Citoesqueleto

FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
INVITED SPEAKERS
-1-
Self-Organization Principles and Mitotic Spindle Morphogenesis
Karsenti Eric
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
The principles invoked to explain cellular morphogenesis are usually restricted to

simple rules of molecular interactions governed by the principle of mass action under
conditions of thermodynamic equilibrium. However, this is not how a cell works.
Energy is consumed and dissipated all the time through nucleotide triphosphate
degradation into diphosphate for most cellular processes and in particular for the
formation of complex cellular organelles like the mitotic spindle. The problem is
therefore to understand how the dissipation of chemical energy in the cell results in
the formation of such complex structures. In my seminar, I will show that assembly
of the mitotic spindle involves two self-organization principles: the formation of
regulatory gradients around chromosomes as a result of reaction-diffusion processes,
and the collective behavior of microtubules and their associated motors. The gradient
generated by chromosomes coordinates spatially microtubule nucleation, dynamics
and organization. The whole process evolves towards a steady state in which
microtubules are organized into two anti-parallel arrays with the chromosomes on
the equator. Energy is dissipated in the form of GTP hydrolysis during formation of a
Ran-GTP steady-state gradient around chromosomes and for microtubule dynamics.
It is dissipated in the form of ATP hydrolysis by the activity of molecular motors that
move and organize microtubules as well as by phosphorylation/dephosphorylation
reactions that are also required in this self-organization process. Therefore
robustness of spindle assembly derives from the fact that all the components
involved will interact in such a way that they will always end up in a dynamic regime
of interactions that corresponds to what we call a mitotic spindle. Mathematicians
call this an attractor. It is likely that similar principles are responsible for most of the
patterns observed at the cell and organism level as proposed long ago, in a principle
form, by Turing and Waddington and more recently by Stuart Kauffman.
-2-
Dynamic Polymorphism of Actin Molecules in the Filament
Kozuka J., Yokota H., Arai Y., Ishii Y. and Yanagida Toshio
Graduate School of Frontier Biosciences, University of Osaka, 1-3, Yamadaoka, Suita, Osaka,
Japan
Actin filament dynamics is crucial in cell motility. We observed the dynamics of actin
conformation by monitoring individual molecules in the actin filaments using single
molecule FRET. The results showed that actin exists in two major conformational
states between which it converted in a time scale of seconds. The equilibrium was
shifted to one state when interacting with myosin and to another when chemically
cross-linked to inhibit myosin motility. Thus, in the absence of an external signal,
the actin switches cyclically between an active and inactive conformation.
References and Notes

Funatsu T., Y. Harada, M. Tokunaga, K. Saito and T. Yanagida (1999). Imaging of
single fluorescent molecules and individual ATP turnovers by single myosin molecules
in aqueous solution. Nature 374: 555-559
Ishii Y., T. Yoshida, T. Funatsu, T. Wazawa and T. Yanagida (1999). Chem. Phys.
247: 163
-3-
Can We Ever Determine the Structure of Intermediate Filaments

at Atomic Detail ?
Strelkov S.1, Herrmann H.2, Kreplak L.1 and Burkhard P.3 and Aebi Ueli1
1
M.E. Mueller Institute for Structural Biology, Biozentrum, University of Basel, Switzerland
2
Division of Cell Biology, German Cancer Research Center, Heidelberg, Germany
3
The Institute of Materials Science, University of Connecticut, Storrs, Connecticut, USA
In contrast to actin and tubulin which are globular proteins, intermediate filament
(IF) proteins are fibrous proteins harboring a >300-residue long segmented -helical
rod domain. Whereas even >100-residue long -helical coiled-coil segments may be
crystallized and their structure determined to atomic detail [cf. Burkhard et al. (2000)
Structure 8: 223-230], it is the non -helical linkers within and end domains flanking
the central rod domain which render crystallization of IF proteins difficult. Hence, we
have developed a ‘divide and conquer’ approach to identify and crystallize a range of
IF protein fragments [Strelkov et al. (2001) J. Mol. Biol. 306: 773-781]. To date, we
have solved the crystal structure of several IF rod fragments including coil 1A which
occurs as a monomer -helix, and coil 1B and coil 2B which both form 2-stranded -
helical coiled coils [cf. Herrmann et al. (2000) J. Mol. Biol. 298: 817-832; Strelkov et
al. (2002) EMBO J. 21: 1255-1266; Strelkov et al. (2003) BioEssays 25: 243-251;
Strelkov et al. (2004) J. Mol. Biol. 343: 1067-1080]. In these structures we have
identified residues which are critically involved in distinct intra- or inter-helical salt
bridges, some of them causing disease phenotypes when miss-sense mutations are
introduced. While we succeeded to crystallize fragments that include linker and end
domain sequences, as yet, we have been unable to interpret the parts of the
electron density maps representing these sequences at atomic detail. Moreover, we
have prepared N- and C-terminal human lamin rod end fragments which, when
mixed in equimolar amounts, form heterologous tetrameric complexes mimicking the
head-to-tail interaction of lamin dimers. While these heterologous complexes are
soluble and reveal symmetrical peaks at 2.4 S by analytical ultracentrifugation, so far
they have resisted crystallization. Nevertheless, we have gone ahead and modelled
this interaction based on the atomic structures of the two fragments, and imposing
mapped crosslinks and point mutation data as constraints [Strelkov et al. (2004),
ibid.]. Also, we have shown recently that mutation of Lys 139 to Cys in linker L1 of
the human vimentin rod arrests IF assembly at the unit-length-filament (ULF) stage
when carried out at 22°C [Mücke et al. (2004) J. Mol. Biol. 340: 97-114]. For the
time being, while being soluble to about 10 mg/ml, evidently these ULFs are too
heterogeneous in terms of their number of subunits per cross-section for large
crystals to grow. We have also engaged these ULFs in small-angle X-ray scattering
(SAXS) experiments. By fitting the emerging scattering profiles we have been able to
model the lateral packing of dimers and tetramers within the ULFs including the tilted
orientation of the dimers relative to one another. While our progress has been much
slower than anticipated, we are confident that by a massively ‘hybrid’ structural
approach that includes X-ray crystallography, SAXS, cryo-electron tomography,
scanning force microscopy, various biophysical methods, and lots of modelling, we
will ultimately succeed to describe IF structure, assembly and dynamics at atomic
detail. Obviously, for this hybrid approach to be successful, we have to further refine
the design of IF protein fragments, particularly in terms of linker and end domain
sequences to be included in our dimer forming rod fragments to yield more stable
and homogeneous homo- and heterotypic teramers, octamers and ULFs.
-4-
Molecular Mechanisms of the Kinesin Motor
Schliwa Manfred
ABI Cell Biology, University of Munich, Munich, Germany
Most forms of movement we encounter in the living world are generated by motor
proteins that use the energy derived from the hydrolysis of ATP to take nanometer-
scale steps along a cellular track. Three classes of motor proteins, each comprised of
several families of motors of different makeup and function, are known to generate
linear movement: myosins, which move on actin filaments, and kinesins or dyneins,
which use microtubules. One class of kinesins, termed kinesin-1, are dimers in which
the two motor domains are coordinated in such a way that the motor can move
processively for several hundred steps in a hand-over-hand fashion. Initial events in
force generation involve the transmission of small structural changes triggered by
ATP hydrolysis in the globular motor domain to structural elements that translate
these initial events into a large conformational change. In kinesin, these elements
include a short, flexible region at the C-terminus of the motor domain, termed neck-
linker, a coiled-coil neck domain consisting of 4-5 heptads, and a highly flexible
region of 40-50 amino acids that links the neck to the central stalk of kinesin. To
study the contributions of, and the functional interplay between, these domains, we
are generating mutants of these motors and study their biochemical, biophysical and
motile properties using cell biological, enzymatic, microscopic, two-hybrid, and single
molecule assays. Our observations suggest a complex network of interactions
between these domains that is dependent on key residues within the neck and
adjacent domains. We propose a novel regulatory mechanism where the neck region
regulates the catalytic activity of the motor domain and the flexible hinge affects
neck dimerization, thus allowing for processive movement.
-5-
Defining the In Vivo Functions for Enterocyte Class I Myosin,

Myosin-Ia (Myo1a)
Mooseker Mark
Department of Molecular Cellular Developmental Biology, Yale University, New Haven,
Connecticut, USA
Myo1a is one of 8 class I myosins expressed in vertebrates. Myo1a is a major

component of the intestinal brush border (BB) cytoskeleton. Myo1a forms a spiral
array of lateral tethers between the microvillus (MV) membrane and the MV actin
core. The tail domain of Myo1a is necessary and sufficient for localization to the MV
membrane through its interaction with the cytoplasmic domain of the lipid raft-
associated hydrolase, sucrase-isomaltase (SI). Disruption of Myo1a-SI interaction by
dominant-negative (DN) - Myo1a tail expression in the intestinal cell line, Caco2-BBe
(BBe), results in a loss of SI from the BB. DN-Myo1a-tail expression also results in a
loss of MV length control and displacement of the minus-end directed motor, Myo6
from the subapical interMV domain where this motor is thought to play a role in
clathrin-dependent endocytosis. Indeed, endocytosis of lactoferrin is inhibited in DN-
Myo1a-tail expressing BBe cells. The initial phenotypic characterization of the
enterocyte of the Myo1a KO mouse has revealed a remarkably complex array of
defects at the structural and molecular level. This includes a variety of perturbations
of the BB membrane, including protrusions of MV membrane, large cytoplasmic
herniations, both presumably due to loss of Myo1a tethers, as well as extensive MV
vesiculation, presumably due to the loss of Ca2+ buffering by Myo1-a associated
calmodulin light chains. MV packing and terminal web organization are also
disrupted. There are also marked compositional changes in the BB. SI expression
levels are reduced, and there is significant mistargeting of SI to the basolateral
domain. As in Myo1a-DN-tail expressing BBe cells, Myo6 is lost from the interMV
domain. Remarkably, the structurally related class I myosin, Myo1c is ectopically
recruited from the BL to BB domain, a clear demonstration of partial functional
redundancy among class I myosins. There is also a marked increase in TW-
associated cytokeratins 8/18, possibly reflective of increased cellular stress levels. A
model integrating the impacts of Myo1a loss of function, including the role of
Myo1a-mediated plus- end tension on the MV membrane will be discussed.
References
Tyska, M.J., A.T. Mackey, J.D. Huang, N.G. Copeland, N.A. Jenkins and M.S.
Mooseker (2005). Myosin-1a is critical for normal brush border structure and
composition. Mol Biol Cell. 16: 2443-57
Tyska, M.J. and M.S. Mooseker (2002). MYO1A (brush border myosin I) dynamics in
the brush border of LLC-PK1-CL4 cells. Biophys J. 82: 1869-83
Tyska, M.J. and M.S. Mooseker (2004). A role for myosin-1A in the localization of a
brush border disaccharidase. J Cell Biol. 165: 395-405
-6-
Control Mechanisms of Actin Polymerisation
Carlier Marie-France
Laboratoire d'Enzymologie et Biochimie Structurale (LEBS), CNRS, Gif-sur-Yvette, France
In living cells, actin is assembled at a steady state. In the absence of regulators,

filament pointed ends depolymerize while barbed ends undergo net assembly
(treadmilling). Motility and protrusive force are generated by controlled assembly of
filaments at barbed ends. We now know a variety of regulators that act at barbed
ends in different ways : either they block assembly (capping proteins), thus
regulating length and life time of growing filaments ; or they branch filaments at a
membrane (WASP-Arp2/3) ; or they facilitate actin assembly at barbed ends (profilin
and WH2 domains) ; or they act as weak (low affinity) cappers, or as leaky cappers
that allow growth at a slower pace ; or they catalyze site-directed rapid processive
growth (formins in association with profilin). Complexity in this regulation is
increased as new capping proteins are discovered that can be controlled by
signalling, thus enhancing motility in a spatially and temporally defined fashion
(Eps8). Other proteins may cumulate sequestering and capping activities. Hence a
variety of motile processes proceed at specified rates, simultaneously, in different
regions of a cell.
-7-
Coordinated Regulation of the Actin Cytoskeleton and Microtubule

