clear role in neurite
initiation has been proposed until now. We are currently performing follow-up studies,
which employ time-lapse microscopic imaging to characterize the dynamics of neurite
initiation in control and knockdown conditions.
10

Richard ARNOLDI : Richard.Arnoldi@unige.ch
Dpt of Pathology and Immunology, Faculty of Medicine, CMU
Geneva, Switzerland
SMOOTH MUSCLE ACTINS: TWO ISOFORMS FOR TWO DISTINCT FUNCTIONS?
Richard Arnoldi and Christine Chaponnier
Dpt of Pathology and Immunology, Faculty of Medicine, University of Geneva, CMU,
Geneva, Switzerland
a-SMA and g-SMA, the two smooth muscle actins which predominate in vascular and
enteric smooth muscle, respectively, are highly conserved across species, differing in
their sequence by only three amino acids. a-SMA has been extensively studied and
recognized for its “contractile” activity in several cell types including smooth muscle,
myofibroblasts, and myoepithelial cells. Anti-a-SMA (clone 1A4) has proven to be a
precious tool in pathology and basic research. Whether g-SMA is its equivalent in the
enteric system is not known mainly because of the lack of tools for an appropriate
investigation. We developed a new specific monoclonal antibody against g-SMA and
confirmed that g-SMA predominates in the enteric system and is minor in the vascular
system, although more expressed in highly compliant veins than in stiff arteries.
Contrary to a-SMA, g-SMA is completely absent from myofibroblasts within various
fibrosis and stromal reactions, as well as in vitro. g-SMA is present in myoepithelial
cells, but heterogeneously distributed. When g-SMA and a-SMA are coexpressed, our
experiments suggest that their subcellular locations are partly distinct, with a-SMA
contractile fibres extending throughout the entire cell and g-SMA fibres being restricted
to its central part. We raised the hypothesis that, whereas a-SMA is responsible for the
“contractile” activity (active tension), g-SMA would be involved in the “controlled
dilation” (passive tension) in SM and SM-like cells. Several models seem to support
this hypothesis, namely artery vs. vein, and the physiological modifications occurring in
the uterus and mammary glands during pregnancy and lactation. Our results suggest
that, in addition to enteric smooth muscles, g-SMA is expressed in all the tissues
submitted to an important dilation including veins, gravid uterus, and lactating
mammary glands.
11

Katrin BENAKOVITSCH :
Katrin.benakovitsch@unibas.ch
Center for Biomedicine; University Basel, Switzerland
NEW TOOLS FOR SYSTEMS BIOLOGY ANALYSIS OF SPATIO-TEMPORAL
SIGNALING DURING POLARIZED CELL MIGRATION
Polarized cell migration is critical to development, the immune system but its
regulation is compromised in pathologies such as metastatic cancer or vascular
diseases. Cell migration processes are highly spatio-temporal regulated and involve
cellular dynamics such as changes in the cytoskeleton, generation of polarity or
adhesion dynamics.
In order to elucidate the highly spatial organized signaling components and networks,
we applied to novel technologies to enable the study of prototypical modes of
polarized cell migration.
First, we were able to separate pseudopods from their cell bodies and performed
biochemical analysis as well as large scale shotgun proteomics. These informations
about proteomic dynamics in pseudopods and cell bodies will be used in a second
approach performing live cell imaging in a microfluidic system.
This system enables to apply chemokine gradients in a very robust way to stimulate
cells growing on integrated line patterns. One specific application of this tool will be the
tracking of live cells after gene silencing in a highly standardized fashion.
Together, these two techniques will help to resolve spatio-temporal signaling dynamics
during polarized cell migration.
Timo BETZ : Timo.Betz@curie.fr
Laboratoire P.C.C. UMR 168, Paris, France
ATP-DEPENDENT MECHANICS OF RED BLOOD CELLS
Red blood cells are amazingly deformable structures able to recover their initial
shape even after large deformations as when passing through tight blood capillaries.
The reason for this exceptional property is found in the composition of the membrane
and the membrane-cytoskeleton interaction. We investigate the mechanics and the
dynamics of RBCs by a unique noninvasive technique, using weak optical tweezers
to measure membrane fluctuation amplitudes with µs temporal and sub nm spatial
resolution. This enhanced edge detection method allows to span over >4 orders of
magnitude in frequency. Hence, we can simultaneously measure red blood cell
membrane mechanical properties such as bending modulus, tension and an effective
viscosity that suggests unknown dissipative processes. We furthermore show that
cell mechanics highly depends on the membrane-spectrin interaction mediated by
the phosphorylation of the interconnection protein 4.1R. Inhibition and activation of
this phosphorylation significantly affects tension and effective viscosity. Our results
show that on short time scales (slower than 100 ms) the membrane fluctuates as in
thermodynamic equilibrium. At time scales longer than 100 ms, the equilibrium
description breaks down and fluctuation amplitudes are higher by 40% than predicted
by the membrane equilibrium theory. Possible explanations for this discrepancy are
influences of the spectrin that is not included in the membrane theory or
nonequilibrium fluctuations that can be accounted for by defining a nonthermal
effective energy that corresponds to an actively increased effective temperature.
12

Thomas BORNSCHOGL: Thomas.Bornschloegl@curie.fr
PhysicoChimie Curie :UMR CNRS 168, Paris, France
Filopodia are small cell protrusions playing important mechanical and sensory roles for
a variety of different cell processes occurring for example during pathogenesis, cell
migration, wound healing or during the dorsal closure in the embryonic development.
Filopodia have also been observed pulling invasive bacteria towards the host cell
before infection occurred. However the exact mechanics and the functionality behind
the filopodial retraction mechanism are far from being understood. We want to shed
light onto the molecular principles behind the filopodial retraction mechanism when it is
triggered by pathogens such as Shigella. To measure the mechanics we will use an
optical trap combined with confocal microscopy. We will quantify the time resolved
force that a single filopodium exerts onto beads that are coated with different bacterial
ligands or other effectors inducing retraction. By simultaneously observing the
movement of fiduciary marks placed on the filopodial actin core and by locally
perfusing drugs that interfere with cytoskeletal components inside the filopodium we
intend to decipher the underlying molecular principles. The local perfusion of drugs
that affect the early stage of filopodial adhesion to beads or to the Shigella bacterium
may also help to define the signaling pathway leading to retraction.
Msham BRITTO : mishanv@gmail.com
Dublin City University, School of health and human performance
Dublin, Ireland
MOESIN IS A PIVOTAL CYTOSKELETAL MODULATOR IN VASCULAR CELLS
DURING MECHANOTRANSDUCTION, GLOBALLY REGULATED BY MICRORNAS
The cytoskeleton recruits adapter, linker and scaffolding proteins that modulate cell
plasticity with haemodynamic forces. The FERM domain containing ERM (Ezrin,
Radixin, Moesin) family are key actin binding proteins. Cardiovascular diseases are a
major cause of morbidity and mortality, which results in remodeling of the vasculature.
This remodeling is effected by cell fate decisions such as migration, proliferation etc,
effected by aberrant blood flow.
Our studies show moesin in contrast to ezrin is artheroprotective and regulated by
mechanotransduction in vasculature. Moesin unlike ezrin is found sensitive to
thrombosis and haemostatic stimuli. Moesin through cyclic strain is also demonstrated
to mediate αVβ3/α5β1 integrins, G-coupled protein FPRL1 (Formyl peptide receptor
1), urokinase-uPAR interaction. These interactions were elucidated through the
investigation of cell motility. Proliferation, angiogenesis, permeability and
microparticles in vascular cells showed the same paradigm. Moreover, microRNA is
demonstrated to globally regulate these major functions in endothelial cells, including
endothelial cell and actin realignment with with blood flow.
For cell motility/mechanics/mechanotransduction among other functions, we find a
requirement for moesin dephosphorylation, regulated by microRNA and RhoA. These
interactions, their effect on functions and global regulation by microRNAs provide a
platform of novel targets during cardiovascular diseases.
13

Matthieu CARUEL : caruel@lms.polytechnique.fr
Laboratoire de Mécanique des Solides, Ecole Polytechnique, Orsay,
France
FAST FORCE RECOVERY IN SKELETAL MUSCLE
Muscle contraction is an ATP driven process that causes relative sliding of actin and
myosin filament. Sliding is at least partially due to the power stroke: a conformational
change in the heads of myosin II , that are attached to actin. This conformational
change, however, is the main player in the response of a myofibrlil submitted to a fast
length change.
In 1971, A.F Huxley and R.M Simmons [Huxley, A. and Simmons, R. (1971). Proposed
mechanism of force generation in striated muscle. Nature , 233:533–538. ] proposed a model that has
been since that time the main reference regarding fast force recovery. They assumed
that the head can be viewed as a particle in a 2 state energy landscape coupled with a
linear elastic element. They also assumed that the rate constants of the power stroke
depend on the mechanical potential energy of the system and thus on the strain
applied to the myofibrils.
In this now classical framework, the tension response to a shortening step has two
main components : 1) an instant elastic drop to an extreme value (T1) and 2) a quick
recovery due to power-stroke in the attached heads that leads to a plateau in the
force-time response (T2). The time scale of fast recovery is such that no attachementdetachement
process can occur. The rate constant of the fast recovery was shown by
Huxley and Simmons to have an exponential dependence on the size of the step.
Recently the model of Huxley and Simmons has been reconsidered in the paper of
Marcucci and Truskinovsky (2009, submitted). They proposed a new mechanical
model of fast recovery which assumes a continuous energy landscape and which
links the tension response to length step with solving a system of stochastic
Langevin's equations. In this poster presentation, we discuss the new step in the
development of this model. The development consists in a systematic account of the
passive elasticity of the filaments. We show that such augmentation of the model leads
unambiguously to two unusual conclusions:
1) The rate of fast force recovery cannot depend on the step size exponentially as
it is widely assumed
2) The fast recovery is highly non symmetric with respect to shortening and
stretching which suggests that the response in stretching cannot be explained
as a reverse of the power-stroke as proposed, for instance, by G. Piazzesi et al
[ Piazzesi, G., Linari, M., Reconditi, M., Vanzi, F., and Lombardi, V. (1997). Cross-bridge
detachment and attachment following a step stretch imposed on active single frog muscle fibres.
JOURNAL OF PHYSIOLOGY-LONDON , 498(1):3–15. ].
14

Leif DEHMELT : leif.dehmelt@mpi-dortmund.mpg.de
Max-Planck-Institute for molecular Physiology and TU Dortmund,
Germany
MECHANISMS IN NEURITE INITIATION: DIRECT OBSERVATION OF
MICROTUBULE PUSHING BY CORTICAL DYNEIN/DYNACTIN KOMPLEXES.
In neuronal and non-neuronal cells, MAP2c decorated microtubule bundles are under
the influence of a dynein-dependent pushing force. If such microtubule bundles are
free to move, they are rapidly transported through the cell. At the cell periphery, such
bundles can exert an outward force that is opposed to an actin mediated inward
contractile force. Changes in the balance of these forces can lead to the induction of
new neurite-like cell protrusions. Here, we directly observed cortical dynein/dynactin
complexes and motile microtubule bundles via wide-field and TIRF microscopy.
Cortical dynein/dynactin complexes are preferentially associated with microtubule
bundles that come in close proximity with the plasma membrane in the TIRF field. We
find that at least two different dynein/dynactin populations exist, that are either
stationary or associated with the minus ends of motile microtubule bundles. Our
results support a model in which free dynein complexes are transported towards the
microtubule bundle minus-end, while stationary, cortex associated complexes can
push microtubules directionally with leading plus-ends, thereby focusing a dyneinmediated
force to locally induce neurite-like cell protrusions.
Olivier DESTAING : destaino@ujf-grenoble.fr
CRI Inserm,La Tronche, France
INTERACTIONS OF BETA1 AND BETA3 INTEGRINS IN THE CONTROL OF THE
DYNAMICS OF INVADOPODIA.
Invadopodia or podosomes are adhesion structures able to degrade the extracellular
matrix and are implicated in tissue invasion by mobile cells such as osteoclast or
metastatic cells. Thus, these structures appear as an interface between actin
polymerization, adhesion and membrane trafficking implicated in invasion. In order to
explore the interaction between invadopodia and extracellular matrix, we investigate
the functions of the integrin family, the class of adhesion receptors composed by
heterodimers of noncovalently associated alpha and beta subunits. Previous works
strongly suggested that beta3 integrins were the main adhesion receptors regulating
these structures. Experiments on micropatterned substrates and TIRF microscopy
revealed an original association between beta1 and beta3 integrins in invadopodia. To
explore the specific functions of each of these class of integrins in invadopodia, we
induced them in beta3 -/- cells or in beta1 conditionnal knock-out cells. Surprinsingly, it
appeared that beta1 integrins, but not beta3, are essential to invadopodia formation.
Using a reverse genetic approaches, we determined the essential reisdues of the
cytoplasmic domains of this class of integrin. In particular, we suggest that beta1
activation is essential to recruit beta3 integrins and induce the formation of
invadopodia. More surprinsingly, modulating beta1 integrins activity results in
pertubation in the coupling between adhesion, actin remodelling and the degradation
of the extracellular matrix. We proposed that invadopodia are the site of a complex
cross talk between integrins and initiated by beta1 integrins.
15

Leah EDELSTEIN-KESHET : keshet@math.ubc.ca
Dept of Mathematics, University of British Columbia
Vancouver, Canada
The biochemical regulation of cell motility is a complex but fundamental process,
without which cell polarization and directed motion would be impossible. We have
explored aspects of eukaryotic motility regulation centered on the roles of small
GTPases such as Cdc42, Rac, and Rho. We have shown the importance of the
membrane-cytosolic cycling of such proteins in generating an internal “map” that
defines the front and the back of the cell, and that signals to the assembly of
cytoskeletal elements that produce forces of protrusion at the front and retraction at
the rear of the cell. I survey our modelling efforts in this presentation.
Horatiu FANTANA : fantana@mpi-cbg.de
MPI of Molecular Cell Biology and Genetics, Dresden, Germany
TOWARDS MEASURING THE CENTERING FORCES ACTING ON THE MITOTIC
SPINDLE IN THE C. ELEGANS EMBRYO.
The mitotic spindle is a highly dynamic, microtubule-based structure, which is
responsible for chromosome segregation and cleavage plane specification during cell
division. At the beginning of mitosis, the spindle is stably positioned at the center of the
cell and returns to the center if displaced by fluctuations.
In this project, we want to measure the magnitude of the restoring forces acting on the
spindle in the one-cell C. elegans embryo by using magnetic tweezers. With our setup,
we can apply calibrated forces of several 100 pN on 1-micron-diameter magnetic
beads, which we introduce into the embryos by microinjection into the gonad of adult
worms. To apply the force on the spindle, we push the beads against the spindle
poles. Preliminary experiments indicate that the pole structure can withstand forces of
at least a few tens of pN.
Absolute force measurements will allow us to relate the mechanical properties of
microtubule arrays in vivo with the properties of single molecules measured in vitro
and to estimate the number of the force generators involved in centering, contributing
to a better understanding of the centering process.
16

Sebastian FUERTHAUER : bastian@pks.mpg.de
Max Planck Institut für Physik komplexer Systeme, Dresden, Germany
ANTISYMMETRIC STRESS AND THE ROLE OF ANGULAR MOMENTUM
CONSERVATION IN COMPLEX FLUIDS.
The stress tensor of a Newtonian fluid is symmetric in the hydrodynamic limit.
However, in complex fluids, such as nematic liquid crystals, the director field can exert
a torque if it is locally rotated away from its undistorted configuration.
This produces a reactive antisymmetric contribution to the stress tensor.
Here, we provide the derivation of a hydrodynamic theory for a complex fluid based on
identifying the entropy production rate from the rate of change of the free energy.
Analyzing the angular momentum balance, reveals that an additional dissipative
contribution to the antisymmetric stress exists. We obtain an expression for the
antisymmetric dissipative stress by expanding thermodynamic fluxes in terms of
thermodynamic forces, which is crucial in understanding the non-equilibrium dynamics
of chiral complex fluids, such as the acto-myosin cytoskeleton or a fluid
driven by beating cilia.
17

Thomas GREVESSE : thomas.grevesse@gmail.com
Laboratory InFluX Complex fluids and interfaces, Mons, Belgium
RHEOLOGICAL BEHAVIOUR OF NERVE CELLS
Following big head shocks (car crash, falls, blast, ,,,) the brain is severed, leading to
dysfunctions called traumatic brain injury (TBI). It is well known that following TBI,
neurons undergo mechanical damages, e.g. beading of axons, due to mechanical
shearing and stress forces, underlying the importance of the mechanical properties of
neurons. However how neurons behave and sustain mechanical loads is still poorly
understood. Therefore, in this work, we caracterized the mechanical properties of
isolated neuronal cells in order to undesrtand and quantify their mechanical behaviour,
Among other cells neurons show a very complex polarized structure with hundreds of
processes (dendrites and axons) arising from the cell body (the soma). These sub
parts show very different structures and physiological properties. It was therefore
necessary to be able to probe the mechanical properties of these sub parts
independently of the others, hence looking at the sub cellular level. For this purpose
we develloped an experimental apparatus, the magnetic tweezers. Via a
electromagnet filled with a sharpened ferromagnetic tip, we are able to apply local
magnetic field gradient on micrometer sized (5 µm) paramagnetic beads fixed on
nerve cells, hence applying forces to cells at the subcellular level.
Using the magnetic tweezers apparatus we performed creep experiments on both the
soma and neurites (dendrites and axons) of PC 12 cells. We applied a constant force
on a small time scale (~10 s) and observed the strain response of the neurites and the
soma. At small time scales neurites exhibit a purely elastic response with Young
modulus of 5 kPa whereas the cell body shows a viscoelastic solid behaviour with a
rigidity of 1,5 kPa. These results show that when subjected to short impulses of great
forces, the cell body can dissipate a part of the stress trough its viscous component.
For the neurites however, all the stress is used to deform the neurite. This suggests
that when subjected to short impulses of force, the neurite will undergo more damage
and be more subject to rupture than the soma.This could bring some elements to
explain the mechanical damage of axons during TBI.
In future work we will probe the mechanical properties of structured networks of
neurons and the influence of forces on nerve impulse transmission
18

Philip GUTHARDT TORRES :
p.guthardt.torres@tphys.uni-heidelberg.de
Heidelberg University, Institute for Theoretical Physics, Heidelberg,
Germany
FAILURE OF NETWORKS OF BIOMOLECULAR BONDS UNDER FORCE
Structural stability of biological cells is provided by the cytoskeleton, a crosslinked
polymer network that is mechanically connected to the environment through adhesion
sites. Although often modeled as static, the cytoskeleton is highly dynamic, with
connections, so called bonds, continuously breaking and forming in time. In this
contribution the cytoskeleton is modeled as a two dimensional network consisting of N
massless nodes and L massless links. The simplest assumption for the mechanical
nature of a cytoskeletal link is a harmonic spring, but a more realistic model is a cable,
which represents the polymeric nature of the link. Cables differ from springs by the
asymmetry of their force-extension curves. We analyze spring networks as well as
cable networks.
In order to introduce the possible rupture of links we adapt an earlier model for the
stochastic rupture dynamics of adhesion clusters. This leads to breaking of links with
force-dependent rates which is different from the usual approach to fracture in material
science. The fracture of hard macroscopic materials, e.g. concrete, is often modeled
using spring networks with randomly distributed force thresholds for the network links
(random spring model).
In our work, we first compare the equilibrium states for different mechanical models
and network topologies. Then stochastic rupture with force-dependent rates is used to
compare the statistical properties of fracture obtained using this model to those
obtained using a random spring model.
19

Florian HUBER : florian.huber@uni-leipzig.de
University of Leipzig, Germany
LINKER INDUCED ACTIN NETWORK FORMATION UNDER CELL-SIZED
CONFINEMENT
Cross-linked actin networks are decisively involved in the overall mechanical
properties of cells. The networks’ architecture ranges from densely packed bundles to
networks with high crossing angles and is typically assigned to specific linker proteins.
Recently, however, it was found that several cross-linkers give rise to both extended
networks and bundles. We used multivalent ions as model-linkers to study actin
filament aggregation in cell-sized geometries. Small droplets close to cellular sizes
were filled with actin filaments. We slowly increased multivalent ion concentration and
at a critical threshold the ions’ potential turns attractive. This implies a phase transition
from isotropic or nematic f-actin solutions to cross-linked actin networks.
In addition to the well known bundle formation, we obtained regularly spaced networks
of star-like astern pattern. These networks display many features of cellular networks
in the actin cortex and may serve as a model system for the cortical actin layer.
Observed phase transitions are fast (seconds to few minutes) which is of high interest
concerning the known ability of living cells to quickly modify their morphology.
Robiya JOSEPH : rubsjo.jmj@gmail.com
CEDAD-IMPRS, Institute of Immunology, Munster, Germany
THE INTRACELLULAR ROLE OF MRP8 AND MRP14 IN CELLULAR DYNAMICS
OF PHAGOCYTES
The migratory properties of phagocytes allow their rapid accumulation at sites of injury
and infection. The complex transmigration processes are not fully understood,
especially the ability of phagocytes to rapidly reorganize their cytoskeleton. Calciumbinding
proteins are key elements in these signal transduction processes and we
investigated the role of the two phagocyte specific calcium-binding proteins S100A8
(MRP8) and S100A9 (MRP14). Previously we could already show, that in activated
monocytes complexes of S100A8/S100A9 co-localize with microtubules (MTs) in a
calcium and phosphorylation dependent manner. Furthermore, phagocytes from
S100A9 deficient mice show altered migratory properties compared to wild-type cells.
To investigate these in more detail we established stable transfected HEK293 cell
lines, which express S100A8/S100A9, S100A8, S100A9, S100A8/S100A9(N69A)
(unable to form tetramers) and S100A8/S100A9(T113A) (mutated phosphorylation
site) and determined the individual contributions of the subunits and mutations to
cellular dynamics. Using S100A8/S100A9 expressing HEK cells we found a similar
pattern of co-localization of the complex with microtubules comparable to monocytes.
Remarkable differences in S100A8/S100A9 transfected HEK cells compared to mock
transfected ones were seen during breakdown and rebuilding of the microtubule based
cytoskeleton indicating that S100A8/S100A9 affects the dynamics of MTs. Cells from
S100A9 deficient mice show altered characteristics in migration rates. Investigating
this in more detail we found that S100A9 knockout cells are more adherent than wildtype
cells. A similar pattern has also been observed for S100A8/S100A9 transfected
and non-transfected HEK cells. These effects seem to be mediated by S100A8 but not
by S100A9. Furthermore, the effects observed are possibly dependent on
phosphorylation of S100A9. Our results provide evidence, that the S100A8/S100A9
complexes fulfil a pivotal role in remodelling cytoskeletal structures necessary for the
migration of leukocytes.
20