Dynamics by mDia1 Formin
Bershadsky Alexander D.1 Ballestrem C.1, Zilberman Y.1, Carramusa L.1, Shtutman
M.1, Gilquin B.2, Khochbin S.2, Shemesh T.3 and Kozlov M.M.3
1
Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100,
Israel;
2
Equipe Chromatine et Expression des Gènes. INSERM U309 - Institut Albert Bonniot -
Faculte de Medecine - Domaine de la Merci - 38706, La Tronche Cedex, France
3
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv
University, Tel Aviv 69978, Israel
Coordination of actin polymerization, myosin-driven contractility and microtubule

dynamics is essential for a broad class of cell motility events. We describe here
several novel mechanisms of such coordination that depend on the function of
formin homology proteins. Formins bind to the barbed (fast growing) ends of actin
filaments but, in contrast to the standard capping proteins, allow for actin
polymerization in the barbed direction. Our model of such "leaky" capping is based
on an assumption of elasticity of the formin/barbed end complex and predicts that
formin-mediated actin polymerization should be greatly enhanced by application of
external pulling force. This proposed force-driven polymerization mechanism could
be a key element in a variety of cellular mechanosensing devices [Kozlov and
Bershadsky (2004) J. Cell Biol. 167: 1011-1017]. Further studies revealed a link
between actin cytoskeleton and microtubules mediated by interaction between
mDia1 formin and alpha-tubulin deacetylase HDAC6. Expression of constitutively
active mDia1 formin homology domains decreased microtubule plus end growth rate
by 50%. Microtubule growth inhibition required intact actin filaments and was
abolished by cytochalasin D or latrunculin B. Interestingly, however, we also found a
more direct relationship between mDia1 and microtubules. In latrunculin B-treated
cells, micron-sized patches containing active mDia1 moved rapidly along
microtubules. HDAC6 cooperates with mDia1 in its microtubule interaction; i.e.
HDAC6 co-immunoprecipitated with mDia1, and both proteins associated with the
plus-end microtubule tracking proteins, EB-1 and the dynactin component, Arp1.
Pharmacologically blocking HDAC6’s deacetylase activity mimicked mDia1’s effect on
microtubule dynamics. HDAC6-siRNA knockdown showed that HDAC6 is necessary
for mDia1 to decrease microtubule growth, and overexpressing wild-type, but not
catalytically inactive, HDAC6 partially restored microtubule growth in mDia1-
expressing cells. Thus, HDAC6 with inhibited deacetylase function mediates mDia1’s
effect on microtubules. We conclude that mDia1 and HDAC6 are constituents of a
molecular complex responsible for a dynamic link between growing ends of
microtubules and actin filaments.
-8-
Root Hairs: a Model System for Studying Cytoskeletal Dynamics

and Cell Expansion
1
Ketelaar Tijs1, Sieberer B.1, Esseling J.J.1, Hussey P.J.2, Emons A.M.C.
1
Laboratory of Plant Cell Biology, Wageningen University, Wageningen, The Netherlands
2
Integrative Cell Biology Laboratory, University of Durham, Durham, UK
Plant cells are walled compartments. Together with the number and location of cell
divisions, their direction of expansion determines plant stature. Since the cell wall
volume increases at the location of cell expansion, exocytosis of cell wall matrix
materials in plant cells has to occur at the location where growth occurs. Root hair
cells are tip growing cells. They expand over a very small surface area, resulting in
the formation of a tubular structure. The small surface area over which exocytosis
occurs requires a polarized intracellular organization that delivers growth materials to
this area and retains them there. Both the actin and microtubule cytoskeletons are
involved in these processes. Microtubules determine directionality of root hair
growth, but are not involved in growth itself, whereas filamentous actin is involved in
delivering exocytic vesicles to, and retaining them at the cellular surface where
growth occurs. Several mutants that are mutated in cytoskeleton organizing proteins
show the importance of the cytoskeleton in root hair cell growth and plant cell
growth in general. Moreover, these mutants pinpoint how cytoskeleton interacting
proteins regulate the dynamics of the cytoskeleton in live cells.
-9-
WASP versus WAVE: Differential Subcellular Distribution,

Dynamics and Function
Rottner Klemens1, Lai F.P.L.1, Steffen A.1, Benesch S.1,2, Polo S3, Scita G.3, Small
J.V2, Wehland J.1 and Stradal T.E.B.1
1
German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig,
Germany
2
Institute of Molecular Biotechnology, Dr. Bohr-Gasse 3-5, A-1030 Vienna, Austria
3
IFOM, the FIRC Institute for Molecular Oncology Foundation, Via Adamello 16, 20139,
Milano, Italy
Cellular actin polymerisation is catalysed by actin filament nucleation or amplification

mediated for instance by the Arp2/3-complex. Prominent Arp2/3-activators in
mammals include WASP and WAVE subfamily members. While the haematopoietic
WASP and the ubiquitous N-WASP can interact directly with the small Rho-family
GTPase Cdc42, WAVE-mediated Arp2/3 activation is thought to be linked indirectly to
Rac1 activation. Here, recent insights into specific functions of WASP and WAVE
proteins are discussed. Microinjection combined with epi-fluorescence time-lapse
microscopy established that the ubiquitous WAVE2 and its constitutively associated
partners Abi-1, Nap1 and the Rac interactor Sra-1 co-translocate to the tips of
lamellipodia upon Rac1 activation. Suppression of Sra-1 or Nap1 expression by RNA
interference prevented Rac-induced WAVE2 positioning at the cell periphery and
abolished lamellipodia protrusion. In contrast, N-WASP was not observed to localize
to these structures and cell lines lacking both WASP subfamily proteins (N-WASP null
fibroblasts) displayed no detectable defects in lamellipodia or filopodia protrusion.
Instead, TIRF microscopy experiments revealed more recently that N-WASP and
associated proteins - but not WAVE2 – can specifically accumulate at sites of
endocytic activity such as clathrin coated pits (ccp) shortly before their invagination
and/or internalization. Moreover, N-WASP null cells showed a reduction of actin
assembly frequency at these sites and an impairment of receptor mediated
endocytosis. Interestingly, Arp2/3 sequestration in the cytosol abolished actin
assembly at ccp entirely, suggesting that N-WASP and (an) additional factor(s)
cooperate in Arp2/3 activation at these sites. Together, these results indicate that
the specificity of Arp2/3 activation by WASP versus WAVE proteins is brought about
by differential subcellular positioning of these factors possibly mediated by assembly
into differential protein complexes. Current experiments are aimed at extending our
analyses to determining the turnover of key protein complexes regulating Arp2/3
activation at specific subcellular locations, for instance by FRAP or photo-activation
approaches.
- 10 -
Studying the Coupling of ER Exit to Microtubules in Living Cells
Pepperkok Rainer
Transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex is
mediated by the sequential action of two vesicular coat complexes. COPII
concentrates cargo for secretion at ER exit sites, COPI is subsequently recruited to
transport carriers and acts in retrieval of recycling proteins back to the ER. These
COPI coated carriers then move towards the Golgi along microtubules, driven by the
dynein/dynactin microtubule motor complexes.
Using different live cell imaging techniques and mathematical modeling of the
experimental data we show that the presence of transport competent secretory
cargo and the interaction of the Sec23/24p COPII sub-complex with the dynactin
complex component p150Glued stabilize COPII at ER exit sites. This prevents
premature COPII disassembly and provides the time to enable cargo sorting,
concentration and subsequent carrier formation. Together, our data suggest a
mechanism by which membranes of the early secretory pathway can be linked to
motors and microtubules for subsequent organization and movement to the Golgi
apparatus.
- 11 -
Coupling Cytoskeletal Dynamics to the Regulation of

Gene Transcription
Guettler S., Miralles F., Vartiainen M. and Treisman Richard

Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London
WC2A 3PX, UK
The transcription factor SRF controls both growth factor-regulated and muscle-
specific genes. Its activity is controlled by two families of regulatory cofactors, the
TCFs and the MRTFs. These cofactors interact with SRF in a mutually exclusive
manner and are primarily activated by MAP kinase signalling (TCFs) or Rho GTPase
signalling (MRTFs). The cofactors involved in the Rho signalling pathway, MAL/MKL1
and MAL16/MKL2, are related to a constitutively active muscle-specific SRF
coactivator, Myocardin. MAL and MAL16 are controlled via a novel pathway in which
depletion of the G-actin pool in response to Rho signalling potentiates their activity.
The MAL proto-oncogene is rearranged in t(1;22)(p13;q13) AML.
In fibroblasts MAL is predominantly cytoplasmic, but upon activation of Rho it rapidly
accumulates in the nucleus. MAL binds G-actin through its N-terminal RPEL domain,
and treatment of cells with stimuli or drugs that disrupt this interaction cause its
nuclear accumulation. The mechanism of MAL-actin interaction will be discussed.
Curiously, although the RPEL domain is conserved in Myocardin, this protein is
nuclear in fibroblasts, and experiments to address the molecular basis for this will be
discussed. MAL is also subject to multiple post-translational modifications, including
phosphorylation, and experiments to address their role in MAL regulation will be
presented.
- 12 -
Lamina Proteins in Nuclear Architecture, Dynamic Chromatin

Organization and Cell Cycle Control
Dorner D., Gajewski A., Makolm C., Vlcek S., Gotzmann J. and Foisner Roland
Max F. Perutz Laboratories, Department of Medical Biochemistry, Medical University of
Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria
The nuclear lamina and nucleoplasmic lamins organize nuclear architecture and
higher order chromatin structure. Numerous mutations in lamina proteins have been
linked to various human diseases (laminopathies), affecting heart, skeletal muscle,
bone, neuronal, and fat tissue, or causing premature ageing. We have been studying
the cell cycle-dependent dynamics and potential functions of the lamin A-interacting
protein lamina-associated polypeptide 2a (LAP2a) in order to unravel molecular
mechanisms of laminopathic diseases. LAP2a is a unique non-membrane bound LAP2
isoform that forms complexes with A-type lamins in the nucleoplasm and has a dual
function in cell cycle dependent chromatin organization and in cell proliferation and
differentiation. During mitosis, LAP2a translocates from the cytoplasm to telomeric
chromosome regions in anaphase and forms stable chromatin-associated structures
with the DNA-crosslinking protein BAF. Thus, LAP2a may be involved in the
establishment of higher order chromatin structure in G1 phase nuclei. LAP2a is also
required for targeting a subfraction of lamins A/C to the nucleoplasm. Nucleoplasmic
lamins A/C-LAP2a complexes interact with the cell cycle regulator protein
retinoblastoma (Rb). Over-expression of LAP2a in cultured fibroblasts delayed growth
restimulation after serum-starvation, whereas RNAi-mediated down-regulation of
LAP2a in lamins A/C-expressing cells interfered with cell cycle withdrawal upon
serum starvation. Furthermore, expression of LAP2a in proliferating pre-adipocytes
initiated partial differentiation into adipocytes in the absence of hormons, suggesting
that lamins A/C-LAP2a complexes regulate the balance between proliferation and
differentiation. This regulatory function requires Rb and affects E2F transcriptional
activity. We propose that lamins A/C-LAP2a complexes control differentiation of adult
stem cells during tissue regeneration and that this novel function of lamina proteins
is highly relevant for the cell- and tissue phenotypes in laminopathy patients. Work
was supported by grants from the Austrian Science Research Fund (FWF).
- 13 -
Dictyostelium, a Model to Assess the Function of

Cytoskeletal Proteins
Noegel Angelika
Institute for Biochemistry I, Medical Faculty University of Cologne, Cologne, Germany
The Dictyostelium genome sequence is now complete and in the public domain. This
is a doubly important event; because it is the first amoebozoan genome sequence to
be determined and because Dictyostelium has become the model system of choice in
several key areas of cell biological research. The genome sequence has revealed its
share of surprises, including: a remarkably high total number of genes, genes that
are present in Dictyostelium and mammals but absent from lower metazoans and a
large number and diverse array of microfilament components. Dictyostelium cells
harbor a dynamic actin cytoskeleton whose rearrangements drive cell movement,
cytokinesis and phagocytosis. The cells are fast and excellent movers, properties
required by their life-style. The exquisite level of control Dictyostelium thereby exerts
over its cytoskeleton is beautifully reflected by the gene content. We now know that
a total of 176 genes contribute to and/or modulate the Dictyostelium actin
cytoskeleton. This number corresponds to 1.4% of the proteome and includes 30
actin genes and 11 genes encoding actin related proteins of which three are
founding members of a new class. Representatives of all classes of actin-binding
proteins (ABPs) are present in the genome and Dictyostelium’s repertoire of ABPs is
most similar to metazoa followed by fungi and then plants. The close reationship to
metazoa is one of the reasons why Dictyostelium is such a valuable model for
studying cell motility.
- 14 -
Bacterial Pathogenesis: Signaling to the Actin Cytoskeleton
Wehland Jürgen
Department of Cell Biology, German Research Center for Biotechnology (GBF), Braunschweig,
Germany
During their long lasting co-existence with their hosts, microbial pathogens have
evolved a variety of strategies to survive in the host, for instance by avoiding or
resisting various immune defence mechanisms or by replicating in protected niches
such as the host cell cytoplasm. Many bacterial pathogens accomplish this by
employing an arsenal of highly sophisticated mechanisms that subvert the host cell
actin cytoskeleton which is pivotal for these pathogens to enter cells, to disseminate
within and between infected tissues, to prevent their uptake by phagocyte cells or to
promote intimate attachment to the host cell surface. To do so these pathogens
have evolved common as well as unique strategies to modulate actin dynamics, not
only at the plasma membrane, but also within the cytoplasm of host cells.
Upon contact with host cells, some pathogens deliver distinct effector proteins
directly into the host cell cytoplasm (e.g. Salmonella and Shigella). These effectors
exert their activity either by directly intruding on actin polymerisation or by activating
cellular upstream regulators of actin polymerisation. Other pathogens like Listeria
can induce their uptake into non-phagocytic cells through ignition of a unique
combination of signaling pathways. Another group of pathogens (pathogenic
Escherichia coli and Helicobacter pylori) do not invade their host cells, but subvert
the actin cytosekeleton from outside with the aim of colonising niches in the stomach
or gut and to take advantage of commensals with less sophisticated adhesion
mechanisms. Over the years, these events have been extensively studied and
therefore now belong to the best-understood facets of host-pathogen interaction.
Recent progress in our understanding of the molecular regulation of bacteria-host
cell interactions driving local actin reorganizations will be presented.
- 15 -
LIM Proteins: Molecular Scaffolds for Cytoskeletal Regulation

and Signaling
Beckerle Mary
Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84103 USA
Proteins involved in regulating complex cellular behaviors such as signal

transduction, cytoskeletal dynamics, cell proliferation, and cell death are typically
comprised of modular domains that define functional attributes and confer temporal
and spatial control. One such modular domain is the LIM domain, which was first
identified in three transcription factors, Lin-11, Isl-1, and Mec-3. The LIM domain is a
cysteine and histidine-rich sequence comprised of about 60 amino acids. A LIM
domain coordinates two Zn ions to generate a double zinc finger structure that
serves as a protein binding interface. LIM domains can regulate protein activity,
partnerships, and location. There are 58 LIM proteins that have been identified in the
human proteome. Many LIM proteins are associated with both cytoskeletal elements
and the nucleus, suggesting a role in communication between these two cellular
compartments. A number of constituents of focal adhesions display LIM domains,
including paxillin, CRP, PINCH, ALP/Enigma, Testin, and Zyxin. Recent studies that
illustrate the involvement of zyxin in nuclear-cytoplasmic communication, cell
motility, actin remodeling, and mechanosensation will be described.
- 16 -
Talin: an Essential Link between Integrins and the Cytoskeleton
Tanentzapf Guy and Brown N.H.

Gurdon Institute, Tennis Court Road, Cambridge, CB2 1QR, UK
Cellular adhesion and communication are vital during the development of

multicellular organisms. These processes use cell surface proteins that can either
mediate adhesion or transmit signals from outside the cell to the interior, so that the
cell can respond to its environment. Integrins, members of one such family of cell
surface receptors, can perform both of these activities, and therefore provide a
molecular link between cell adhesion and signalling. Integrins bind to extracellular
matrix ligands on the outside of the cell and link to the cytoskeleton inside the cell.
The cytoplasmic protein talin has been suggested to act as a linker between integrins
and the cytoskeleton and we have tested this within the developing Drosophila
embryo. Genetic analysis of talin function in the Drosophila embryo revealed that it
was essential for linking between integrins and the actin cytoskeleton. Analysis of the
distribution of tagged domains of talin in wild type and mutant backgrounds showed
that different parts of the talin protein mediate interactions with either the
cytoskeleton or with integrin itself. In addition, mutational, analysis of the integrin
and actin binding domains of talin revealed surprising flexibility and functional
redundancy within the talin molecule in its ability to associate with integrins and with
the cytoskeleton. These results suggest that talin associates with either integrin or
the cytoskeleton in multiple ways. This previously unforeseen flexibility could provide
a mechanism by which the diverse array of functions mediated by integrins may be
translated into distinct cytoskeletal behaviours.
- 17 -
Plasma Membrane Organization and Cell Growth Signaling by the

Membrane-Cytoskeleton Linker Ezrin
Arpin Monique
Institut Curie, Biology Division, Paris, France
Ezrin is a membrane-cytoskeleton linker involved in dynamic functions that occur at

the plasma membrane and in signal transduction pathways. In the highly polarized
epithelial cells, where the protein is primarily expressed, ezrin participates to the
assembly of specific domains of the plasma membrane.
Actin-membrane linkage is critical for ezrin functions therefore is highly regulated. In
the cytoplasm, ezrin exists in a closed or inactive conformation due to intramolecular
association between its N-terminal and C-terminal domains. Activation through the
release of this interaction is necessary for ezrin binding to actin filament and
membrane. Ezrin activation occurs through sequential conformational changes that
are triggered by binding to PIP2 followed by phosphorylation at threonine 567
(T567). Moreover, PIP2 binding and T567 phosphorylation are both necessary for the
correct apical localization of ezrin and for its role in epithelial cell morphogenesis.
The correct activation of the protein is also a prerequisite for its signalling functions.
We have shown that ezrin can regulate functions such as proliferation and survival
when cells are grown in a 3D-collagen matrix, in a tubulogenesis assay. Ezrin signals
proliferation and survival through its phosphorylated tyrosine residues 145 and 353
respectively in response to growth factor stimulation and extracellular matrix
adhesion.
In conclusion, regulated conformational activation of ezrin conditions its signalling
functions in the development and maintenance of epithelial cell polarity.
- 18 -
Abi1 Signaling Complexes are Master Regulators of

Actin Dynamics-Based Cell Motility
Scita Giorgio
The FIRC Institute for Molecular Oncology (IFOM), Milano, Italy
Dynamic assembly of actin filaments generates the forces supporting cell motility.
Several recent biochemical and genetic studies have revealed a plethora of different
actin binding proteins, whose coordinated activity regulates the turnover of actin
filaments, thus controlling a variety of actin-based processes, including cell
migration. Additionally, emerging evidence is highlighting a scenario whereby the
same basic set of actin regulatory proteins is also the convergent node of different
signaling pathways emanating from extracellular stimuli, like those from receptor
tyrosine kinases. Within these pathways, Rho GTPases are crucial signal transducers.
Here, emphasizing the role of two signaling proteins, Abi1 and Eps8, we will discuss:
i) how Abi1-based molecular complexes regulate de novo actin nucleation and
branched filament elongation, through WAVE and N-WASP family of proteins, and ii)
how Eps8 and its family members can control the availability of free filaments barbed
ends, by acting as barbed end capping proteins. The integration and spatially
restricted regulation of these activities is crucial for actin-based generation of forces
leading to cell motility.
- 19 -
Biomimetic Systems to Study Actin-Based Movement

and Force Generation
Sykes Cécile
Laboratory of Physical Chemistry UMR 168, Research Section, Curie Institute, Paris, France
Actin polymerizes within cells and can cause movements and deformations. This
phenomenon of force generation by monomer assembly is currently found only in
biological systems. To probe the mechanisms of polymerization-driven force
production, we use biomimetic experimental systems made of inert micrometric
objects that can be propelled in cytoplasm by actin assembly at one side. These
systems allow for versatile handling, since parameters like their size, and their
deformability can be controlled. They can be micromanipulated and we will show
measurements of the force generated during the polymerization process as a
function of the velocity of propulsion. By the use of deformable droplets as propelled
objects, we show that the propulsion mechanism is based on the elastic properties of
the actin network built by polymerization and assembly of actin filaments. Propulsion
of initially spherical objects is preceded by a fracture of the actin shell, which is
reminiscent of shape oscillations displayed by cells in suspension in the absence of
microtubules.
- 20 -
Dynamics of Force-Generating Microtubules

and Microtubule Bundles
Dogterom Marileen
FOM Institute for Atomic and Molecular Physics (AMOLF), Amsterdam, The Netherlands
We perform in vitro experiments aimed at understanding force generation by the

“microtubule polymerization motor” in the absence and presence of microtubule
associated proteins. Using micro-fabrication techniques we create rigid barriers that
are used to measure the piconewton forces that are generated by the growth of
single microtubules. With the help of optical tweezers the molecular details of the
microtubule growth process in the presence of force can be detected. A bead is
attached to a stiff microtubule-nucleating object (an axoneme), which is held with a
time-shared “key-hole” optical trap in front of a micro-fabricated barrier. Growth of
the microtubule results in motion of the bead that can be detected with nanometer
resolution. Using this technique it is possible to study directly the molecular details of
the microtubule growth process as well as the molecular mechanism by which
microtubule associated proteins (MAPs) interfere with microtubule dynamics and
force generation. Results in the absence and the presence of the microtubule
associated protein XMAP215 will be presented.
Our technique can also be used to study the dynamics of up to 10 force-generating
microtubules growing in parallel against a barrier. In cells, dynamic microtubules
often form parallel bundles, for example when interacting with the kinetochores of
chromosomes in the mitotic spindle. First results on force generation and dynamics
of microtubule bundles will be presented.
- 21 -
Mechanical Weakness of Nuclei and Cells in Laminopathies Result

From Both Lamin Disorganisation and Cytoskeletal Abnormalities
Ramaekers Frans1, Peeters E.A.G.2, Kuijpers H.J.H.1, Endert J.1, Baaijens F.P.T.2
and Broers J.L.V.1,2
1
Dept. of Molecular Cell Biology, University of Maastricht, P.O. Box 616 NL-6200 MD
Maastricht, The Netherlands
2
Dept. of Biomedical Engineering, Biomechanics and Tissue Engineering, Eindhoven
University of Technology, Eindhoven, The Netherlands
Laminopathies comprise a heterogeneous group of inherited diseases caused by

mutations in the lamin A/C gene (LMNA). Until now, the underlying mechanisms,
causing these diseases, remain largely obscure. One of the hypotheses, the
mechanical stress hypothesis, was examined by investigating the cellular and nuclear
architecture as well as cellular stiffness in normal and affected cells. Bleaching
studies in CHO cells, transfected with mutated lamins, showed that these mutations
caused lamina instability, as well as a prominent loss of internal nuclear lamin
organization. Using a specially designed cell-loading device we have compressed
normal fibroblasts (MEF+/+) next to cells lacking lamin A/C gene expression
(MEF-/-). These studies showed that MEF-/- cells have significantly reduced
mechanical stiffness, when compared with MEF+/+ cells. Moreover, these cells have
weaker nuclei, as seen by nuclear fragmentation during compression. This phenotype
could not be rescued by transfection of lamin A and/or lamin C of MEF-/- cells,
probably because the transfected cells did no longer go through mitosis. This finding,
combined with our observation that anisotropic deformation was lost in MEF-/- cells,
urged us to more closely examine the cytoskeletal organisation of normal and
diseased cells. While all three types of cytoskeletal proteins (actin, tubulin and
vimentin) had an irregular appearance, especially vimentin seemed to be completely
disorganised. Also, in CHO fibroblasts, transfected with A-type lamins containing
mutations, identical to those found in laminopathies, these irregularities were found.
Studies using the FLIP technique revealed a decreased stability of these mutant
lamin polymers, both in the lamina and the nucleoplasm. Apparently, total cellular
weakness results from the loss of normal function of lamin proteins, causing
cytoskeletal abnormalities.
- 22 -
The Invasion Nature of Mammary Tumors: Insights into

Cell Signaling and Motility
Condeelis John
Albert Einstein College of Medicine, Department of Anatomy and Structural Biology, Bronx NY
10461, USA
Cell motility has been implicated in the spread of cancer cells and is an essential step
in metastasis. Chemotaxis to blood vessels is involved in the escape of cancer cells
from primary mammary tumors. The recent convergence of technologies for
expression profiling and intravital imaging has revealed the identities of the genes
involved in the survival, motility and chemotaxis of cancer cells inside living tumors.
The pattern of expression of these genes is called the invasion signature. The
invasion signature indicates that invasive cancer cells are a population that is neither
proliferating nor apoptotic but highly chemotactic. Of particular relevance to the
chemotactic behavior of invasive cancer cells is the finding that the genes coding for
pathways leading to the minimum motility machine, i.e., the cofilin, capping protein
and Arp2/3 pathways, that regulate actin polymerization at the leading edge, and the
directionality of cell protrusion, are coordinately up-regulated. Key genes in the
invasion signature have been studied for their ability to alter metastatic outcome and
these results confirm the importance of the invasion signature in predicting
metastasis. For example, LIMK1 is a member of a novel class of serine-threonine
protein kinases. Cofilin, the substrate of LIMK1, is essential for the regulation of actin
polymerization and depolymerization during cell migration. Unfortunately, previous
studies have come to opposite conclusions as to the role of LIMK1 in tumor cell
motility and metastasis claiming either an increase or decrease in cell motility and
metastasis as a result of LIMK1 over expression. The invasion signature resolves this
paradox by showing that the effect of LIMK1 expression on migration, intravasation
and metastasis of cancer cells can be most simply explained by its regulation of the
output of the cofilin pathway. LIMK1-mediated decreases or increases in the activity
of the cofilin pathway are shown to cause proportional decreases or increased in the
migration, invasion and metastasis of tumor cells, respectively. The important
conclusion from this study is that looking at the status of expression of a single gene
can be misleading since it is the output activity of the pathway as a whole, of which
that gene product is a part, that determines the metastatic phenotype of a tumor.
- 23 -
POSTER ABSTRACTS
are in alphabetical order with respect to the presenting author (underlined)
- 24 -
Actin Filament Reorganisation during Early Adhesion Formation in