Svenja-Marei KALISCH : s.kalisch@amolf.nl
Group Bio-Assembly and Organisation, FOM-Institute AMOLF
Amsterdam, Netherlands
AGAINST THE WALL: MICROTUBULE DYNAMICS IS REGULATED BY FORCE
AND END-BINDING PROTEINS
The dynamics and organization of microtubules (MTs) are essential for cell
morphogenesis. Microtubule plus-end dynamics is highly regulated by specific endbinding
and end-tracking proteins, as well as by the forces generated when MTs grow
against obstacles such as the cell cortex. However, the molecular mechanisms
underlying the regulatory actions of end-binding proteins, especially under force,
remain largely unknown. We therefore investigate the force-induced behavior of
dynamic MTs in the presence of the fission yeast proteins Mal3, Tea2, and Tip1 in
vitro. Head-on forces are achieved by growing the MTs against a micro-fabricated rigid
barrier. Using TIRF microscopy we show that the protein complex in general enhances
the MT growth speed and induces catastrophes. Moreover, when coming in contact
with the barrier, the force experienced by the MT shortens the catastrophe time further
by nearly an order of magnitude. We use this experiment to investigate whether the
presence of end-binding proteins qualitatively or quantitatively changes the way MT
dynamics responds to force. This should allow us to improve our insight into the
molecular details of (force-enhanced) MT regulation by end-binding proteins.
Nada KHALIFAT : Nada.Khalifat@univ-montp2.fr
LCVN, UMR 5587, cc 26, Université Montpellier II, France
ACTIVATION AND MULTIMER FORMATION OF EZRIN INDUCED BY PIP2
Ezrin protein acts as a crosslinker between the actin network near the cell surface and
the cytoplasmic leaflet of the plasma membrane, and has been shown to exist in a
dormant conformation in the cytoplasm. In its active conformation, the N-terminal
FERM domain binds to the plasma membrane, and the C-terminal region binds to
actin filaments. Transition of ezrin to its active conformation occurs by binding to a
specific lipid, Phosphatidylinositol-4,5-biphosphate (PIP2).
PIP2 is a minor but key phospholipid involved in the regulation of a wide variety of
processes such as membrane trafficking exocytosis and endocytosis. In this current
work, we investigate the interaction of wild-type ezrin or a mutant known to not bind
the membrane, and PIP2 in solution and in lipid bilayer membrane. We identified the
specifity of PIP2: (i) on the conformational transition of ezrin and the importance of
PIP2 alkyl chain in this process; (ii) on the formation of wild-type ezrin multimers.
21

Elena KREMNEVA : kremneva@mappi.helsinki.fi
Pekka Lappalainen’s laboratory, Institute of Biotechnology, Helsinki,
Finland
DISTINCT ROLES OF ADF/COFILIN ISOFORMS IN SARCOMERE ASSEMBLY
AND MATURATION
The actin cytoskeleton is necessary for a large number of cell biological processes,
including morphogenesis, motility, endocytosis, cytokinesis and signal transduction.
The structure and dynamics of the actin cytoskeleton are both spatially and temporally
regulated by an array of proteins that interact with actin filaments and/or monomeric
actin. The actin cytoskeleton in non-muscle cells is highly dynamic and actin filaments
are thus constantly assembled and disassembled in response to various intracellular
and extracellular signals. In contrast, the actin filaments in muscle cells are formed into
well organized structures, sarcomeres, and are believed to be significantly less
dynamic than the ones in other cell-types. However, many proteins promoting actin
dynamics in non-muscle cells are expressed also in muscle cells. Thus, it is possible
that the muscle-specific isoforms of these proteins promote actin dynamics differently
from their non-muscle counterparts. ADF\cofilins are central actin-binding proteins that
enhance turnover of actin filaments by severing and depolymerizing F-actin. This class
of proteins consist of 3 different isoforms in mammals – cofilin-1, cofilin-2 and ADF.
Our Western blot analysis reveald that cofilin-1 and cofilin-2 are expressed in cultured
rat cardiomyocytes. Interestingly, the amount of cofilin-2 increases in cardiomyocytes
during maturation, whereas level of cofilin-1 decreases. Furthermore, my preliminary
studies using isoform specific antibodies revealed distinct localization patterns of these
proteins. Cofilin 1 displays more diffuse distribution in sarcomeres compared to cofilin-
2 which localizes to narrow stripes close to M- and Z-lines. Biochemical analyses
revealed that cofilin-1 and cofilin-2 display different thermal stability and affinity to actin
filaments in the presence and absence of tropomyosin. Together, these data suggest
that cofilin-1 and cofilin-2 play different roles in regulation of actin dynamics in heart
muscle cells.
22

Dorothy KUIPERS : d.kuipers@ucl.ac.uk
The UCL Ear Institute & London Centre for Nanotechnology, London,
UK
CELL DEATH AND ACTIN DYNAMICS IN A MODEL EPITHELIUM
Cell death within an epithelium could lead to loss of the barrier function and damage to
surrounding tissues. Hence, epithelial cells have evolved mechanisms to preserve
epithelial integrity. We are using a monolayer of MDCK cells to investigate the
complex patterns of actin dynamics that occur at sites of cell death in a model
epithelium.
Our imaging of MDCK cells stably-transfected with fluorescent actin reporters
suggests that dying cells are internalised by the coordinated movement of surrounding
cells. This coincides with the formation of an apical actin 'ring' which closes above the
dying cell. In addition, lamellipodial crawling of surrounding cells closes the area
basally, resulting in two distinct planes of actin enrichment. In order to determine which
cells contribute to the actin ring structure, we used mosaics of cells transfected with
fluorescent actin reporter-variants. Analysis of 27 individual events from 4 different
experiments always revealed actin rings when the dying cell was expressing a
reporter. On the contrary, in 9 examples where the dying cell was not expressing a
reporter a there was no clear evidence of actin ring formation in the surrounding cells.
Opposing sides of the actin ring contract towards one another with a speed of ~0.2
microns/min, that remains surprisingly constant over the closure time of 15-30 min
(n=18).
Further live imaging with myosin reporters will reveal the role of myosin contraction in
dying and surrounding cells. Modeling techniques will be employed to investigate the
forces driving the epithelia repair and whether they are due to the dying or surrounding
cells.
23

Anne LEBAUDY : anne.lebaudy@ibgc.cnrs.fr
IBGC-CNRS UMR5095, Université de Bordeaux 2, France
ACTIN BODIES MOLECULAR ARCHITECTURE AND CELLULAR PROPERTIES
Actively proliferating yeast cells display three F-actin containing structures: actin
cables, which serve as tracks for the polarized transport of vesicles, organelles and
mRNA; actin patches that are required for endocytosis; and an acto-myosin cytokinetic
ring. A large set of conserved actin-binding proteins (ABPs) tightly orchestrates the
formation and the maintenance of these highly dynamic F-actin containing structures.
We have recently described that quiescent yeast cells totally reorganize their
actin cytoskeleton and assemble a specific F-actin containing structure that we have
called Actin Bodies. Actin Bodies contain various ABPs, they are rather immobile and
display a very slow actin filaments turn-over. Remarkably, Actin Bodies disassemble
within seconds upon cells re-entry into the proliferation cycle.
We now are interested in deciphering Actin Bodies molecular architecture. For
that purpose, we are using live cell imaging to follow Actin Bodies formation and
disassembly in quiescent cells. We are also studying more in depth the turn-over of
the filaments that are embedded into this specific structure. Finally, we are trying to
decipher the role of some specific ABPs in the formation, maintenance and
dissociation of Actin Bodies. All together our data strongly suggest that Actin Bodies
are just not an aggregate of actin filaments but a rather very organized structure that is
very sensitive to environmental conditions.
Jimmy Le DIGABEL : j
jimmy.ledigabel@univ-paris-diderot.fr
Matière et Systèmes Complexes, Benoit Ladoux, Paris, France
ACTIVE SUBSTRATES TO STUDY MECHANOTRANSDUCTION
Cellular processes imply an important coordination of interactions with the extracellular
medium. Accumulating evidences demonstrate that cell functions can be modulated by
physical factors such as the mechanical forces acting on the cells and the extracellular
matrix, as well as the topography or rigidity of the matrix. These extracellular signals
can be sensed by mechanosensors on the cell surface or in the cell interior to induce
various cell responses. We have developed an original approach based on microfabricated
substrates of PolyDimethylSiloxane (PDMS) to study cell migration. We
used a closely spaced array of flexible micropillars (diameter~1µm) to map the forces
exerted by cells on their substrates. In this case, the micropillars act as passive force
sensors. Here, we propose to analyze the cell response to an external applied stress
by a well-controlled actuation of the substrate. To do so, we used magnetic pillars.
Such substrates allow us to modify dynamic adhesion conditions of cells and to better
understand the coupling phenomena between mechanical sensing and biochemical
activity of a living cell. Using polyacrylamide hydrogels doped with ferromagnetic iron
oxide particles or ferrofluids, we can make magnetic pillars with diameters of 5 to 10
microns while a magnetic field can be locally applied with a magnetic needle. Such a
technique will be helpful to study the mechanical response of cells to an external force
or to local changes in their microenvironment.
24

Chiu Fan LEE : cflee@pks.mpg.de
Max-Planck-Institute for the Physics of Complex Systems, Dresden,
Germany
PROTEIN AMYLOID SELF-ASSEMBLY
Amyloids are insoluble fibrous protein aggregations stabilized by a network of
hydrogen bonds and hydrophobic interactions and they are intimately related to many
neurodegenerative diseases such as the Alzheimer’s Disease, the Parkinson Disease
and other prion diseases. Better characterization of the various properties of amyloid
fibrils is therefore of high importance for the understanding of the associated
pathogenesis. Here, we present our recent results on various equilibrium and
dynamical properties of protein amyloid self-assembly. In particular, we discuss the
length distribution of amyloid fibrils in thermal equilibrium [1], the possibility of
isotropic-nematic phase transition as monomer concentration is increased [2], and the
dynamical processes of nucleation and fibril elongation [3,4]. Our methods of
investigation consist of techniques in statistical mechanics and molecular dynamics
simulations.
References: [1] Lee (2009) Self-assembly of protein amyloid: a competition between
amorphous and ordered aggregation. Phys. Rev. E 80, 031922. [2] Lee (2009)
Isotropic-nematic phase transition in amyloid fibrilization. Phys. Rev. E 80, 031902. [3]
Jean, Lee, Lee, Shaw and Vaux (2010) Competing discrete interfacial effects are
critical for amyloidogenesis. To appear in the FASEB J. [4] Lee, Loken, Jean and Vaux
(2009) Elongation dynamics of amyloid fibrils: a rugged energy landscape picture.
Phys. Rev. E 80, 041906.
Jonathan LEE TIN WAH :
Jonathan.Lee-Tin-Wah@curie.fr
Laboratoire Physico-Chimie Curie, UMR168, Paris, France
COLLECTIONS OF MOLECULAR MOTORS UNDER ELASTIC LOADING
The goal of this project will be to study the collective mechanical properties of a small
group of molecular motors under elastic loading in a simple in-vitro system. A similar
situation can be found in various biological systems, leading, both in-vivo and in-vitro
to spontaneous oscillations. It has already been observed that the mechanosensitive
hair cells of the inner ear are able to oscillate spontaneously depending on the
actomyosin protein complex. We will attempt to shed light on the mechanism behind
the oscillatory activity of the acto-myosin system, in particular by determining the
parameters that control their frequency and amplitude through a number of mechanical
and biochemical means in-vitro. The main objective of this thesis will be to measure
the dynamical responsiveness of molecular motors working in groups to external
stimuli. An important challenge will be to determine conditions under which motor
assemblies can operate at timescales compatible with auditory frequencies ( > 1KHz )
which are much shorter than the ATPase cycle of a single motor. To achieve this, we
plan to make use of conventional and an unconventional myosins, in particular Myosin
1c which is regulated by calcium and is involved in the oscillation of hair cells.
25

Alexandre LEWALLE : a.lewalle@ucl.ac.uk
London Centre for Nanotechnology, UK
NEUTROPHIL MOTILITY IN CONFINED ENVIRONMENTS
There is evidence that the degree of confinement of a motile cell plays a role in
determining how the cell produces the forces that allow it to move. Traditionally, cell
motility is studied on fibronectin-coated surfaces, to which a cell can adhere via focal
contacts. These focal complexes act as anchor points that provide a mechanical
coupling between the cell and its environment. Motility is achieved when forces,
generated within the cell, are exerted on the surface via the focal adhesions. However,
it appears that motility is possible in the absence of focal adhesions when the cell is
placed in a more confined geometry, e.g., (1) in the two-dimensional environment
between a glass surface and a soft agarose gel, or (2) in narrow one-dimensional
channels fabricated from the polymer pdms. In both cases, surfaces are passivated to
prevent the formation of focal adhesions at the cell membrane.
We examine actin dynamics in HL60 cells undergoing chemotaxis in these different
confined environments. The cells contain actin that is doubly labelled: with RFP and
with photo-activatable GFP (PAGFP), which becomes visible once excited with a burst
of violet light applied to a small region of the cell. We monitor the time evolution, in a
moving cell, of the RFP and of the PAGFP intensities simultaneously near the cell’s
leading edge, with a view to measuring the actin turnover in this region, and
understanding the effect of confinement on actin-mediated force generation.
Ofélia MANITI : ofemaniti@yahoo.com
LMGP, CNRS-UMR 5628, Grenoble, France
EZRIN INTERACTIONS WITH BIOMIMETIC VESICLES CONTAINING
PHOSPHATIDYLINOSITOL (4,5) BISPHOSPHATE (PIP2)
The plasma membrane-cytoskeleton interface is a dynamic structure, participating in a
variety of cellular events including cell shape, polarization and motility. Among the
proteins involved in the direct linkage between components of the cytoskeleton and
the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM domain in
their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP2) binding site
responsible for membrane-binding whereas their C-terminus bind actin. In this work,
our aim was to characterize the interaction of ezrin with large unilamellar vesicles
(LUVs) containing PIP2 and with giant unilamellar vesicles (GUVs) containing PIP2.
We first synthesized human recombinant ezrin bearing a cysteine residue at its C-
terminus for subsequent grafting with Alexa488 maleimide. This allowed us to perform
fluorescence spectroscopy and microscopy experiments. The
functionality of labeled ezrin was checked by comparison with that of wild type (WT)
ezrin. The affinity constant between ezrin and LUVs, determined by co-sedimentation
assays and fluorescence correlation spectroscopy, was found to be ~5 µM for PIP2-
LUVs and 20 to 70 lower for PS-LUVs, depending of the experimental technique. We
found that the interaction is not cooperative for PIP2-LUVs. We prepared fluorescently
labelled GUVs using PIP2 analogues used as tracers (0.1% of total lipids) to
investigate ezrin/GUVs interactions by fluorescence microscopy. Zeta potential
measurements confirmed that the effective incorporation of PIP2 in the GUVs. Finally,
the interaction of ezrin with PIP2-containing GUVs was investigated. Using either
labeled ezrin and unlabeled GUVs or both labeled ezrin and GUVs,
we evidenced that clusters containing both PIP2 and proteins are formed.
26

Olga MARKOVA : markova@ibdm.univ-mrs.fr
IBDML – UMR 6216, Institute of Developmental Biology Marseilles
France
HYDRODYNAMIC DESCRIPTION OF TISSUE ELONGATION DURING
DROSOPHILA EMBRYO MORPHOGENESIS: THE ROLE OF ACTIVE
ANISOTROPIC CYTOSKELETAL STRESSES
Olga Markova and Pierre-François Lenne
Institute of Developmental Biology Marseilles‐Luminy (IBDML), 13288 Marseille, France
During tissue morphogenesis, cells are able to change shape and rearrange, yielding
dramatic changes such as tissue elongation. In early Drosophila embryos, the
elongation of the germ band (GBE) results from cell intercalation, a process driven by
tensile actomyosin networks which generate anisotropic forces (Bertet et al. 2004,
Rauze et al. 2008). In an attempt to describe quantitatively the dynamics of GBE, we
used the hydrodynamics theory of viscous fluids (Bittig et al. 2008). We find that during
the fast phase of GBE, cells flow like viscous fluids driven by internal anisotropic active
stresses generated by actomyosin anisotropic contraction. From quantitative analysis
of vector fields of cell motion we estimate the viscosity of the Drosophila germband.
Our work proposes a non-invasive method - based on the analysis of kinetics of tissue
movement in vivo - to determine tissue mechanical properties.
Bertet, C., Sulak, L. & Lecuit, T., 2004. Myosin-dependent junction remodelling controls
planar cell intercalation and axis elongation. Nature, 429(6992), 667-671.
Bittig, T., Wartlick, O., Kicheva, A., González-Gaitán, M., Jülicher, F. 2008. Dynamics
of anisotropic tissue growth. NEW JOURNAL OF PHYSICS, 10.
Rauzi, M., Verant, P., Lecuit, T., Lenne, PF. 2008. Nature and anisotropy of cortical
forces orienting Drosophila tissue morphogenesis. Nature Cell Biology, 10(12), 1401-1410.
27

Carolina MARTINEZ CINGOLANI : cmartinezci@unal.edu.co
CURIE INSTITUT INSERM U 932, Paris, France
PHD DIRECTOR: VASSILI SOUMELIS
Lab Director: Sebastian AMIGORENA
Inserm U932 « Immunité et cancer ». Institut Curie
MOLECULAR MECHANISMS AND TUMORAL MICROENVIRONMENT ROLE IN HUMAN
DENDRITIC CELL MIGRATION IN CONFINED SPACE
Dendritic cells (DCs) have an essential role due to their participation in the innate and
acquired immune system responses. The DCs that reside in the peripheral tissues must be
able to leave this environment and to migrate to secondary lymphoid organs, where they
exert their function as professional antigen presenting cells. (Banchereau et al, 2000). To do
so, they have to pass through very narrow spaces, such as the tight epithelial intercellular
junctions, the extracellular matrix, the basal membrane and the endothelium. The velocity of
the movement must be optimal, because the DCs must induce transitory deformations of the
microenvironment, without leaving collateral lesions, and at the same time, they must sense
and integrate the signals around them. DCs have to be able to maintain their sentinel and
phagocyte functions in the most efficient way (Faure-André, 2008). This migration occurs in
a three-dimensional microenvironment (confined microenvironment). Its direction is
determined mainly by chemokine gradients (Rossi, and Zlotnik, 2000). However, there are
other factors that may be implied in this process. In fact, some physical parameters, such as
the tissue geometry, can affect the type of movement and the necessary conditions for the
migration itself (Lämmermann, Bader, et al, 2008).
Our recent findings show that DCs can be instructed to migrate by the inflamed tissues. This
is, in particular, the case of Thymic Stromal Lymphopoietin (TSLP). This pro-inflammatory
cytokine secreted by epithelial keratynocytes, is responsible for Langerhans cell depletion
that characterizes the Human Papilloma Virus infected tissues. This molecule was revealed
as an inductor of DC cytoskeleton polarization and migration, in a chemokine-independent
and a myosin II-dependent way (Fernandez, et al, Immunity, submitted).
Now, we intend to determine which is the relative contribution of different molecular
(chemokinesis, chemo-attraction) and physical parameters (tissue geometry, confinement
and lymphatic flux) in the DC migration, both in physiological and pathological conditions
with particular attention to cancer. It has been reported that reduced DC numbers and DC
migration alterations occur in solid tumours (melanoma and uterine cervical intraepithelial
neoplasia, Halliday, Le, et al 2001, Fernandez, et al, submitted and Villablanca et al 2010).
We want to evaluate the DC migration alterations in a microenvironment that favours the
acquisition of migratory capacity by the non-immunological metastatic cells (Sahai, 2007).
The relative contribution of different molecular and physical parameters will be determined
after DC treatment with TSLP and other migration stimulating and inhibiting factors. To this
purpose we will use a micro-channel system developed by M. Piel ´s team at the Curie
Institute (Faure-André, 2008). The cytoskeleton rearrangements will be assessed by
immunofluorescence for each condition using anti-actin, anti-tubulin and anti-myosin II
antibodies. To evaluate the effects induced by the tumoral microenvironment we will use the
supernatants derived from both tumoral cell line and primary tumour cultures. The cytokine
composition of these supernatants will be analysed ELISA and immunohistochemistry.
This is a multi-disciplinar project that involves physics, human immunology, tumoral
and cellular biology. We believe that our work will highlight the fundamental mechanisms
used by DC to migrate. Our results could be useful for the establishment of novel therapeutic
strategies to promote antitumoral immunity by favouring DC migration.
28

Helen MATTHEWS : h.matthews@ucl.ac.uk
MRC Laboratory for Molecular Cell Biology, London; UK
When a cell enters mitosis, it dramatically rearranges its actin network to assume a
stiff, rounded shape. Recent work from our lab has shown that mitotic rounding is
driven by ERM-induced cortical stiffening and is essential for proper spindle formation
and successful mitosis (Kunda et al). Although much is known about the regulation of
entry into mitosis by mitotic kinases, it has not been determined how this regulation
could feed into the actin cytoskeleton. We are interested in identifying molecular
regulators of mitotic rounding and, ultimately, understanding how they generate the
mechanical forces required to drive cell rounding.
The spatial and temporal dynamics of mitotic cell rounding were analysed in two
human cell lines, Hela and retinal pigment epithelial (RPE1) cells. Rounding begins
early in mitosis, preceding nuclear envelope breakdown, and continues into
metaphase. It consists of two steps; an initial detachment from the substrate and
retraction of lamellipodia, followed by the construction of a meshwork of cortical actin,
during which stage phosphorylated ERM proteins accumulate at the membrane.
Interestingly microtubules are not required for either stage as nocodozole-treated cells
round normally. Myosin contractility contributes to mitotic rounding, with the inhibition
of myosin/ROK/RhoA slowing the process, although cells do eventually achieve a
rounded shape.
I am currently taking two approaches to identify novel regulators of mitotic rounding.
Firstly, I am using small molecule inhibitors of the mitotic kinases to try to identify the
kinase(s) driving shape change at mitosis. Secondly I am carrying out an RNAi screen
using a targeted library of actin regulators, to identify genes affecting mitotic cell
shape. Here I will present some of the preliminary data from these analyses.
Kunda et al (2008). Moesin controls cortical rigidity, cell rounding and spindle morphogenesis during
mitosis. Current Biology 18(2), 91-101
29

Manos MAVRAKIS : mavrakis@ibdm.univ-mrs.fr
IBDML / CNRS UMR 6216, Marseille, France
AN EMERGING ROLE OF SEPTINS IN TUNING ACTOMYOSIN CONTRACTILITY
The formation of the primary epithelium in the Drosophila embryo involves a dramatic
morphogenetic process during which plasma membrane invaginates between
cortically-anchored nuclei to produce 6000 polarized epithelial cells within less than an
hour. A set of proteins including F-actin, myosin-II, anillin and septins partition into the
tips of the invaginating membrane front. This actomyosin-II network is maintained
coincident with the plasma membrane growth throughout cellularization, and its
perturbation has been reported to disrupt cellularization. However, how the actomyosin
network assembles, how it is stabilized and how its contractile properties are regulated
in space and time so that it is coupled with invagination without premature constriction
are largely unknown. The role of septins during this process is also entirely unclear.
We imaged myosin-II dynamics during cellularization in embryos expressing functional
myosin-II regulatory light chain–GFP. In wild-type embryos myosin-II is initially found
as small myosin-II particles lining the invagination front. This distribution changes
progressively into a continuous ring-like distribution, which is maintainted until the end
of cellularization. When we treat embryos with forchlorfenuron, a drug which disrupts
septin assembly, we observed that myosin-II prematurely constricts, and the
invagination front closes off in an untimely fashion at the very onset of cellularization.
We observed the same effect when we knocked down septin expression using RNAi.
Increasing myosin-II activity with calyculin A, a myosin phosphatase inhibitor, leads
also to an untimely hypercontractile ring phenotype. Our results suggest an interplay
between septin organization and myosin-II activity and/or stabilization on F-actin.
30