Migrating Cells (1)
Alexandrova Antonina1,2, Resch G.P.1 and Small J.V.1

1
Institute of Molecular Biotechnology, Vienna, Austria
2
N.N. Blokhin Cancer Research Center, RAMS, Moscow, Russia
We correlated the dynamics of formation of early substrate adhesions at the leading

edge of fast crawling cells with the organisation of the actin cytoskeleton, using a
combination of live cell imaging and electron microscopy (EM). Adhesion complexes
of living cells were marked with EGFP or RFP-tagged constructs of VASP, paxillin and
zyxin, either alone or in combination. Two types of cells were studied: fibroblasts, in
which protrusion of the leading edge consists of a sequence of local protrusions and
retractions, and B16 mouse melanoma cells (with and without stimulation with
aluminium fluoride), where the lamella can protrude rapidly without retractions at
the cell front. VASP was a useful marker of very early adhesions, appearing 10-30
sec before zyxin in continuously protruding lamellipodia, whereas incorporation of
zyxin or paxillin was connected with local retraction. Negative staining EM showed
the primary VASP dots in lamellipodia corresponded to a focal convergence of actin
filaments in the lamellipodium network, sometimes associated with one or more
loose parallel arrays of a few filaments directed towards the rear of the
lamellipodium. The earliest zyxin-containing focal complexes (0,5–3 min lifetime)
were seen as small, tight bundles of actin filaments which diverged into filament
groups, of up to a total of around 20 filaments, that linked into actin meshwork
behind the lamellipodium. Focal complexes with lifetimes greater than 5min
containing zyxin and paxillin were typically composed of tightly packed actin
filaments continuous with small stress fibre-like bundles. We propose the following
stages for early adhesion formation: 1, a focal increase in density of the actin
meshwork initiated by anchorage; 2, alignment of anchored filaments by ruffling or
retraction of the lamellipodium, coupled with their elongation; 3, recruitment of
filaments of opposite polarity to the adhesion site, followed by contractility via
myosin recruitment. This is followed by maturation into a focal adhesion associated
with a contractile bundle.
- 25 -
The Microtubules Optical Density Analysis Allows to Estimate the

Ratio of Centrosome-Attached and Free Microtubules (2)
Smurova K.M. and Alieva Irina

AN Belozersky Institute for Physical and Chemical Biology, Moscow State University, Moscow,
Russia
In internal cytoplasm the density of microtubules is highest in the centrosome area

and falls to periphery of a cell. The quantity of fluorescent microtubules cannot be
counted up, but it’s possible to estimate microtubules quantity using measuring of
their optical density. To determine appropriateness that circumscribe the reduction
of microtubules optical density from the centrosome to the cell margin, we modeled
cell contours with the certain ratio and interposition of centrosome-attached and free
microtubules in vector schedules CorelDraw program. The decrease of optical density
was analyzed in MetaMorph program as it was described earlier (Smurova et al.,
2002). It was shown that in the system joins only microtubules growing from the
centrosome up to the margin the fluorescent microtubules optical density decreased
exponentially (y=ae-bx). In the case of not all radial centrosome-attached
microtubules reached the margin the curve became smoother, and the carrying of
free microtubules into the system leads to the sharp falling of optical density in the
centrosome area and to its gradual decrease on the cell periphery. After increasing
of free microtubules quantity character of the curve which describe the decreasing of
optical density had changed – microtubule system which included free and
centrosome-attached microtubules in proportions of 5:1 was described by the
equation of linear regression (f=k*x b). Thus, the mathematical dependence
described the microtubules distribution from the centrosome to the cell periphery,
depends on a ratio of microtubules and their relative positioning in the cell volume.
Data obtained on modeling system have coincided with the results of experiments.
The graphs which described the microtubules optical density increasing during
microtubule repolymerization after nocodazole treatment in Vero cells, corresponded
to the modeling cells graphs. Thus, the method we used allow to analyze the
microtubules system in cases when the direct observation of individual microtubules
is difficult.
- 26 -
Description and Studying the Actin Cytoskeleton-Associated Protein

Zyxin in Tumour Suppressor Effect (3)
Amsellem Valérie1, Kryszke M.H.1, Leh H2. and Auclair C.1

1
Laboratoire de Biotechnologie et Pharmacologie Génétique Appliquée, CNRS UMR 8113,
ENS de Cachan, Cachan, France
2
BioAlliance Pharma, Paris, France
Actin cytoskeleton disruption and changes of the cell morphology are common
features accompanying cell transformation. Supporting the involvement of the
microfilament network in tumour cell behaviour, zyxin, a key actin-polymerization
regulatory protein, may play a role in oncogenesis. Zyxin is a multifunctional protein
harboring two functional main domains: an aminoterminal prolin-rich domain and a
three LIM motifs carboxyterminal separated by a central nuclear export signal. We
have previously shown that the overexpression of zyxin in cell lines expressing the
EWS-FLI-1 oncoprotein (derived from a t(11;22) chromosomal translocation which is
a main characteristic of Ewing tumours) acts as a tumour suppressor [Amsellem et
al. (2005) Exp. Cell Res. 304: 443-456]. In fact, the tumour suppressor effect was
characterised by inhibition of anchorage-Independent growth associated with an
impairment of tumour formation in athymic mice for both EWS-FLI1-transformed
fibroblasts and the Ewing sarcoma SK-N-MC cell line. Moreover, we observed the
reorganization of the actin cytoskeleton and the reconstitution of zyxin-rich focal
adhesions and intercellular junctions. To further characterise the tumour suppressor
mechanism of zyxin protein, we have constructed several zyxin mutants and
analysed their effects on EWS-FLI1-transformed fibroblast phenotype. We show that
the tumour suppressor role of the zyxin can be divided into two distinct phenotypic
effects: the inhibition of anchorage-independent growth depends on the three LIM
motifs domain whereas the impairment of tumour formation in nude mice is related
to the aminoterminal prolin-rich domain.
- 27 -
ERM-Dependent Regulation of the Actin Cytoskeleton by

-Dystroglycan (4)
Batchelor Clare and Winder S.J.

Biomedical Sciences, University of Sheffield, Sheffield, UK
Dystroglycan is part of an adhesion receptor complex linking the extracellular matrix

to the actin cytoskeleton. In muscle this complex is crucial for maintaining muscle
integrity, as such mutations in proteins associated with this complex lead to various
muscular dystrophies. In addition to forming an adhesion receptor complex in non-
muscle cells, -dystroglycan has also been localized to microvilli and filopodial
structures where it interacts with the ERM (ezrin, radixin, moesin) family of
cytoskeletal cross-linking proteins. Overexpression of -dystroglycan in non-muscle
cells leads to a dramatic re-arrangment of the actin cytoskeleton with the induction
of numerous peripheral filopodia and microvilli which we can show is dependent
upon its interaction with ERM proteins. We have currently been addressing the
mechanism by which ß-dystroglycan induces filopodial formation in non-muscle cells
by co-expressing various GFP--dystroglycan constructs with members of the
RhoGTPase family and analysing their effects on filopodial formation by
immunofluorscence detection. In addition, we have been employing in vivo and i n
vitro interaction studies and subcellular fractionation to identify which of these
factors associate with the -dystroglycan/ERM complex. Our current data suggests
that the ERM proteins may recruit guanine nucleotide exchange factors to -
dystroglycan in order to locally activate Cdc42 and induce filopodial formation. These
studies reveal novel functions and additional signalling roles for dystroglycan in non-
muscle cells.
- 28 -
Visualization of Unconventional Myosin VIII Isoforms in Plant Cells

via GFP Fusion Constructs of the Tail Domains (5)
Beck Martina1, Wasser K.1, Kretz C.1, Helling D.2, Menzel D.1
1
Institute for Cellular and Molecular Botany, University Bonn, Germany
2
Heidelberg Institute for Plant Sciences, University Heidelberg, Germany
Myosins are a large superfamily of motor proteins which move along actin filaments.
However, their exact roles in actin-based processes are not yet fully understood.
Myosins have three domains in common; a motor domain that interacts with actin
filaments and binds ATP, a neck domain that binds calmodulin light chains and a tail
domain that is thought to interact with the cargo. The structure of the tail domain
varies between classes and even between members in the same class. In the A.
thaliana genome 17 myosin genes have been identified - class VIII myosins with four
isoforms, and class XI myosins with 13 isoforms. With the intention to identify the
cargo organelles, we tried to visualize the tail domains of the four class VIII isoforms
in plant model systems via GFP-fusion constructs. To this end, we fused GFP N-
terminally to the tail domains of ATM1, ATM2, VIIIA, VIIIB and expressed these
constructs transiently in Allium cepa and Vicia faba and by stable transformation in
tobacco BY-2 cells and A. thaliana plants. Our data suggest, that ATM1 and VIIIA are
localized to the plasma membrane with particularly strong labelling at the
plasmodesmata and the maturing cell plate in BY2 and A. thaliana, corroborating our
earlier immunofluorescence study. However, ATM2 and VIIIB are most strongly
localized in the nucleoli and only weakly at the plasma membrane, suggesting their
involvement in nuclear functions, possibly in the transport of nucleolar components.
These results indicate that the two subgroups of class VIII myosins seems to have
distinctive roles plant cells.
- 29 -
Microtubule Plus-End Capture and the Formation of

Non-Centrosomal Microtubule Arrays (6)
Bellett Emma1, Carter J.1, Evans-Gowing R.1, Nathke I.2 and Mogensen M.1
1
School of Biological Sciences, UEA, Norwich, England
2
School of Life Sciences, University of Dundee, Scotland
The classic radial pattern of microtubules focused on a centrally located centrosome

is abandoned in many differentiated cells such as polarised epithelial cells. In these
cells the majority of the microtubules are anchored at non-centrosomal apical sites
and an apico-basal array is evident. The mechanisms involved in the generation of
an apico-basal microtubule array are not fully understood.
We are using cochlear, MDCK and RPE cells as model systems to analyse the
mechanisms responsible for the assembly of non-centrosomal arrays in polarised
epithelial cells. The microtubule minus-ends become anchored at apical sites and the
plus-ends elongate towards the cell base during assembly of the apico-basal arrays.
Gamma-tubulin is concentrated at the centrosome while the microtubule minus-end
anchoring protein ninein is present at the centrosome and at the apical sites. APC is
located along the length of elongating microtubules and gradually accumulate at the
cell base.
Our recent data on nocodazole regrowth experiments suggest that microtubule plus-
end mediated capture at cortical sites may be a vital intermediary step in the
generation of an apico-basal array. An initial aster forms at the centrosome that
elongates towards the cell periphery resulting in an extended radial microtubule
array. Subsequently, in polarised cells an apico-basal array develops and the
extended apical microtubule array diminishes. Immuno-fluorescence and electron
microscopy are being used to study microtubule plus-end cortical interactions. Our
findings suggest that microtubule +TIPs such as CLIP-170, EB-1 and APC associate
at cell-cell junctions in cells displaying an extended array and that actin plays a key
role in the cortical positioning of microtubules.
- 30 -
Nuclear Function of Cytoskeletal Proteins: Coordinating Adhesion

and Transcription by Novel Target Genes of Cadherin-Catenin
Signaling in Colon Cancer (7)
Conacci-Sorrell M.1, Gavert N.1, Brabletz T.2 and Ben-Ze’ev Avri1