Tomas MAZEL : tomas.mazel@mpi-dortmund.mpg.de
Technische Universität Dortmund, Chemische Biologie, Germany
CYTOSKELETON DYNAMICS DURING NEURITE INITIATION - MODELING
APPROACH
Tomáš Mazel, Anja Biesemann, Olga Müller, Leif Dehmelt*
Technische Universität, Fakultät Chemie, D-44227 Dortmund, Germany
e-mail: mazel@mpi-dortmund.mpg.de
Neurites enable targeted, long distance connections in the nervous system.
Many signaling pathways are important for neurite development, but little is known,
how these signals are transformed into morphological features. Deeper understanding
of the underlying mechanisms could lead to new strategies in treatment of stroke,
spinal cord injuries, or neurodevelopmental and neurodegenerative diseases, in which
neurite development or regeneration is often compromised.
In order to get an insight into these complex cellular mechanisms, we started to
develop an in silico model of neurite initiation. This model consists of several layers: 1)
a regulatory network, including plasma membrane receptors and their downstream
signaling cascades, including Rho family GTPases. 2) effectors, controlled by this
regulatory network, 3) the cytoskeleton, microtubuli and actin, which is controlled and
rearranged by these effectors, 4) the cell border, which is under the influence of forces
generated by the cytoskeleton.
We started with a highly simplified system, in which we can directly measure the
behavior of many of the system components directly: the intracellular motility of
MAP2c-induced microtubule bundles. By combining high-resolution TIRF microscopy
with object tracking, we can estimate dynamic properties of microtubules and
interacting motor complexes, which are then integrated into our model system.
Comparison of experimental observation with computer-based simulations is then
used to identify missing components or hidden parameters in our model. We will
subsequently extend both our computational model and our experimental observations
to approach our goal of modeling neurite initiation in a more complex and more
realistic cellular system.
31

Xavier MEZANGES : xavier.mezanges@curie.fr
Institut Curie UMR 168 Physicochimie, Paris, France
SPERM CELL CRAWLING IN THE NEMATODE CAENORHABDITIS ELEGANS
Cell motility is important in biological processes, such as the immune response,
and in pathological states, such as cancer cell metastasis. Actin is implicated in most
amoeboid cell movement, but Caenorhabditis elegans sperm cells lack actin, and their
motility is driven by the Major Sperm Protein (MSP) cytoskeleton. Both MSP and actin
form filament systems, but there is no biochemical or structural similarity between the
two molecules. Little is known as to how MSP polymerization creates movement, or
what biochemical partners are necessary for motility.
In vitro systems, where cellular actin polymerization is recreated on the surface
of a bead, giving actin comet tails and movement, have been useful in defining the
biochemical and physical parameters of actin-based motility. Our goal is to apply this
technology to the MSP motility system. The first step is to identify the activator(s) of
MSP polymerization. The activator(s) appears to be a membrane-bound protein that
contains a phosphorylated tyrosine residue in its active form. Our approach is to
perform immunoprecipitation from sperm cell extracts with an anti-phosphotyrosine
antibody, followed by mass spectroscopy to identify the activator or activator complex.
Once the activator(s) is in hand, we will find conditions for absorbing it to
bead surfaces to produce MSP comets in sperm cell extracts. These comets will
permit a characterization of MSP-based movement (speed, comet structure, comet
elasticity etc). The comets will be further analyzed by mass spectrometry to identify
other components of the MSP motility system. In parallel, we will provide a first
characterization of MSP filaments formed in solution in vitro using TIRF and confocal
microscopy, and microrheology and AFM to measure the elastic properties of MSP
networks and single filaments.
In the long term, the comparison of MSP and actin based movement should
lead to a better understanding of the fundamental principles cell motility.
32

Francesca MILANESI :
francesca.milanesi@ifom-ieo-campus.it
Membrane and actin dynamics in the control of migratory and
invasive strategies Laboratory, Milan, Italy
MOLECULAR BASIS FOR THE DUAL FUNCTION OF EPS8 ON ACTIN
DYNAMICS: BUNDLING AND CAPPING
Actin capping and cross-linking proteins regulate the dynamics and architectures of
different cellular structures by controlling the number of free actin growing ends and
organizing filaments into higher order structures, respectively. EPS8 is the founding
member of a unique family of capping proteins capable of binding and bundling actin
filaments. The structural basis through which EPS8 binds actin filaments remains,
however, unexplored. We combined biochemical and molecular approaches with
electron microscopy image analysis, to dissect the molecular mechanism responsible
for the distinct activities of Eps8. We propose that Eps8 caps by wrapping around
filament ends, inserting its amphiphatic H1 helix into the hydrophobic pocket of the
barbed end unit, acting as a clamp that prevents further addition of monomers.
Whereas, actin crosslinking is primarily mediated by contacts between the filament
and the Eps8 globular helical bundle. Mutantions in either the amphipatic helix or in
the globular helical core of Eps8 permitted to dissect the capping and bundling
activities, respectively, thus validating the model, both in vitro and in vivo. Thus, Eps8
controls actin-based motility through its capping activity, while, as a bundler, is
essential for proper intestinal morphogenesis of developing Caenorhabditis elegans.
Makito MIYAZAKI :
miyazaki@chem.scphys.kyoto-u.ac.jp
Department of Physics, Graduate School of Science, Kyoto University,
Japan
UNCOVERING THE HIDDEN STRUCTURE OF THE PROTEIN USING BAYESIAN
INFERENCE
In single-molecule experiments of proteins, the observable variables are restricted
within a small fraction of the whole degrees of freedom in general.
For instance, in the case of motor proteins, attaching a large probe particle to the
molecule is a widely adopted strategy. In this case, the motion of the probe is the only
observable while the motion of the protein itself is completely hidden.
Therefore, in order to investigate the protein in detail, we need to infer the internal
structure of the protein (ex. shape of the Interaction potential between the motor
protein and the “track”), only referring to the motion of accessible degrees of freedom.
We formulated this problem on the basis of Bayesian framework, which can be
applicable for various complex systems in non-equilibrium, and we obtained the simple
and general evidence that this framework actually works. From careful numerical
studies, although we confirmed that the performance of our method is better than the
conventional method by orders of magnitude, we found that there is a critical line in the
original parameter space below which the precision of the estimate is abruptly lost.
We will illustrate the basic feature of the proposed method including the property of
“loss-of-precision transition” using a simple but nontrivial model and we will propose
the application for the single molecule experiments.
33

Nikola OJKIC : nro207@lehigh.edu
Physics Department, Bethlehem, USA
Kinetics of self assembly of the contractile ring from a broad band of cortical nodes
During cytokinesis of fission yeast, a contractile ring forms through the condensation of a
broad band of ~ 65 cortical “nodes”. Each node is a protein complex which contains myosin-
II motors and formins. A mechanistic model describing the aggregation of nodes into a ring
is the search, capture, pull and release model (Vavylonis et al. Science 97:319, 2008).
According to this model, formin polymerizes actin filaments in random directions along the
cell cortex. These filaments establish transient connections among nodes and serve as links
for myosin to exert the force required to pull the nodes towards one another. Simulations
and experiments with mutant cells (Hachet, Simanis, Genes. Dev. 22, 2008) have
suggested that nodes may form clumps instead of ring when the parameter values are
different to those corresponding to wild type cells. To better understand the conditions for
successful ring assembly, we used scaling arguments, coarse grained stability analysis of
homogeneous node distributions, and Monte Carlo simulations. We found that initial uniform
distribution of nodes is stable over short times due to randomness of connection among the
nodes. Poisson fluctuations in the initial node distribution lead to clump instabilities at long
times. Uniform ring forms when the width of the broad band is less or approximately equal
to the average length of actin filaments: in this case the lateral shrinking occurs faster than
clump formation. We used scaling arguments to calculate the timescales for clump
formation and for lateral shrinking. We used these theoretical results to describe clump
formation process observed in the formin mutant cells.
34

Didier PORTRAN : didierportran@aol.com
IRTSV/LPCV , Grenoble, France
STUDY OF DYNAMIC ASSEMBLY AND ORGANIZATION OF MICROTUBULE
BUNDLE ARRAYS IN VITRO
In eukaryotes, microtubule (MT) arrays provide a molecular framework for
various cellular processes including cell morphogenesis, establishment of cell polarity
and cell division. Powering these cellular functions often hinges on the ability of the MT
arrays to auto-organize in higher order structures. This is particularly true for noncentrosomal
cells, such as plant cells, polarized epithelial cells, neural cells,.... where
MT arrays do not emanate from a centrosome. Instead, they are mainly self-organized
as MT bundles highly dispersed within the cell cortex. Thus, assembly of these cortical
MT bundles is a key feature of cortical array organization and function in numerous
cells. To understand physical laws and molecular mechanisms that underlie the
dynamic assembly that control the spatial organization of these MT arrays, we are
going to reconstitute MT bundle arrays in minimal biochemical systems (with zippering
MAPs such as Arabidopsis MAP65 members wich are homolog to PRC1 and Ase1p
proteins respectively in mammals and in yeast). Observations of MT dynamic, bundle
growth, bundle encountering… will use Total Internal Reflection Fluorescence
Microscopy (TIRFM). The biomimetic assays will be further complexify by adding other
molecules (stabilizing proteins such as CLASP and/or kinesins and/or +Tips proteins
such as EB1). This biomimetic system will be further completed by controlling the
spatial self-organization networks of MTs using micro- and nano-surface technic
(micro-pattern) allowing spatial constrained architectures. The aim is to be able to
define rules that govern MT behaviour when growing in specific environments or when
encountering other MTs, and also to get insights into the links between MT bundle
organization and physical constraints.
Amsha PROAG : amsha.proag@univ-paris-diderot.fr
Laboratoire Matière et systèmes complexes UMR 7057, Paris, France
MICROSTRUCTURED CHAMBERS TO STUDY THE SENSITIVITY OF CELLS
TO THE GEOMETRY AND STIFFNESS OF THEIR ENVIRONMENT
In order to study the behaviour of a living cell constrained in a tridimensional
environment, we designed microstructured substrates with controlled geometry and
stiffness.Using microfabricated moulds, we produce arrays of elastomer and hydrogel
microwells (poly(dimethylsiloxane), polyacrylamide, agarose), in which we can culture
cells.The size of one well (typically from 20μm to 250μm in diameter) determines the
numberof cells per well and the extent of their spreading; its height restricts the
number of possible layers. The shape of the well can be adapted to model the cell
physiological environment, depending on the cell type, which makes it possible to
examine in vitro the behaviour of cells in their natural geometrical configuration.
In addition, the Young modulus of the substrate material can be tuned over several
orders of magnitude (from 0.1kPa to 1MPa): we may thus observe the response of
cells to the substrate stiffness.
We also investigate surface treatment protocols to enable protein grafting on the
substrates before adding cells. We are currently developing a method for differential
coating of the bottom surface, inside walls and top surface of the microwells.
35

Thomas PUJOL : thomas.pujol@espci.fr
PMMH UMR 7636, ESPCI, Paris, France
Mechanical characterization of actin gels by a magnetic colloids technique.
The mechanism of the branched actin gel growing at the leading edge of moving cells
is a major topic in biophysics. Understanding the mechanical properties of actin
networks can give insights into cytoskeleton rigidity and cell migration.
We synthetize a branched actin network around colloids using the Arp2/3 protein
machinery. The particles are super-paramagnetic and display an important dipole
moment when an external magnetic field is applied. They attract each other via dipoledipole
interaction and form chains. By increasing the magnetic field, we increase the
force between the colloids and therefore apply a increasing stress on the actin gel
between two beads. The gel deformation is measured optically by observing the
distance between particles. We obtain stress-strain curves for actin networks, and
measure its Young modulus. This technique can also be used as a micro-rheomoter
by applying a sinusoidal stress with different frequency to obtain the dynamic modulus.
As each pair of bead in a chain is a sensor in itself, this technique allows us to monitor
a large number of gels and acquire convincing statistics.
We plan to characterize different actin networks by varying the concentration of the
capping and branching proteins and by using other actin binding proteins.
Michal REICHMAN-FRIED : mreichm@uni-muenster.de
Institute of Cell Biology, ZMBE, Munster, Germany
MIGRATION OF PRIMORDIAL GERM CELLS IN ZEBRAFISH EMBRYSO
The migration of Primordial Germ Cells (PGCs) in zebrafish relies on directional cues
provided by the chemokine SDF1a and its receptor CXCR4b. These cells serves as an
excellent model system for understanding long-range cell migration in development
and disease. Using time-lapse microscopy we have been able to monitor the
behaviour of PGCs from early stages of development to later stages when they have
reached their target. We show that following their specification the cells undergo a
series of morphological alterations that precede the acquisition of motility and
responsiveness to attractive cues. The cells then migrate towards their target while
correcting their path following phases of loss of morphological cell polarity. In the
following stages the cells gather at specific locations and move as cell clusters
towards their final target. In all of these stages, zebrafish PGCs individually respond to
alterations in the shape of the sdf-1a expression domain by directed migration towards
their target, the somatic part of the gonad.
To determine the molecular basis for PGC polarization and migration we have followed
the distribution of cytoskeletal elements in the migrating cells. Interestingly, unlike
findings in some other cell types, this analysis did not support the idea that actin
polymerization propels cellular protrusions. Rather, we could demonstrate that
zebrafish primordial germ cells generate bleb-like protrusions that are powered by
cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where
activation of myosin contraction occurs. Separation of the acto-myosin cortex from the
plasma membrane at these sites is followed by a flow of cytoplasm into the forming
bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in
free calcium that in turn activates myosin contraction in the part of the cell responding
to higher levels of the ligand SDF-1. The biased formation of new protrusions in a
particular region of the cell in response to SDF-1 defines the leading edge and the
direction of cell migration.
36

Derek REVILL : phy5djr@leeds.ac.uk
Contractility Group, Astbury Centre for Structural Molecular Biology,
University of Leeds, UK
MECHANICAL PROPERTIES OF MYOSIN LEVERS
Our work studies the mechanical properties of myosin lever domains, using myosin 5
as a model system. Myosins are motor proteins that participate in key cellular
functions including muscle contraction, intracellular cargo transport and cell migration.
Identified by a motor domain that binds actin and hydrolyses ATP, myosins share the
ability to translocate or move cargo along actin filaments. Their structure is
characterised by three domains: motor, lever and tail. Mechanical properties of the
lever are especially important in function. This domain, a single α-helix stabilised by
light chains that bind IQ motifs, must be both rigid enough to rotate as a lever and
generate force, whilst being flexible enough to accommodate functionally important
strained conformations. Variety across the myosin family suggests evolution of lever
structure to match mechanical function. Despite its functional importance, we lack a
detailed understanding of the mechanical properties of myosin levers and how they
derive from internal substructure. Work is presented that combines negative stain
electron microscopy and single-particle image processing to examine thermally-driven
lever conformations of myosin 5 molecules. Preliminary results show averages with
smoothly varying conformations, suggesting isotropic flexibility, but contrastingly, a
smaller subset of conformations were also found with sharp bends in the lever
indicating potential pliant points. A Metropolis Monte Carlo simulation technique was
developed to validate mechanical lever models against EM image averages. Future
plans are outlined to investigate the relationship between myosin lever mechanics and
substructure using various experimental and analytical techniques.
37

Anne-Cécile REYMAN : anne-cecile.reymann@cea.fr
iRTSV-LPCV,Bat C2, pièce 227D, CEA Grenoble, France
Physique du Cytosquelette et de la Morphogenese
GEOMETRICAL CONTROL OF ACTIN NUCLEATION GOVERNS ACTIN
NETWORK ARCHITECTURE
Cellular architecture needs to perform highly complex mechanical transformations in
order to achieve efficient morphogenesis, cell motility or any cell shape changes.
Perpetual dynamics, organization, regulation or rapid reconstruction are only a few of
the properties required for these morphological features which are supported by the
actin cytoskeleton. To achieve their cellular functions, actin filaments can assemble
into a large variety of super-structures The formation of these structures has been
studied biochemically however, how the localization of the nucleation zones affects the
structure of the actin networks is still an open question. Here we have combined UV
based substrate micro-patterning techniques and in vitro actin polymerization to design
a versatile and powerful biomimetic model system allowing actin dynamics study at the
cellular scale. Interestingly, actin polymerization occurs on this innovative biomimetic
device with conventional biochemical properties. Significantly, actin filament networks
assembled on these nucleating devices adopt a collective and highly reproducible
organization. Our study demonstrated that the spatial distribution of the boundary
conditions of actin nucleation sites controls the dynamics and mechanics of actin
assembly. Consequently, these boundary conditions give rise to specific network
architectures and hence affect the force production location. Through extensive
analysis of actin networks assembled on various geometries of nucleation zones, we
conclude that basic physical and probabilistic laws govern the spatial arrangements of
anti-parallel and filopodia-like parallel filaments. Importantly, the geometry of different
nucleation zones results in an entanglement of actin filaments into networks that can
control their length
Olena RIABININA : oriabinina@googlemail.com
UCL Ear Institute, London, UK
TUNED BY LOVE: MATING SONGS AND EARS OF FRUIT FLIES.
Drosophila melanogaster is an established model species in the studies of genetic
basis of sensory processing and behaviour. Here we investigate the components of
the auditory communication in the flies of 9 Drosophila species. Auditory
communication constitutes a crucial part of the flies mating ritual: while a male fly
pursuits a female, he vibrates his wings in a characteristic manner to produce a
species-specific “love song”. The frequency content of the songs differs greatly even
between flies of closely related species. The spectral variability of the songs suggest
corresponding differential tuning of the flies’ ears. Here we are bringing the signal and
the receiver aspects of this story together. Employing the laser-Doppler vibrometry
technique, we characterise the tuning of the antennal ears of the flies, and compare
the peaks of the hearing sensitivity to the maxima in the males’ songs. We
demonstrate, that the positive correlation holds between the characteristics of the ears
of the awake, but not sedated, flies. In the sedated state, the active amplification of the
hearing processes is likely to be affected. Thus, it is the active processes in the ears,
rather than the passive characteristics like mass and size, that has been shaped
during the evolution.
38

Serge RINCON : Sergio.Rincon@curie.fr
Institut Curie UMR 144, Anne Paoletti / Phong Tran Lab, Paris, France
SPATIO-TEMPORAL CONTROL OF CELL DIVISION BY MEDIAL CORTICAL
NODES IN FISSION YEAST CELLS.
Schizosaccharomyces pombe is a unicellular eukaryotic organism whose easy
genetics and reproducible cell shape make of it a good model for studying cell polarity
and division. Fission yeast cells are rod shaped, grow by tip extension and divide at a
constant length of about 14 μm by the formation of a contractile ring and septum in the
middle of the cell. Recent work has revealed that cortical nodes organized at the
medial cortex by the Wee1 regulatory kinase Cdr2 play a key role in the spatiotemporal
control of cell division (Moseley et al. 2009; Almonacid et al. 2009; Martin et
al. 2009). These nodes recruit the anillin-like protein Mid1 and influence the position of
the division plane in parallel to Mid1 export to the nucleus. In addition, medial cortical
nodes contain a second Wee1 regulatory kinase Cdr1 as well as a fraction of Wee1
kinase. The distribution of medial cortical nodes and the activity of Cdr2 kinase are
negatively regulated by Pom1 kinase. This polarity factor forms a gradient of
concentration from the cell tips to the cell middle. As cells grow, Pom1 concentration in
the medial region decreases, alleviating Cdr2 inhibition and promoting Wee1 inhibition
and entry into mitosis.Therefore, Cdr2-mediated assembly of cortical nodes and their
restricted localization to the medial cortex are key factors for the correct coordination
of cell size and mitotic entry and for the definition of the division plane. We are now
investigating how Cdr2 associates with the cortex to promote cortical nodes assembly
and how anchoring is inhibited by Pom1. These studies will help understanding how
the medial cortical region controlling cell division is defined.
Bibliography
- Moseley, J. B., A. Mayeux, et al. (2009). "A spatial gradient coordinates cell size and mitotic
entry in fission yeast." Nature 459(7248): 857-60.
- Almonacid, M., J. B. Moseley, et al. (2009). "Spatial control of cytokinesis by Cdr2 kinase
and Mid1/anillin nuclear export." Curr Biol 19(11): 961-6.
- Martin, S. G. and M. Berthelot-Grosjean (2009). "Polar gradients of the DYRK-family kinase
Pom1 couple cell length with the cell cycle." Nature 459(7248): 852-6.
39

Jérémy ROLAND : roland.jeremy@gmail.com
Laboratoire TIMC/IMAG, CNRS UMR 5525, La Tronche, France
HOW FAR DO WE UNDERSTAND ADF/COFILIN-DEPENDENT ACTIN FILAMENT
SEVERING?
Direct visualization of ADF/cofilin-decorated F-actin shows that most actin filament
severing events occur at the boundaries between decorated and non-decorated
sections of actin filaments. Consistent with this behavior, bare actin filaments and
those fully saturated with cofilin sever less easily than those partially saturated with
cofilin. However, the mechanical basis for preferential filament fracture at boundaries
has not been identified. It has been hypothesized that severing arises from the intense
and localized shearing of the filament due to an accumulation of stress at boundaries
between ADF/cofilin-decorated and non-decorated segments.
We have previously characterized the existence of twist-bend coupling in actin
filaments and hypothesize that twist-bend coupling in actin filaments is a plausible
candidate for converting bending filament undulation into stress accumulation at
boundaries that generates filament severing.
In the present work, we investigate the classical equations for elastic rods
supplemented with twist-bend coupling terms. By simulating actin filament dynamics in
different conditions (terminal load or twist; random forces) and in the presence or
absence of ADF/cofilin bound to the filament, we demonstrated that twist-bend
coupling is directly responsible for localized and important twist shear at boundaries of
ADF/cofilin decorated domains. Additionally, more than half of the elastic energy
density is associated with twist-bend coupling. Finally, numerical simulations of
filaments subjected to random forces provide a coherent qualitative and qualitative
description of the mechanical events leading to filament severing.
In conclusion, we suggest that actin filaments are nanomachines that convert long
range bending fluctuations into intense and localized twist shearing that contribute to a
greater probability of filament severing. This energy processing, which consists of
transforming elastic energy into work to break actin subunit bonds, is of crucial
importance in the dynamics of complex filament architectures, such as the
cytoskeleton.
40