1
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel
2
Department of Pathology, University of Erlangen-Nurenberg, 91054 Erlangen, Germany
Aberrant -catenin signaling is prevalent in most colorectal cancer patients. -

catenin, an important cell-cell adhesion protein binding cadherin receptors to the
cytoskeleton, is also a key co-transcriptional activator of target genes (together with
LEF/TCF factors) in the nucleus. Hyperactive -catenin induces genes regulating the
cell cycle and the invasion and metastasis of cancer cells. Using human colon cancer
cells, we found that sparse cultures mimic cells at the invasive front of colorectal
tumors displaying low E-cadherin levels, but highly active nuclear -catenin that
transactivates Slug , a negative transcriptional regulator of E-cadherin. These cells
also express abundant ErbB1/2 and active ERK. In contrast, dense cultures have
distinct membranal E-cadherin and -catenin, only limited -catenin signaling, no
Slug, and therefore high E-cadherin levels, but low levels of ErbB1/2 and MAPK
signaling. This cell density-regulated phenotypic conversion is reminiscent of the
plasticity in -catenin and E-cadherin in the invasive versus the differentiated
colorectal carcinoma tissue. We identified two members of the L1 cell adhesion
family (normally expressed in nerve cells), as novel target genes of -catenin, and
detected them in sparse cell cultures and exclusively in the invasive front of colon
carcinoma tissue. Forced expression of L1 and Nr-CAM in fibroblasts induced motility,
transformation, conferred tumorigenicity in mice, and liver metastasis in colon cancer
cells. Suppression of L1 levels in colon cancer cells reduced their motility and ECM
invasion. We suggest that these “neuronal” adhesion molecules, when aberrantly
induced by hyperactive -catenin, are exploited opportunistically by colon cancer
cells to promote metastasis. Since the extracellular domains of L1 and Nr-CAM are
often shedded by metalloproteinases, they could serve as diagnostic markers and as
anticancer therapy targets. We identified L1 and the metalloproteinase ADAM10 (that
cleaves the ectodomain of L1) at the invasive front of human colorectal cancer
tissue.
Relevant references
Gavert et al., (2005). L1, a novel target gene of -catenin, transforms cells and is
expressed at the invasive front of human colon cancer. J. Cell Biol. 168: 633.
Conacci-Sorrell et al., (2003). Autoregulation of E-cadherin expression by cadherin-
cadherin interactions: the roles of -catenin signaling, Slug, and MAPK. J. Cell
Biol. 163: 847.
Conacci-Sorrell et al., (2002). Nr-CAM is a target gene of the -catenin/LEF-1
pathway in melanoma and colon cancer and its expression enhances motility and
confers tumorigenesis. Genes Dev. 16:2058.
Conacci-Sorrell et al., (2002). The cadherin-catenin adhesion system in adhesion,
signaling and cancer. J. Clin. Invest. 109: 987.
- 31 -
In Silico Model for Actin Filament Assembly Based on

Molecule Population Dynamics (8)
Berro Julien and Martiel J.L.

Laboratoire TIMC, UMR CNRS 5525, Faculty of Medicine, La Tronche, France
During the last decade, mathematical models for actin polymerization have been
analysed and tested to account for the dynamics of nucleation and/or elongation of
filaments and the control of polymerisation by proteins. However, since most of
these models use continuous deterministic differential equations, they cannot
account directly for the specific role of actin, i.e. the production of mechanical forces
to move cellular endosomes or the membrane at the micrometer scale. We propose
a stochastic model in which we consider a population of individual proteins (actin
monomers, Arp2/3 complexes, profilin…) and filaments. Our approach resembles that
of the molecular dynamics, but in our model the reactions obey the action-mass law
and we use macroscopic rate constants to determine the probability of a reaction to
occur. We develop a new efficient simulation algorithm which accounts for spatio-
temporal processes (diffusion, reaction, collision with intracellular endosomes, the
membrane, …). First, we partition the physical space into sub-domains that serve to
define the molecules neighbourhood and their subsequent encounters. Second, we
adapt the Gillespie algorithm to take advantage of the sub domain partition, so that
simulations with large populations of molecules and filaments are possible in realistic
time. We can simulate actin polymerisation in a space equivalent to a 2D-virtual test
tube. Since we can track each molecule, we take into account all position-dependent
reactions explicitly and we are able to compute the mechanical force exerted by
growing filaments against a macroscopic target. Currently, we constrain our model
with biophysical and biochemical data to simulate the generation of forces on
structures, like membranes, cell endosomes or latex beads.
- 32 -
The Role of Vav Family Proteins in Macrophage Migration (9)
Bhavsar Parag and Ridley, A.J.

Ludwig Institute for Cancer Research, UCL Branch, London, UK
The ability to migrate is essential to leukocyte function. In response to inflammatory

stimuli these cells adhere to, and move through the vascular endothelial wall.
Thereafter, they migrate towards the focus of inflammation. There they carry out
functions such as the phagocytosis of pathogens, antigen presentation and the
potentiation of the inflammatory response. Migration is the product of coordination
between changes in cell morphology and regulation of adhesion to the substratum. It
has been demonstrated that the Rho family of small GTPases are critical regulators
of the cell’s cytoskeletal organization. In particular Rac1 has been shown to be
required for the formation of lamellipodia and ruffles. The Vav family of proteins
have been shown to act as tyrosine phosphorylation-dependent guanine nucleotide
exchnage factors (GEFs) for Rho/Rac GTPases. Three isoforms exist in mammalian
cells: Vav1, Vav2 and Vav3, of which Vav1 has haematopoeitic-specific expression.
Though their roles in lymphocyte development and function has been extensively
studied, less is known of their function in myeloid lineage cells such as neutrophils
and macrophages. In this study, bone marrow-derived macrophages (BMM) from
vav1 -/-, vav2 -/- and vav3 -/- mice are being used to investigate the role of these
proteins in macrophage migration. Using timelapse microscopy, immunofluorescence
and biochemical techniques, it has been found that: Vav1-/-, Vav2-/- and Vav3-/-
BMM are all capable of a normal acute response to the chemoattractant CSF-1
(colony-stimulating factor-1), producing F-actin-rich membrane ruffles and
lamellipodia, and activating Erk1/2 with a normal time-course. However, Vav1-
deficient BMMs have reduced a migration speed in CSF-1-containing medium
compared to wild-type BMMs. Vav1 is tyrosine phosphorylated downstream of the
CSF-1 receptor, potentially rendering it active as a GEF for Rho/Rac GTPases. The
contribution of Vav1 to CSF-1-stimulated Rho GTPase activity is therefore being
investigated.
- 33 -
Identification of Proteins Interacting with the N-Terminal Domain of

Periplakin, a Cytoskeletal Linker Protein (10)
Boczonadi Veronica, Long H. and Maatta A.

School of Biological Sciences, University of Durham, Durham, UK
Periplakin is a cytoskeletal linker protein that has been implicated in the formation of
epidermal barrier. However, ablation of periplakin does not impair barrier function of
the skin and the function of periplakin in other epithelia has not been studied in
detail. We are studying the function of periplakin in both stratified and simple
epithelia and show that periplakin co-localises with both desmosomal and adherens
junction proteins. Periplakin is targeted to cellular junctions by its N-terminal domain
but so far only one protein-protein interaction for periplakin N-terminus has been
discovered. To identify proteins interacting with periplakin, we have generated stably
transfected cell lines carrying either periplakin N-terminus or the intermediate
filament-binding C-terminus. Co-immunoprecipitation experiments with the N-
terminal domain suggest that there are several proteins interacting with periplakin N-
terminus and we are currently characterising these interactions further.
- 34 -
p120 Catenin Beyond Cell-Cell Junctions: Association with Dynamic

Actin Structures and Involvement in Cell Motility (11)
Boguslavsky Shlomit, Grosheva I., Landau E., Cohen M., Arnold K., Shtutman M.,
Feinstein E., Geiger B. and Bershadsky A.
The Weizmann Institute of Science, Rehovot, Israel
Armadillo family protein p120 catenin (p120) binds juxtamembrane domains of

classical cadherins and localizes to cell-cell junctions. In this study, we further
dissected the localization and function of p120. In addition to cell-cell junctions, p120
was found in ruffles and lamellipodia at the cell edge, and in actin-rich ‘halos’
associated with moving endocytotic vesicles. Besides actin, all these structures are
enriched in cortactin, and we demonstrated that p120 binds the cortactin N-terminal
domain. Down regulation of p120 by siRNA prevented formation of cortactin-
containing lamellipodia in Balb-3T3 fibroblasts and inhibited migration and spreading
of these cells. The velocity of intracellular vesicle movement increased upon p120
overexpression and decreased upon its siRNA-induced down regulation. In epithelial
MCF-7 cells, siRNA-induced down regulation of p120ctn resulted in destabilization of
cell-cell junctions in agreement with previous studies. This destabilization was
accompanied by dramatic disappearance of cortactin and its partner Arp3 from the
junctions. In addition, p120 appeared to be required for protrusional activity and cell-
matrix adhesion in these cells. Control MCF-7 cells respond to stimulation with
neuregulin (a ligand for ErbB-3 and ErbB-4 receptors) by intense production of
cortactin- and Arp3-rich lamellipodia and subsequent formation of focal adhesions.
The lamellipodial stability, focal adhesion formation, and cortactin-Arp3 recruitment
to the cell periphery were significantly reduced in MCF-7 cells with down regulated
p120ctn. We propose that a common mechanism underlying all p120ctn functions is
based on its involvement in the local regulation of actin polymerization via
modulation of the cortactin-Arp2/3 complex localization.
- 35 -
Actin Cytoskeletal Responses Induced by the Helicobacter pylori

CagA Protein and Tir from Enteropathogenic Escherichia coli (12)
Brandt Sabine, Kwok T., König W. and Backert S.

Otto von Guericke University Magdeburg, Department of Medical Microbiology, Magdeburg,
Germany
The host cell actin cytoskeleton is a common and recurring target of pathogens
harnessed to promote their attachment, entry or movement within and between host
cells. Thus, pathogens provide a useful tool for studying many aspects of cytoskeletal
function. Here, we investigated the effects of two tyrosine-phosphorylated bacterial
effector molecules, CagA of Helicobacter pylori and Tir of enteropathogenic
Escherichia coli (EPEC), in actincytoskeletal rearrangements. These two pathogens
target the host actin cytoskeleton from outside the cell. The CagA protein is injected
into gastric epithelial cells by type IV secretion. Translocated CagA is then tyrosine
phosphorylated by Src. Phosphorylated CagA causes the dephosphorylation of
cortactin and ezrin, two central regulators of the host actin cytoskeleton, followed by
global changes of the actin cytoskeleton that lead to host cell elongation. By
comparison, Tir from EPEC is translocated by type III secretion followed by tyrosine-
phosphorylation (by Abl and Src kinases) and serine-phosphorylation (by protein
kinase A). Phosphorylated Tir then binds the adapter protein Nck to recruit the
Arp2/3 complex, which is critical for the induction of local actin polymerization and
pedestal formation. In this study, we asked if we could identify possible synergistic
or antagonistic effects on CagA-induced signaling by the presence of Tir. In order to
obtain further insight into this signaling, we investigated the phenotypical outcome
induced by both virulence factors (wild-type and mutant constructs) using
fluorescence and scanning electron microscopy. Interestingly, we found that Tir can
efficiently block the CagA-induced host cell elongation. Our data indicate that
phosphorylation of Tir at serine residues 434 or 463, but not at tyrosine 474, is
crucial for the ability of Tir to block CagA-induced phenotypical outcome. Our data
indicate that phosphorylated CagA and Tir trigger antagonistic signaling pathways.
Implications of these observations for novel functional aspects of both important
virulence factors will be discussed.
- 36 -
Integrin-Mediated Expression of Cyclooxygenase 2 in a

Non-Transformed Intestinal Cell Line (13)
Broom Oliver and Sjölander A.

Experimentell Patologi, UMAS, Malmoe, Sweden
The link between cyclooxygenase 2 (COX-2) and cancer (especially of the colon) has
been established firmly for many years. Expression of this inducible enzyme can be
achieved through various means, from cellular stress to signalling cascades
stimulated by inflammatory mediators. Cellular migration is a key part in the
spreading of cancer, not least for the local environment but also for metastasis to
other sites in the body. Migration of any cell is mostly regulated by the actions of
integrins, through both physical and signalling capacities. The signals generated by
integrin activation have become increasingly important and are now thought to play
important roles, for example in cell survival. In this study we examine the importance
of integrin signalling on the regulation of COX-2 expression and the intermediate
proteins involved. We have found that upon integrin activation an increase in COX-2
protein is observed and the pathway leading to this is dependent on several key
proteins although being independent of erk1/2.
- 37 -
Effect of Shear Stress and Thrombin on

Endothelial Cell Responses (14)
Christodoulou Maria and Ridley A.J.