Florian RUCKERL : Florian.Ruckerl@curie.fr
Institut Curie, CNRS, UMR 168, Paris, France
DYNAMICS OF ACTIN ASSEMBLY PROMOTED BY FORMIN
The shape and function of a cell is mainly determined by its cytoskeleton, especially by
the networks and bundles of polymerized actin. Force generation by actin
polymerization is, together with the contraction by myosin motors, essential for cell
motility. The branched actin networks are assembled by the Arp2/3 complex, and are
already well understood. Other structures like contractile rings and filapodia, which are
both polarized actin bundles, are organized by the multi domain protein formin. It acts
as a so-called leaky capper and remains bound to barbed end of the filament during
the polymerization process.
While the general mechanism of actin filament assembly by formin is known, the data
on binding rates, potential dead times and stall forces is still scarce.
Using functionalized microbeads in a precise optical tweezer setup with high lateral
and temporal resolution enables to measure the addition of single or multiple
monomers to a bundle or filament. This allows to measure the rate and frequency of
the assembly and gives insight to the molecular workings of the formin complex.
Especially the stepsize is interesting, as it is unclear whether formin adds single
monomers or pre assembled oligomers to the filament. Applying additional force to the
fibre with the optical trap enables to measure the stall force . This is of particular
interest, as the force created by formin mediated actin assembly might exceed the
normal polymerization forces.
41

Sara SADI : sara.sadi@wgi.su.se
Department of Cell Biology, Stockholm University, Suède
THE ROLE OF PROFILIN FOR SRF/MAL CONTROLLED GENE EXPRESSION
Actin filaments form the basic structure of the force-generating microfilament
system, which is essential for fundamental cellular processes such as cell motility,
adhesion and cytokinesis. Profilin is a key regulator of actin polymerization; it binds to
actin monomers and forms the profilin:actin (P:A) complex and delivers actin
monomers to the growing (+)-end of the filament via nuclear promoting factors like
Ena/Vasp and the formins. Profilin binds a number of other proteins and also lipids of
the phosphatidylinositol family. It has been reported that both actin and profilin are
present in the nucleus, however it is less clear to what extent the P:A complex is
formed and functions in the nucleus.
Actin is implicated in gene expression, e.g. the transcription factor SRF which
controls a large number of genes, including many encoding microfilament associated
proteins like actin itself and profilin, ‘senses’ cytoplasmic changes in actin dynamics
through its co-activator MAL. MAL is an actin monomer binding protein under constant
nucleocytoplasmatic shuttling. It competes with profilin for actin binding and upon
serum stimulation it dissociates from actin and accumulates in the nucleus. (Vartiainen
M).
In this project I am studying the connection between SRF/MAL and actin
dynamics with respect to the role of profilin and P:A and I have observed that downregulation
of profilin expression using siRNA interferes with the nuclear accumulation
of MAL fused to GFP. Furthermore, depletion of profilin also represses SRF/MALcontrolled
transcription as seen using a plasmid reporter system. It remains to be seen
if this is a concequence of the competition between profilin and MAL for actin or if
other mechanisms are involved. Other projects in my laboratory focus on microfilament
organization and dynamics at the leading edge in non-muscle cells.
42

Isabelle SAGOT : isabelle.sagot@ibgc.u-bordeaux2.fr
IBGC-CNRS UMR5095, Université de Bordeaux , France
ACTIN BODIES COMPOSTION: A COMPLEX PROBLEM
Actin Bodies are F-actin containing structures that are specifically assembled in
quiescent yeast cells. Actin Bodies are remarkably stable structures but disassemble
within seconds upon cells re-entry into the proliferation cycle. Actin Bodies formation
and molecular properties can result either to the specific expression in quiescent cells
of one or more ABPs or can be due to the specific post-translational modification of
one or more ABPs that lead to a change in its/theirs biochemical properties. In order to
decipher the molecular basis of Actin Bodies formation and maintenance, colocalization
studies have been undertaken and have shown that specific Actin Binding
Proteins are associated with these structures. Further genetic approaches have
demonstrated that some of these ABPs are required for Actin Bodies formation and/or
maintenance. However, these two approaches are not exhaustive and some critical
components of the Actin Bodies could have been missed. Although the actin filaments
that are embedded in Actin Bodies have a rather slow turn-over in living cells, Actin
Bodies are extremely sensitive structures and their purification turn out to be rather
difficult. To circumvent this problem, we have undertaken two proteomic approaches;
one based on 2D-DiGE and the other based on 2D BN / SDS – PAGE followed by
Mass Spectromerty. Partial preliminary results show that indeed, one ABP that colocalize
with Actin Bodies and is required for their formation and/or maintenance is
specifically modified in quiescent yeast cells. Our global approach may lead to the
deciphering of the complex interplay between ABPs that causes actin cytoskeleton
reorganization in quiescent cells.
Carsten SCHULDT : schuldt@physik.uni-leipzig.de
University of Leipzig Faculty of Physics and Earth Science, UK
DYNAMICS OF FORMIN PROMOTED ACTIN POLYMERIZATION
In vivo the semiflexible polymer actin is organized preferentially in networks or
bundles. These structures contribute to the cytoskeleton, whose inherent properties
determine the cell’s morphology, both mechanically and functionally, and facilitate
motility via protrusions and contractions. The assembly of large and polarized
cytoskeletal actin bundles (contractile ring, filopodia) far from thermodynamic
equilibrium is driven by a multi-domain protein called formin. This ’leaky capper’ is
known to remain bound to the growing barbed ends of filaments and is capable of
accelerating the polymerization rate.
We employ an optical tweezer setup in interaction with functionalized microbeads to
measure formin’s stall force and step size in vitro. Determining the stall force will yield
further insight into formin’s ability to produce forces from biochemical energy. In
particular, formin may be able to override the force limit of normal actin polymerization.
Measuring the step size may elucidate the assembly process itself. Does formin
preform actin oligomers and insert them to the existing filament at once?
The application of the sophisticated force clamp method seems to be an apropriate
technique to measure step size and examine the behavior of formin with and without
external applied tension.
43

Rama SHESHKA : sheshka@lms.polytechnique.fr
Laboratoire de mécanique des solides, École polytechique, Orsay, France
THE ROLE OF POWER STROKE IN MOLECULAR MOTOR MYOSIN II
The motor protein such as myosin II produce directional motion and force by
consuming the chemical energy stored in ATP. We try to understand the mechanism
used by molecular proteins to convert this chemical energy directly into the mechanical
work. We propose the mathematical model based on directional Brownian motion.
The hydrolysis of ATP plays an essential role in creating the directed motion
observed in motor proteins. Many experimental observations demonstrate the coupling
between ATP hydrolysis and the force-displacement generation. The binding of ATP to
the actin-myosin complex leads to rapid dissociation of the crossbridge from actin,
then myosin hydrolyze ATP and forms a stable ADP-Pi complex, the myosin attaches
to the actin filament. As ADP is realized, the motor protein passed in the rigor state in
which it is locked in its position on the filament until one other molecule of ATP is
bound. Binding cause a myosin- actin complex change its state so as to move the
actin approximately 5 nm, it is the powerstroke.
The attachment-detachment process can be viewed as a model of Brownian
ratchets. The motor (myosin) rectify the randomly fluctuating stochastic forces using
the periodic asymmetric potential which we model the actin filament.
The Power Stroke we presented entirely in mechanical framework by using a
bistable element. We combine the bistable element with the motor based on brownian
ratchets and also we try to explain and to reconstruct the Lymnn-Taylor cycle of
muscle contraction with four states model.
We apply this mathematical framework in the case of cooperative motor for understanding
the experimental curves during isometric and isotonic contraction using the numerical
computation of correspond system of Langevin equations
44

Tomita Vasilica STIRBAT :
tstirbat@lpmcn.univ-lyon1.fr
Laboratoire de Physique de la Matière Condensée et Nanostructures,
Lyon, France
The interest in the rheological and mechanical characterization of embryonic
cell aggregates is due to the fact that these “spheroids”, through their complex
properties are proven to be models of embryonic tissues. Their fluid like behavior can
help for the understanding of the process of tissue organization in the fields of
embryology, oncology and tissue engineering.
The team is interested in studying the physical properties of aggregates by
measurements of aggregate’s surface tension (using a compression tissue
tensiometer based on the Laplace formula applied to compressed aggregates), by
measurements of apparent viscosity (knowing the fusion time of two aggregates) and
by measurements of the elastic, viscous and plastic timescales (aggregates
compression-relaxation experiments).
We now want to go further in the rheological characterization of these aggregates.
The stress-strain relationship will be studied in details using a parallel-plate rheometer.
Moreover, in order to test their potential not-Newtonian properties we plan to measure
the velocity profiles of cells by forcing the aggregates to flow in microfluidic channels.
Björn STUHRMANN : stuhrmann@amolf.nl
FOM Institute for Atomic and Molecular Physics , Amsterdam,
NetherlandS
BIOMIMETIC MODELLING OF CELLULAR MORPHOGENESIS
Migration and division of living cells are ultimately generated by the coupled
morphogenesis of the cell cytoskeleton and the plasma membrane. Despite
impressive advances in the identification of the cell molecular inventory, the underlying
processes are still poorly understood. We strive to discern biophysical principles of
cytoskeletal and cell morphogenesis. To this end, we construct a biomimetic model
system of the cytoskeleton by confining to liposomes cross-linked actin biopolymers
driven by the active processes of polymerization and motor sliding. The key innovation
of this project lies in its systematic biomimetic approach alongside quantitative
morphological and mechanical examination and theoretical modelling.
45

Cristian SUAREZ : suarezcristian38@yahoo.fr
iRTSV-LPCV,Bat C2, pièce 227D CEA Grenoble, France
Physique du Cytosquelette et de la Morphogenese
Cell motility, based on actin polymerisation, requires a tight regulation of the dynamics
of Actin Binding Proteins (ABPs). ADF/cofilin is one of three ABPs that precisely
choreograph actin polymerization and organization to generate “comet-tail” motility in
vitro. ADF/cofilin severs actin filaments and then enhances disassembly of actin
filaments. During actin polymerisation, hydrolysis of the nucleotide (ATP to ADP-Pi)
bound to actin subunits occurs rapidly (k - = 0.35 s -1 ) whereas Pi release is much more
slower (k - = 0.0019 s -1 ). ADF/cofilin has a strong affinity only for ADP-actin, by
consequence its binding is excluded from newly polymerised zone of an actin filament.
We use TIRF (Total Internal Reflection Fluorescence) microscopy to follow in real time
the association of fluorescently-labeled ADF/cofilin on growing actin filaments. Our
results demonstrate that labeled ADF/cofilin is a marker of the nucleotide state of actin
filaments. We show also that fragmentation occurs near the interface between actin
subunits free of ADF/cofilin or bound to it. Finally, FRAP experiments allow us to
measure directly the dynamic of interaction of ADF/cofilin with actin filament.
Olga SYTINA : sytina@amolf.nl
Laboratory of Bio-organization, FOM Institute AMOLF, Amsterdan,
Netherlands
MEASURING SINGLE MICROTUBULE DYNAMICS IN VITRO WITH NEAR
MOLECULAR RESOLUTION.
OLGA SYTINA, SVENJA-MAREI KALISCH AND MARILEEN DOGTEROM
Microtubules are one of the main cytoskeletal polymers in all eukaryotic cells.
The experimental method to study molecular mechanisms of microtubule (MT) selfassembly,
force generation and regulation by MAPs (microtubule associated proteins)
with nanometer resolution will be presented. The technique integrates optical
tweezers with line optical trap coupled to the DIC high-resolution imaging microscope
and microfluidic cell with incorporated microfabricated barriers. In the experiment a
microbead with bound to it axoneme, serving as a nucleation site for MT growth, is
trapped in the laser beam and can be directed against a rigid barrier. MT growth from
axoneme is initiated by injection of the reaction mix and the displacement of the bead
upon MT length change is monitored with nanometer resolution. The new special
feature being introduced now is a force feedback system to understand how MT
dynamic properties change under constant load. Preliminary results of the MT growth
against rigid barriers and shrinkage dynamics will be presented.
46

Nessy TANIA : ntania@math.ubc.ca
Department of Mathematics The University of British Columbia
Vancouver, Canada
A Mathematical Model of Cell Motility Regulation by the Cofilin Pathway
Directed cell movement is an integral part of a wide range of physiological
processes. Cofilin is an important regulator of the actin cytoskeleton. Recent
experimental data from our collaborator, John Condeelis, on mammary
carcinoma cells reveal that upon stimulation by epidermal growth factor
(EGF), a pool of active cofilin is generated leading to the production of new
barbed-ends of actin filaments. These fast growing barbed-ends lead to
protrusive forces as the filaments extend. In carcinoma cells, cofilin thus plays
a primary role in initiating cell protrusion and determining cell direction. In this
presentation, we discuss and show results from a mathematical model of
cofilin regulatory pathway and its response to stimulation by EGF. The model
consists of a set of differential equations describing the dynamics of various
cofilin forms due to regulations by membrane lipids (PIP2), F-actin binding,
and inactivation/phosphorylation by LIM kinase (LIMK). In addition, F-actin
density is tracked and cofilin-induced barbed end generation is quantified.
Results from our model capture the dynamics of barbed end generation upon
the first minute of EGF stimulation as observed experimentally on mammary
carcinoma cells. Interestingly, we find that increasing cofilin inactivation via
LIMK promotes barbed end production. We show how this effect can be
explained in terms of the resting level of the membrane-bound cofilin form.
Hirokazu TANIMOTO :
thirokazu@daisy.phys.s.u-tokyo.ac.jp
Sano Laboratory, Department of Physics, Graduate School of Science,
the University of Tokyo, Japan
CELLULAR SHAPE, MOTION AND TRACTION FORCES ON VARIOUS ADHESIVE
SURFACES
Although many biophysical studies about cell migration have been done, the
quantitative relationship between cellular shape, motion and its forces is still unclear.
To elucidate the relationship, experiments with controlling physical conditions are
needed.
We used Dictyostelium cells as the model of fast moving cells and controlled the cellsubstrate
adhesion strength by changing collagen density on the poly-acrylamide gels.
With this setup, we measured cellular shape pattern, centroid motion and traction
forces exerted to the substrate. The results are as follows.
(1): Dictyostelium’s velocity takes its maximum value at the intermediate adhesion
strength.
(2): Cellular shape becomes ordered and oscillating in time with increasing adhesion
strength.
(3): The magnitude of the traction forces becomes larger with increasing adhesion
strength.
In this poster session, we will show these results and discuss how cells
coordinate their shapes, motions and forces in space and time.
47

Ulrike THEISEN : U.Theisen@warwick.ac.uk
Cytoskeletal Organization Centre for Mechanochemical Cell Biology
Warwick Medical School, UK
KIF1C, A KINESIN-3 FAMILY MEMBER, IS INVOLVED IN CELL MIGRATION,
POLARITY AND FUSION
Upon differentiation, myoblasts leave the cell cycle and change their morphology to a
bipolar spindle shape before they fuse predominantly at their tips to form a muscle
fiber. Microtubules are crucial in this process, forming a parallel array with the plus
ends extending towards the tips of elongating cells. Maintenance of cell polarity and
acquisition of fusion competence is therefore likely to involve kinesin-mediated
transport of important, as yet unknown factors towards the cell tips.
In an RNAi screen highly expressed Kinesins were depleted in C2C12 cells, and
monitored these cells for morphological defects upon differentiation. In this assay, 10
Kinesins were needed for proper cell elongation and subsequent cell-cell fusion.
Among these is the poorly characterized Kinesin-3 family member Kif1c. In addition to
causing defects in cell elongation and fusion, Kif1c depleted cells exhibit difficulties in
establishing or maintaining cell polarity and cell motility. In undifferentiated and moving
cells, Kif1c-GFP localizes to the centrosomal region and the cell periphery, especially
cell extensions. Upon differentiation, Kif1c concentrates at the tips of the elongating
cells, and when cells fuse redistributes to the tips of the recently fused myofibres.
Although initial studies indicated an involvement of Kif1c in cell adhesion in
macrophages, information on Kif1c’s function in cell polarity and fusion is lacking.
Current work to identify Kif1c’s cargo will hopefully reveal novel factors that are
important for the establishment and/or maintenance of cell polarity, and will help to
understand how their correct spatial distribution supports changes in cell morphology.
Anastasiya TRUSHKO trushko@mpi-cbg.de
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany
INTERACTION OF XMAP215 WITH DYNAMIC MICROTUBULES STUDIED WITH
OPTICAL TWEEZERS
Anastasiya Trushko, Erik Schäffer 2 , Jonathon Howard
Microtubules have a crucial organizing role in all eukaryotic cells. They are long
and relatively stiff hollow tubes of protein that consist of tubulin dimers and can rapidly
disassemble in one location and reassemble in other. Cell can regulate the
microtubule growth rate and their location with help of MAPs (microtubule associated
proteins). One member of this large family is XMAP215.
The protein XMAP215 binds to the end of growing microtubules and changes the
polymerization rate, promoting faster elongation. To investigate the addition of tubulin
dimmers to the plus end of the microtubule by XMAP215 and the dependence of the
addition on the applied force, XMAP215 is tethered to a microsphere held in optical
trap. Because XMAP215 remains at the microtubule end for several rounds of tubulin
addition, one can use it as a handle to hold the microtubule tip. In this way one can
assess the force exerted by polymerizing microtubule and explore changing its growth
dynamics under applied load with high temporal and spatial resolution.
48

Feng-Ching TSAI : tsai@amolf.nl
Biological Soft Matter Lab, Amsterdam, Netherlands
SYNTHETIC CYTOSKELETAL NETWORKS IN SOFT CONFINEMENT
Konstantinos TSEKOURAS : Konstantinos.Tsekouras@curie.fr
Institut Curie, UMR 168, Paris, France
ACTIN FILAMENT BUNDLE PUSHING AGAINST A WALL
Motivated by recent experiments of Israelachvili et al., we construct a theoretical
model of a bundle of parallel actin filaments growing together from a nucleating
location towards a wall upon which they exert force. We incorporate in our model actin
polymerization and depolymerization as well as hydrolysis of ATP-actin directly to
ADP-actin. A phase diagram is derived and the dynamics of the system theoretically
investigated. All results are validated with extensive Monte-Carlo simulations and
areas of agreement or deviation with experimental results discussed.
49

Stanilas VINOPAL : vinopal@img.cas.cz
Institute of Molecular Genetics ASCR, Prague, Czech Republic
LOCALIZATION OF GAMMA-TUBULIN IN NUCLEOLUS AND ITS DYNAMICS
Gamma-tubulin is the key protein for nucleation of microtubules. We have shown
previously that in human gliomas and glioblastoma cell lines γ-tubulin aberrantly
accumulated in the cytoplasm (J. Neuropathol Exp. Neurol. 65: 465, 2006).
Immunofluorescence microscopy of samples prepared under various fixation
conditions disclosed that beside microtubule organizing centers, γ-tubulin was also
located in the cytoplasm in the form of large aggregates. Moreover, under some
fixation conditions, bright staining for γ-tubulin was observed in nuclei with anti-peptide
monoclonal antibodies against γ-tubulin. Immunofluorescence with nucleolar markers
and marker of Cajal bodies demonstrated accumulation of γ-tubulin in nucleoli but not
in Cajal bodies. Nucleolar staining was inhibited when antibodies were preabsorbed
with immunizing peptides. Surprisingly, nucleolar staining of interphase cells was not
limited to glioblastoma cells, but was also detected in the other tested cultured cells.
The presence of nucleolar γ-tubulin was further confirmed by TEM, immunoblotting
and immunofluorescence of isolated nucleoli. To assess whether exogenous γ-tubulin
is associated with nucleoli, various tagged versions of human γ-tubulin were prepared.
In all tested cases exogenous γ-tubulin was detected in the nucleoli. After treatment
with leptomycin B, γ-tubulin did not accumulate in the nucleus suggesting the
independence of its nuclear export/transport on the RanGTP/Crm1 pathway. Using
green-to-red photoconvertible γ-tubulin-DendraII fusion, it was shown that γ-tubulin
does not rapidly shuttle between cytoplasm and nucleus. Confocal imaging of mitotic
cells revealed the association of γ-tubulin with nucleoli remnats in prophase and with
newly forming nucleoli in telophase as well. These results suggest that γ-tubulin might
get into the nucleolus during mitotis. In conclusion, we have shown the novel γ-tubulin
localization in the nucleolus. Our findings indicate that γ-tubulin can fulfill, in
transformed as well as non-transformed cells, other functions in addition to
microtubule nucleation.
50

Simone WIEGAND : s.wiegand@fz-juelich.de
Research center Juelich, IFF - Soft condensed Matter, Germany
MOVEMENT OF BIO- AND SOFT MATTER IN A TEMPERATURE GRADIENT
In order to explain how the concentration problem of early evolution in the highly
diluted ocean could have been solved often the importance of temperature gradients is
discussed as an origin of life concept [1]. Also in the reproduction process of
mammalians temperature gradients guide the sperms from the cooler reservoir site,
towards the warmer fertilization site [2]. Very recently it could be shown the convective
flow both drives the DNA replication polymerase chain reaction while concurrent
thermophoresis accumulates the replicated DNA in bulk solution. In order to gain a
deeper understanding of the underlying mechanism we study systematically bio- and
soft matter in a temperature gradient.
Various low molecular weight structures such as sugar molecules, sugar surfactant
and microemulsions we studied by a holographic grating method called infrared
thermal diffusion forced Rayleigh scattering (IR-TDFRS) setup [3]. Additionally we
developed a thermal diffusion cell in order to study synthetic and biological colloids
under a microscope. The new developed set-up is presented and its strength and
weakness in comparison with other methods is discussed. As first system we
investigate colloidal suspensions of silica particles and of fd-virus in a temperature
gradient. Open questions such as the radial dependence of the thermal diffusion
coefficient and its relation with the interfacial tension are considered. Additionally,
empirical correlations with the ratio of the thermal expansion coefficient and the
kinematic viscosity for polar and non-polar substances are discussed.
Marija ZANIC : zanic@mpi-cbg.de
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany
EB1 RECOGNIZES THE NUCLEOTIDE STATE OF TUBULIN IN THE
MICROTUBULE LATTICE
Plus-end-tracking proteins (+TIPs) are localized at the fast-growing, or plus end, of
microtubules, and link microtubule ends to cellular structures. One of the best studied
+TIPs is EB1, which forms comet-like structures at the tips of growing microtubules.
The molecular mechanisms by which EB1 recognizes and tracks growing microtubule
ends are largely unknown. However, one clue is that EB1 can bind directly to a
microtubule end in the absence of other proteins. Here we use an in vitro assay for
dynamic microtubule growth with two-color total-internal-reflection-fluorescence
imaging to investigate binding of mammalian EB1 to both stabilized and dynamic
microtubules. We find that under conditions of microtubule growth, EB1 not only tip
tracks, as previously shown, but also preferentially recognizes the GMPCPP
microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the
GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount
of salt in solution. The ability to distinguish different nucleotide states of tubulin in
microtubule lattice may contribute to the end-tracking mechanism of EB1.
51

Lecturers and organizers
e-mail
Laurent BLANCHOIN
Enrique DE LA CRUZ
Jonathon HOWARD
Fanck JULICHER
Marko KAKSONEN
Gijsje KOEDERING
David KOVAR
Pekka LAPPALAINEN
Matthias RIEF
Phong TRAN
Mike SIXT
Cécile SYKES
laurent.blanchoin@cea.fr
enrique.delacruz@yale.edu
howard@mpi-cbg.de
julicher@mpipks-dresden.mpg.de
kaksonen@embl.de
gkoenderink@amolf.nl
dkovar@uchicago.edu
pekka.lappalainen@helsinki.fi
mrief@ph.tum.de
tranp@mail.med.upenn.edu
sixt@biochem.mpg.de
cecile.sykes@curie.fr
52

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