Ludwig Institute for Cancer Research, London, UK
Fluid shear stress induces remodelling of the actin cytoskeleton of endothelial cells
leading to their polarization and in the direction of flow, allowing the vessels to adapt
to different levels of fluid flow. The involvement of integrins and small Rho GTPases
in mechanotransduction has been proven, but the actual mechanism of signal
transduction is not yet fully understood. Immunofluorescence and western blotting
are being used to observe shear stress-induced changes in the localization of
proteins implicated in integrin signalling, including FAK, paxillin and talin. Endothelial
cells are exposed to more than one stimulus at the same time. It is therefore of
interest to observe the effect of a combination of stimuli on endothelial cells. For
example; the cytokine thrombin can be activated in atherosclerotic plaques. Just like
shear stress, thrombin induces remodelling of the actin cytoskeleton of endothelial
cells via Rho GTPases. The effect of thrombin on FAK, talin and paxillin is being
compared with shear stress. Thrombin induced a stronger and more rapid tyrosine
phosphorylation of FAK and talin than shear stress. Both thrombin and shear stress
induce the activation of calcium channels. The activity and role of calpain, a protease
whose activation is calcium dependent and whose substrates include FAK and talin, is
being investigated in each response. Preliminary results indicate that calpain
inhibitors alter the morphological response of endothelial cells to thrombin.
- 38 -
TRPM7, a Novel Regulator of Actomyosin Contractility and

Cell Adhesion (15)
Clark Kristopher1, Langeslag M.2, van Leeuwen B.3, Ran L.3, Figdor C.G.1,
Moolenaar W.H.3, Jalink K.2 and van Leeuwen F.N.1
1
Department of Tumour Immunology, Nijmegen Centre for Molecular Life Sciences (NCMLS),
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
2
Division of Cell Biology,
3
Division of Cellular Biochemistry and Center for Biomedical Genetics, the Netherlands
Cancer Institute, Amsterdam, The Netherlands
Actomyosin contractility regulates various cell biological processes including

cytokinesis, adhesion and migration. While in lower eukaryotes -kinases control
actomyosin relaxation, a similar role for mammalian -kinases has yet to be
established. Here, we examined whether TRPM7, a cation channel fused to an -
kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by
bradykinin leads to a Ca2+- and kinase-dependent interaction with the actomyosin
cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain.
Accordingly, ectopic expression of TRPM7 increases intracellular Ca2+ levels
accompanied by cell spreading, adhesion and the formation of focal adhesions.
Activation of TRPM7 induces the transformation of these focal adhesions into
podosomes by a kinase-dependent mechanism, an effect that can be mimicked by
pharmacological inhibition of myosin II. Collectively, our results demonstrate that
regulation of cell adhesion by TRPM7 is the combined effect of kinase-dependent and
-independent pathways on actomyosin contractility.
- 39 -
In Vivo Cytoskeleton Dynamics Quantification by

Pulsed UV Laser Nanosurgery (16)
Colombelli Julien, Reynaud E.G., Rietdorf J., Pepperkok R. and Stelzer E.H.K.
We report on the manipulation of intracellular filaments using a nanosurgery system

based on a short pulsed UV laser optimized for the localized severing of biological
polymers. By inducing artificial catastrophe of selected microtubules, we perform
shrinkage rate measurements in interphase Ptk-2 cells throughout the entire cell. We
quantify the impact of two labelling methods and three fluorescent markers, showing
a 25% faster depolymerization with Alexa 488 tubulin compared to Rhodamine and
YFP tubulins and a 20% higher variability induced by microinjection compared to
stable transfection. Using EB3-GFP tip marker, we establish a new protocol to
measure shrinkage rate, growth rate and rescue frequency simultaneously with high
temporal and spatial specificity. As our analysis shows, laser induced microtubule
dynamics are physiologically relevant. The high statistical efficiency that the method
offers in terms of numbers of measured events and, therefore, reduced standard
deviations represents an important quantitative improvement in the measurement of
dynamic instability parameters in vivo. We extend the application of the method by
demonstrating the possibility to monitor MTs directionality after laser nanosurgery
towards cellular components like the ER exit sites and also demonstrate actin stress
fibers severing. We propose the method as a new and regional way to quantify
cytoskeleton dynamics.
- 40 -
Localization and Characterization of Alpha-Actinin

in Neurospora crassa (17)
Cotado-Sampayo Marta1, Ojha M.1, Ortega Perez R.1, Chappuis M.-L.1, Seum C.2
and Barja F.1
1
Department of Botany and Plant Biology, University of Geneva, Jussy-Geneva, Switzerland
2
Department of Zoology, University of Geneva, Jussy-Geneva, Switzerland
The presence of a spectrin superfamily protein was examined in the crude extracts
from exponentially growing Neurospora crassa with polyclonal and monoclonal
antibodies against -spectrin and -actinin, respectively. Analysis of SDS-PAGE gels
showed the presence of a single band of about 100 kDa. The immunofluorescence
and immunogold labeling of the protein with these two antibodies in the germ tube
and hyphae showed its predominance in the tip region and along the plasma
membrane. The absence of classical sequences of spectrin gene(s) in N. crassa
raises the question about the identity of the immunological reactions. The conserved
sequences of spectrin from different organisms was used to search for its homologue
in the N. crassa database and a hypothetical protein of 1027 amino acids
(NCU06429.1) with a similarity of about 35% was found. This protein contained
characteristic domains of spectrin superfamily, i.e. two calponin homologue domains,
two spectrin repeats and an EF-hand motif, but lacks other conserved motifs found
in spectrins, i.e. plekstrin homology-, SH3-domain and extended repeats. The
classical domain structure of -actinin contains at least four spectrin repeats,
however, some organisms seem to have only one or two repeats. Therefore,
NCU06429.1 encoded protein is closer to -actinin, a member of the spectrin
superfamily, than the classical spectrin of the higher eukaryotes. Contrary to the
previous results published, the presence of a homologue of higher eukaryote spectrin
in N. crassa could not be established and does not find support in the light of our
results.
- 41 -
Nuclear and Junctional ERM (Ezrin, Radixin, Moesin) Proteins

in Human Skin (18)
Campbell A.D.1, Batchelor C.L.1,2 and Crouch Sam1

1
Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School,
Dundee, Scotland DD1 9SY, UK
2
Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S
Yorks, England S10 2TN, UK
The ERM (ezrin, radixin, moesin) proteins act as molecular linkers between the actin
cytoskeleton and the plasma membrane. They localise to actin-rich areas of the cell
which rapidly undergo changes in shape. In addition to their cytoskeletal roles, the
ERM proteins also act as signalling molecules in important biological processes, such
as immunological synapse formation, cell growth and apoptosis. More recently, our
lab proposed a novel function for the ERM proteins in the nucleus [Batchelor et al.
(2004) Exp. Cell Res. 296: 208-222]. All three ERM proteins can localise to the
nucleus and contain a sequence that regulates their nuclear localisation. Using
human skin as an in vivo model system, we now show that the ERM proteins localise
to discrete, punctate junctions in the skin. The ezrin-containing junctions qualitatively
and quantitatively differ in the different layers of the skin, suggesting different
functions throughout the epidermis. Not surprisingly, the junctional ERM proteins are
phosphorylated in the actin-binding domain. Whilst the exact composition of these
novel junctions is not known, they appear to be related to adherens junctions, since
they contain E-cadherin, rather than other cell:cell junctions such as desmosomes.
Significantly, we also show that ezrin localises to the nucleus in a spatially regulated
manner in the epidermis, with nuclear ezrin found primarily in the suprabasal layers.
Nuclear ezrin has been confirmed using multiple independently derived ezrin
antibodies, suggesting that the nuclear staining is not an artefact of staining. We
also find nuclear actin, but not moesin or CD44 in these cells, showing a selectivity in
the localisation of ERM proteins to the nucleus. Importantly, nuclear ezrin is also
seen in endothelial cells and smooth muscle cells. These tissues are either growth
arrested or terminally differentiated, leading us to suggest that nuclear ezrin
correlates with growth arrest. The nuclear function of ERM proteins will be discussed.
- 42 -
Cdc42 is not Essential for Filopodia Formation, Directed Migration,

Cell Polarization and Mitosis in Fibroblastoid Cells (19)
Czuchra Aleksandra1, Wu X.1, Meyer H.1, van Hengel J.1, Rottner K.2 and
Brakebusch C.1
1
Max Planck Institute of Biochemistry, Junior Group "Regulation of Cytoskeletal
Organization", Am Klopferspitz 18, 82152 Martinsried, Germany;
2
German Research Centre for Biotechnology (GBF), Cytoskeleton Dynamics Group,
Mascheroder Weg 1, 38124 Braunschweig, Germany
Cdc42 is a small GTPase involved in the regulation of the cytoskeleton and cell
polarity. To test whether Cdc42 has an essential role in the formation of filopodia or
directed cell migration, we generated Cdc42-deficient fibroblastoid cells by
conditional gene inactivation. We could show that loss of Cdc42 did not affect
filopodia or lamellipodia formation and had no significant influence on the speed of
directed migration nor on mitosis. In Cdc42-deficient cells, protrusion formation was
stably polarized during migration, while re-orientation of the Golgi apparatus into the
direction of migration was decreased by approximately 50%. However, expression of
dominant negative Cdc42 in Cdc42-null cells resulted in strongly reduced directed
migration, complete loss of Golgi polarization and of directionality of protrusion
formation towards the wound, as well as membrane blebbing. Thus, our data show
that besides Cdc42 additional GTPases of the Rho-family, which share GEFs with
Cdc42, are involved in the establishment and maintenance of cell polarity during
directed migration.
- 43 -
Actin Nucleators in Plants (20)
Deeks Michael, Kaloriti D., Machesky L.M., Malhó R., Davies B. and Hussey P.J.
ICBL, University of Durham, UK
The dynamic nature of the eukaryotic actin cytoskeleton is essential for a range of
processes within plant cells. Key regulators of the actin cytoskeleton include actin
nucleating factors, two forms of which are conserved in plants: The Arp2/3 complex
and formins. The F-actin nucleating/branching activity of the Arp2/3 complex plays a
key role in plant cell morphogenesis. A protein complex of SCAR, PIR121, NAP1, ABI,
and HSPC300 is required for Arp2/3 regulation by cell signalling pathways, but the
exact nature of this interaction is controversial and represents a continually evolving
model. Recent genetic observations in Arabidopsis are compatible with a scenario in
which SCAR stimulation of the plant Arp2/3 complex is supported by NAP1 and other
members of the SCAR complex. In animals SCAR is required for both lamellipodia
and filopodia formation. Structures homologous to these are absent from plant cells,
possibly implicating the SCAR complex in novel cell processes that affect plant cell
morphogenesis. At least 21 formin isoforms exist within the plant genome and can
be grouped into a hierarchy of clades based upon sequence similarity. Formin 4, a
members of group Ie, localises to cell-to-cell contacts, interacts with plant isoforms
of profilin, and shows actin nucleating activity. Unlike the Arp2/3 complex, regulation
pathways for plant formins have not been identified. Together the actin nucleators
are adding to a growing network of plant actin regulation.
- 44 -
Phosphotyrosine Proteomics of the Epithelial

Mesenchymal Transition (21)
de Graauw Marjo1, Tijdens I.1, Smeets M.2, Deelder A.M.2, van de Water B.1
1
Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University,
Leiden, The Netherlands
2
Department of Parasitology, Center of Infectious Diseases, Leiden University Medical
Center, Leiden, The Netherlands
The epithelial to mesenchymal transition (EMT) is an important process in metastasis

formation and is characterized by loss of epithelial cell polarity, which occurs as a
result of F-actin cytoskeleton reorganization and the disappearance of cell-cell
interactions. EMT is under the tight control of (non)-receptor tyrosine kinases such
as Src kinase. This results in changes in the tyrosine phosphorylation (pTyr) status of
cytoskeletal and cell adhesion proteins. Here, we used temperature sensitive ts-v-Src
MDCK cells, which exhibit an epithelial phenotype at the non-permissive temperature
for Src kinase activity (40°C), but rapidly loose cell-cell interaction and acquire a
mesenchymal-like morphology at the permissive temperature (35°C). This shift
correlates with an increased pTyr content of several proteins. To identify these
proteins we used phosphotyrosine-proteomics, which includes 2D electrophoresis
followed by 2D immuno-blotting techniques and MALDI-TOF-MS. In total, we
identified 18 different proteins with a changed pTyr during EMT, including the
adhesion and cytoskeletal proteins ezrin, radixin, vinculin, annexin A1 and A2.
Annexin A2 is phosphorylated at Y23 upon activation of Src kinase. Our studies using
EGFP-tagged annexin A2 wt or Y23E, which mimics the Y23 phosphorylation, indicate
an important role for this Tyr residue in cell attachment, adherens junction
formation, migration and tubulogenesis. Expression of Y23E resulted in enhanced cell
adhesion and migration. In addition, the tubulogenesis process was less well
organized in cells Y23E cells. Since Y23E-expressing cells contained less F-actin
stress fibers compared to wt-expressing cells we propose a direct role for Src-
mediated phosphorylation of Y23 in the regulation of actin dynamics and the control
of cell-cell interactions, most likely through regulation of Rho-family members.
Altogether, phosphotyrosine-proteomics is a powerful tool to identify differentially
phosphorylated proteins. Identification of such proteins in relation to EMT will
provide better insight into the molecular mechanisms involved in tumor metastasis
formation.
- 45 -
Integrin-Dependent Actomyosin Contraction Regulates

Epithelial Cell Scattering (22)
de Rooij Johan, Kerstens A., Danuser G., Schwartz M.A. and Waterman-Storer C.M.
The Netherlands Cancer Institute (NKI), Amsterdam, The Netherlands
Scattering of MDCK cells in vitro mimics key aspects of epithelial-mesenchymal

transitions during development, carcinoma cell invasion and metastasis. Scattering is
induced by hepatocyte growth factor and involves disruption of cadherin-dependent
cell-cell junctions. The extracellular matrix has been show to regulate this process
suggesting possible crosstalk between integrins and cell-cell junctions. We use an
automated multi-well, phase-contrast microscopy assay and custom-developed cell-
tracking software to analyze scattering under different matrix conditions. We find
that scattering efficiency correlates with stronger integrin adhesion. We show that
HGF does not trigger any decrease in E-cadherin function, but increases integrin-
mediated adhesion. High resolution time-lapse imaging suggests that contractile
forces on cell-cell junctions may disrupt cell-cell adhesion. Interestingly, the effect of
HGF on phosphorylation of the regulatory myosin light chain depends the ECM and
correlates with its ability to induce scattering. To directly test the role of integrin-
dependent traction forces, substrate compliance was varied. Rigid substrates that
promote high traction forces promoted scattering compared to more compliant
surfaces. We conclude that integrin-dependent acto-myosin traction force mediates
the disruption of cell-cell adhesion during epithelial cell scattering.
- 46 -
Overexpression of Thymosin Beta 4, Thymosin Beta 10 and

Neuroblastoma Thymosin Beta Affects Cell Migration and
Induces Collagen Type I Invasion (23)
Dhaese Stien1, Bruyneel E.2, Bracke M.2, Vandekerckhove J.1, Ampe C.1 and
Van Troys M.1
1
Department of Medical Protein Chemistry (VIB09) and of Biochemistry, Ghent University,
Belgium
2
Department of Radiotherapy and Nuclear Medicine, Ghent University, Belgium
The concerted, balanced activity of actin modulating proteins (AMPs) determines

actin dynamics and consequently all associated features of cellular motility. AMPs
often have altered expression levels in malignant cancer cells that display abnormal
migratory behaviour [Lambrechts et al. (2004) Int. J. Biochem. Cell Biol. 36: 1890-
1909]. We hypothesize that these altered expression levels cause a disturbed
regulation of actin dynamics and this is, in part, responsible for the aberrant
migration and invasive potential of cancer cells. Beta-thymosins influence actin
dynamics by sequestering actin monomers, hereby providing a pool of polymerizable
ATP-G-actin [Huff et al. (2001) Int. J. Biochem. Cell Biol. 33: 205-220; Van Troys et
al. (1996) EMBO J. 15: 201-210]. We performed an in silico study to compare the
divergence of beta-thymosin isoforms across three species: the beta-thymosin family
in mouse was found to be more diverse than in human and rat. We study three
human beta-thymosin isoforms: thymosin beta 4, thymosin beta 10 and
neuroblastoma thymosin beta (TbNB). At the start of our studies, the latter isoform
was uncharacterised. Therefore we analyzed its actin binding and sequestering
properties as well as its reported neuroblastoma specific expression [Yokoyama et al.
(1996) DNA Res. 3: 311-320]. Biochemical and cell biological analysis showed that
TbNB is a bona fide beta-thymosin. Intriguingly, we found TbNB to have a much
broader expression, not limited to neuroblastoma tissue. Currently, our research on
thymosin influenced physiological processes comprises the study of cell migration
and invasion. We show that overexpression of each of the beta-thymosin isoforms
increases cell migration in Boyden chambers. Analysis of wound healing and random
migration is ongoing. The influence on invasion was investigated using a collagen
type I invasion assay, we found that overexpression of all three isoforms induces
collagen type I invasion in several inherently non-invasive cell lines.
- 47 -
Plant LIM Proteins and their Interacting Partners (24)
Dieterle Monika1, Hoffmann C.1, Thomas C.1 and Steinmetz A.1,2

1
Laboratory of Plant Molecular Biology, CRP-Santé, Luxembourg
2
IBMP-CNRS, Strasbourg, France
Plant LIM domain proteins are short proteins of approximately 200 aminoacids
related to the animal Cysteine-Rich Proteins (CRPs). This subgroup of LIM proteins is
characterised by the presence of two LIM domains which are separated by a spacer
region of 40 to 55 residues. Plant LIM proteins differ from the animal CRPs in that
they lack the glycine-rich region and they have an unusually structured second LIM
domain. At least one tobacco LIM protein (NtWLIM1) was found to co-localise with
the actin cytoskeleton in tobacco BY2 cells and in epidermal cells from Nicotiana
benthamiana leaves (see poster by C. Hoffmann). Recent data from our laboratory
suggest that this protein has both a stabilizing as well as a cytoskeleton-organising
function. The aims of these studies are to understand how plant LIM proteins
interact with actin filaments and to identify partner proteins participating in LIM
protein-mediated actin cytoskeleton-associated functions. To isolate and identify
proteins which bind to NtWLIM1 in vivo we use affinity-purification,
immunoprecipitation, as well as the yeast two hybrid assay. First results of these
approaches will be presented.
- 48 -
Cytoplasmic Actins are Differentially Distributed in Normal,

Displastic and Carcinoma Cells of Human Breast (25)
Dugina Vera1, Tchypysheva T. 2, Ermilova V.3, Vasiliev J.2, Gabbiani G.4 and
Chaponnier C.4
1
Belozersky Institute of Physical and Chemical Biology of Moscow State University, Russia
2
Laboratory of Cancerogenesis Mechanisms, and
3
Department of Tumor Pathology, Cancer Research Center, Moscow, Russia
4
Department of Pathology and Immunology, CMU, University of Geneva, Switzerland
Our laboratory has shown that - and -cytoplasmic actins are segregated in distinct
cellular compartments. -actin is organized in parallel filaments such as in stress
fibers, filopodia and in bundles at cell-cell contacts, whereas -actin is formed at
branched filament meshworks in cortical and lamellipodia structures. This different
distribution was observed in cultured normal fibroblastic, epithelial and endothelial
cells, by means of newly developed monoclonal antibodies specific to - and -
cytoplasmic actin. As the -actin network was prominent during the processes of cell
spreading and motility in culture conditions, we asked the question as to whether -
actin is predominantly involved in tumorigenic processes in vivo. We have therefore
compared the distribution of - and -cytoplasmic actins in normal cells compared
with displastic malignant breast epithelial cells.
We have observed that: 1) in normal mammary gland, -cytoplasmic actin has an
apical distribution and -actin is localised at the baso-lateral domain of epithelial
cells; 2) in benign lesions, the polarized distribution of actin isoforms is partially lost;
3) in ductal and lobular in situ carcinoma cells, -actin filamentous structures are
absent, but there is increased of -cytoplasmic actin network throughout the
cytoplasm.
It is well accepted that the enhanced motility of cancer cells compared to the non
malignant situation is crucial in the process of cancer invasion. Our results suggest a
role for -cytoplasmic actin during epithelial cell malignant transformation.
- 49 -
A New Role for the Axial Budding Protein Bud3 in Regulating Septins
Network for Cytokinesis in Saccharomyces cerevisiae (26)
Eluere Raissa, Cabantous S. and Simon M.N.

IBSM-CNRS, Marseille, France
In Saccharomyces cerevisiae , proper coordination of the cell cycle events is

orchestrated by only one Cyclin Dependent protein Kinase (CDK) encoded by CDC28.
Cdc28 drives the entire cell cycle by successively complexing with a set of nine
cyclins. The cyclins Clb1 and Clb2 are present from the end of S phase until
cytokinesis that takes place at the mother bud neck. The cortex at the mother bud
neck is organized by a family of evolutionarily conserved filament-forming proteins
called the septins. Clb2 is the only mitotic cyclin to be localized to the mother-bud
neck. We showed that bud neck localization of Clb2 is specifically abolished in BUD3
deleted cells. The protein Bud3 is best known for its role in the generation of an axial
budding pattern. However, the requirement of Bud3 for anchoring Clb2 to the neck
and a 2-hybrid interaction between Clb2 and the C-terminal part of Bud3, suggested
another function of Bud3 in cell cycle progression. Our data on either null mutation
or truncation of the Bud3 protein shed light on a new role of Bud3 in cytokinesis.
This function depends on the C-terminal part of the protein, which is required for
Clb2 targeting to the bud neck but not for the function of Bud3 in bud site selection
[Bailly et al. (2003) J. Cell Sci. 116: 4110-4130]. We showed that the septin network
is desorganized in BUD3 deleted cells at the time or just before cytokinesis. Bud3
appears to function with the anillin homolog Bud4, and similar septin disorders are
observed in bud3D, bud4D as well in clb1D clb2D cells. Both proteins Bud3 and Bud4
are in vitro substrates of Clb2/Cdc28 [Ubersax et al. Nature 425: 859-864]. We will
further discuss a potential unanticipated function of the Clb1, 2/Cdc28 kinase in
preparing the septin network for cytokinesis.
- 50 -
Functional Characterization of Arabidopsis thaliana

Gamma-Tubulin Complex Components (27)
Evrard Jean-Luc, Seltzer V., Herzog E., Janski N., Altanoury Z., Canaday J. and
Schmit A.C.
Institut de Biologie Moléculaire des Plantes, CNRS, Strasbourg, France
Plants display complex microtubular arrays that are involved in specific intracellular
functions. Even the assembly of these arrays occurs probably at different places in
the plant cell, the nucleation process itself seems to be conserved like in the other
eukaryotes. To support this idea, ortholog genes for GCP1 to 6 (Gamma-tubulin
Complex Proteins) have been identified in the Arabidopsis thaliana genome. Our aim
is to characterize AtGCPs functionally. We have shown that AtGCP1, 2 and 3 interact
with each other in vitro and are involved in same protein complexes in vivo. Using
eGFP-tagging and immunolocalization in tobacco BY-2 cells, we have localized
AtGCP1, 2 and 3 at the nuclear envelope, a known plant microtubule organizing
center. Sub-domains of AtGCP2 and 3 have been shown to be probably involved in
the targeting to the nuclear envelope of these proteins and maybe of the nucleation
complexes themselves. Clearly, recruitment and activation of gamma-tubulin
nucleation complexes under the cell cycle control are key processes for the
regulation of microtubules nucleation in plant cells.
- 51 -
Nck Promotes N-WASP-Mediated Actin-based Motility

of Shigella flexneri (28)
Fabian Anke1, Lommel S.2, Stradal T.E.B.2, Rottner K.1 and Wehland J.2*
1
Cytoskeleton Dynamics Group and
2
Department of Cell Biology, German Research Centre for Biotechnology (GBF), Mascheroder
Weg 1, 38124 Braunschweig, Germany
* corresponding author: jwe@gbf.de
The human pathogen Shigella flexneri exploits Arp2/3 complex-mediated actin

assembly to drive its intra- and intercellular motility. As opposed to Listeria
monocytogenes, which is capable of recruiting Arp2/3-complex directly, Shigella has
evolved to usurp the Arp2/3-complex indirectly by interaction of the Shigella surface
protein IcsA with the host cell Arp2/3-regulator N-WASP. N-WASP is further activated
by additional interacting proteins, such as the SH2/3 adaptors Nck1 and 2, which
recruit to and co-localise with N-WASP at the Shigella surface. Nck1 can activate N-
WASP-mediated Arp2/3 activation in vitro, which is mediated by direct interaction of
its three SH3-domains with the poly-proline domain of N-WASP, also capable of
binding to profilin. In this study we have examined the relevance of these
interactions for Shigella motility. Employing peptide scan analyses, we have
determined the interaction surfaces within the poly-proline domain of N-WASP for
both Nck adaptors and profilin and identified five consensus P5XR-motifs as
interaction sites for SH3-domains. Systematic mutational analysis within these motifs
allowed us to generate a N-WASP mutant, the binding of which to SH3-domains was
strongly impaired in vitro. Re-expression of this mutant in N-WASP-defective cells
restored the velocity of Shigella only to 70% as compared to wildtype N-WASP, while
deletion of the entire poly-proline domain restored movement to 50%, indicating an
additional contribution of profilin binding. Finally, re-expression of full length Nck1 or
Nck2 in Nck1/2 double knockout cells increased Shigella velocity by app. 30% as
compared to non-transfected controls, while expression of a single SH3-domain of
Nck1 in these cells increased bacterial velocity only by 10%. Together, these data
indicate that Nck proteins promote Shigella motility by direct and cooperative binding
of their SH3-domains to the multiple interaction surfaces located within the poly-
proline domain of N-WASP.
- 52 -
The Fragile X Mental Retardation Protein Shows a Granular

Localization and a Cytoskeleton Association in Primary Neurons (29)
Ferrari Francesca1,2, Mercaldo V.1,2, Cannata S.1, Sala C.3 and Bagni C.1,2
1
Dipartimento di Biologia. Università di Roma “Tor Vergata”, Roma, Italy
2
Istituto di Neuroscienze Sperimentali, Fondazione Santa Lucia, IRCCS, Roma, Italy
3
CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of
Pharmacology, University of Milano, Milano, Italy
The Fragile X syndrome is the most common form of inherited mental retardation
and is caused by the absence or reduction of function of the Fragile X mental
retardation protein (FMRP). FMRP is an RNA-binding protein that plays a key role in
activity-regulated localization and translation of mRNA in dendrites and at synapses.
Using a highly specific antibody, we describe a particular granular localization of
FMRP in the dendrites of primary neurons reminiscent of mRNA transport granules.
Moreover, we find that FMRP and Staufen, an RNA-binding protein involved in
transport of messenger RNAs, are concentrated in the same biochemical subcellular
fraction and mildly associated, in comparison with tubulin and MAPK, to the
cytoskeleton. These data support the notion that a fraction of FMRP and Staufen are
in the same ribonucleoprotein complexes, regulating the trafficking and translation of
mRNAs.
- 53 -
Subcellular Localisation Studies of the Tobacco

LIM Domain Protein NtWLIM2 (30)
Gatti Sabrina1, Shen W.H.2, Grausem B.1, Dieterle M.1, Hoffmann C.1, Thomas
C.1 and Steinmetz A.1,2
1
2
The coding sequence of NtWLIM2 was isolated from tobacco BY2 cells following
screening of an expression cDNA library with a radioactively labeled H4 promoter
probe. The protein is 189 aminoacids long and differs considerably from the other
tobacco LIM protein NtWLIM1 (which is 200 aminoacids long and with which it
shares 49% sequence identity plus 14% sequence similarity). It is therefore
expected that the functions of the two proteins diverge at least to some extent. As a
first step towards a functional identification we compared its cellular localisation in
undifferentiated and in differentiated tobacco cells to that of NtWLIM1. To this end
the protein was expressed as a GFP-fusion under the control of an inducible
promoter following Agrobacterium-mediated transformation of tobacco BY2 cells, and
in epidermal leaf cells of Nicotiana benthamiana following agroinfiltration.
Surprisingly, the NtWLIM2-GFP-fusion protein-expressing cells showed a
predominatly diffuse nuclear and cytoplasmic fluorescence, occasionally associated
with thin filaments. These observations contrast considerably with those made in the
case of the NtWLIM1-GFP fusion which always showed a strong and sharply defined
fluorescence associated with actin cables. Also, the binding of NtWLIM2 to actin
filaments was not enhanced by oxygen deprivation of the cells, contrary to NtWLIM1.
Mobility shift experiments confirmed the binding of the protein to the H4 promoter
fragment used in the isolation of the coding sequence. These data suggest that
NtWLIM2 is involved in gene transcription and that its cytoskeleton-associated
function differs from that of NtWLIM1.
- 54 -
The PAFAH1b Subunits And Neuronal Migration (31)
Ghosh Indraneel
The Weizmann Institute of Science, Rehovot, Israel
PAFAH1B is a heterotetrameric enzyme composed of 1 (30 kDa), 2 (29 kDa), and

(45 kDa) subunits. The subunit gene is the LIS1 gene. PAFAH1B has been
implicated in the central nervous system and the reproductive system. The 1
protein is expressed only in migrating neurons in the embryonic brain and not in glial
cells. In contrast, 2 and proteins are detected both in neuronal and non-neuronal
tissues. Here we show that both 1 and 2 subunits interact with the dynamic
microtubule network through the plus-end tracking protein EB1. EB1 is a 30 kDa
protein associated with the plus end of microtubule tips, where it regulates
microtubule polymerization. Loss of the 2 subunit leads to an increase in the rate of
dynamic microtubule polymerization in fibroblasts without any effect on neuronal
migration in the cerebral cortex.
- 55 -
Viscosity and Protein Osmotic Pressure are the Indissoluble Partners

of Muscle Contraction (32)
Grazi Enrico and Di Bona C.

Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Italy
A simple model is presented where, by an iterative procedure, the forces delivered

by the power strokes are summed up to overcome the load. The system is
moderated by the viscous hindrance. The model reproduces the features of muscle
contraction as defined by the data of He et al. (1999) on rabbit psoas muscle fibres.
The model recalls the concept of impulsive force first introduced by Wortington
(1962) and elaborated by Wortington and Elliot (1996). The force generated by the
power stroke is transmitted to the zeta disk and promotes contraction. To raise a
load force summation is required. This occurs when the persistence of the force
generated by the power stroke is longer than the time elapsing between two power
strokes. Now we introduce viscous hindrance to account for contraction velocity,
heat dissipation and energetic yield. An hyperbolic form, defined by the constant 1/k
(s^-1), is assigned to contraction velocity. contraction velocity = k (driving
acceleration) t / (k+t) 1/k increases with the load thus indicating that viscous
hindrance also increases with the load. This is in keeping with the observation that
both cross-bridge attachment and stretching decrease water activity thus increase
the elastic energy of the cross-bridges and the viscosity of the medium. From the
one side the decrease of water activity allows the attached cross-bridge to withstand
to and to deliver larger forces, from the other side the decrease of water activity
increases viscosity, thus promotes heat dissipation, hinders the energetic yield and
decrease the velocity of the contraction. We conclude that there is no muscle
contraction without viscosity.
- 56 -
Omega-Shaped Structures in the Pre-Synaptic Active Zone Caused

by Non-Dissociable Profilin:Actin (33)
Grenklo Staffan1,2, Tomilin N1, Evergren E.1, Brodin L.1, Karlsson R.2 and
Shupliakov O.1
1
Center of Excellence in Developmental Biology, Department of Neuroscience, Karolinska
Institute, Stockholm, Sweden
2
Department of Cell Biology, WGI, Stockholm University, Sweden
Previously, the actin sequestering agent latrunculin A has been shown to alter
neurotransmitter release, indicating an involvement of actin dynamics in synaptic
exocytosis. In agreement with this, actin has been identified in synaptic regions. To
further investigate the role of actin dynamics during synaptic signaling we interfered
with its polymerization in the synapse by microinjecting a non-dissociable variant of
profilin:actin (PxA), which blocks actin polymer-forming complexes operating at
filament (+)-ends. The giant reticulospinal synapse in lamprey was used as an
experimental model for these experiments. After microinjection and subsequent
stimulation of the synapses at 5 Hz for 30 min, a significant increase in the number
of omega-shaped structures was observed at active zones as compared to control
synapses. Most of these structures had a size corresponding to synaptic vesicles,
although larger profiles were also observed. These omega-shaped structures might
represent vesicles undergoing fusion or being at a stage of “kiss-and-run” retrieval,
and could reflect a highly dynamic stage in the presynaptic membrane-vesicle
interplay captured by the introduced PxA. We subsequently purified neuronal
lamprey profilin:actin, and found that it has similar in vitro polymerization properties
as mammalian profilin:actin. The lamprey profilin was also used to generate
antibodies, which partly co-distributed with the synaptic marker SV2 in the lamprey
spinal cord, suggesting that profilin can accumulate in synapses. Together these
observations indicate a role for profilin:actin in the nerve synapse, possibly in
connection with synaptic vesicle trafficking in the active zone.
- 57 -
A Functional Association between Merlin and HEI10,

a Cell Cycle Regulator (34)
Grönholm Mikaela#1, Muranen T.#1, Toby G.G.2, Utermark T.3, Hanemann C.O.3,
Golemi E.A.2 and Carpén O.1,4
#
equal contribution
1
Neuroscience Program, Biomedicum Helsinki, Department of Pathology, University of
Helsinki and Helsinki University Central Hospital, 00014 Helsinki, Finland
2
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, USA
3
Department of Neurology, University of Ulm, 89081 Ulm, Germany
4
Department of Pathology, University of Turku and Turku University Hospital, 20520 Turku,
Finland
Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to

multiple tumors of the nervous system, mainly schwannomas and meningiomas. The
mechanism by which the cytoskeletal protein merlin, the NF2 gene product, functions
as a tumor suppressor is not known although participation in events related to cell
cycle regulation has been suggested. In a yeast two-hybrid screen we identified a
novel merlin binding partner, the cyclin B-binding protein HEI10, involved in the
regulation of progression from G2 to M-phase. The interaction was mapped to the
beginning of the alpha-helix in merlin and to the HEI10 coiled-coil domain and
requires conformational opening of merlin. Colocalization of the two proteins was
dependent on cell cycle and adhesion states and on merlin expression levels. Merlin
and HEI10 were present at the plasma membrane but also targeted to the nucleus in
early G1 and localized to the mitotic spindle and contractile ring early G1. The
subcellular localization of HEI10 in Schwann cells was different from schwannoma
cells. A constitutively open merlin construct bound HEI10 in transfected cells and the
binding induced degradation of HEI10. Further, merlin and HEI10 affected cell cycle
progression in a coordinated manner. These results link merlin to the regulation of
cell cycle progression and may help to resolve its tumor suppressor function.
- 58 -
G12/13 is Essential for Directed Cell Migration and Localized

Rho Function (35)
Goulimari P., Kitzing T., Knieling H, Brandt D.T., Offermanns S. and Grosse Robert
Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120
Heidelberg, Germany
Scratch-wound assays are frequently used to study directed cell migration, a process
critical for embryogenesis, invasion and tissue repair. The function and identity of
trimeric G-proteins in cell behaviour during wound healing is not known. We found
that G12/13, but not G q/11 or Gi, is indispensable for coordinated and directed cell
migration. In mouse embryonic fibroblasts (MEFs) endogenous Rho activity is
present at the rear of migrating cells but also at the leading edge whereas it is
undetectable at the cell front of G 12/13-deficient MEFs. Spatial activation of Rho at
the wound edge can be stimulated by LPA. Active Rho colocalizes with the
Diaphanous-related formin Dia1 at the cell front. G12/13-deficient cells lack Dia1
localization to the wound edge and are unable to form orientated, stable
microtubules during wound healing. Knock-down of Dia1 reveals its requirement for
microtubule stabilization as well as polarized cell migration. Thus, we identifed
G12/13 proteins as essential components linking extracellular signals to localized Rho-
Dia1 function during directed cell movement.
- 59 -
RNAi of FACE1 Protease Results in Growth Inhibition of

Human Cells Expressing Lamin A; Implications for
Hutchinson-Gilford Progeria Syndrome (36)
Gruber Jens, Lampe T., Osborn M. and Weber K.

Max-Planck-Institute for Biophysical Chemistry, Department of Biochemistry and Cell Biology,
Goettingen, Germany
FACE 1 is the endoprotease responsible for cleavage of prelamin A to lamin A.

Transfection of HeLa cells with siRNA for human FACE 1 results in a strong
phenotype. Protein and mRNA levels for FACE 1 are knocked down and cell division
stops abruptly. Two populations of cells are detected. The first form aberrant mitotic
spindles, arrest in mitosis and later enter apoptosis. The second show dramatic
changes in nuclear morphology with extensive formation of lobulated nuclei and
micronuclei. Using antibodies that specifically recognize prelamin A, but not lamin A,
we show that prelamin A accumulates at the nuclear lamina in FACE1 silenced cells,
whereas in control cells prelamin A is found in many small nuclear dots, but not at
the nuclear lamina. In double knockdown experiments with FACE 1 and lamin A
siRNAs, the results depend on which protein is knocked down first. FACE1
knockdown 24 hours prior to lamin A knockdown gives results similar to the single
FACE1 knockdown. In contrast lamin A knockdown 24 hours prior to FACE1
knockdown results in none of the changes described above. Knockdown of FACE1 in
HL60, a cell line that lacks lamin A, also has no effect. The combined results suggest
that prelamin A is a poison in cells subjected to FACE 1 knockdown. Finally we draw
attention to similarities in phenotype between FACE1 silenced HeLa cells and
fibroblasts from patients with Hutchinson-Gilford Progeria Syndrome containing
prelamin A mutations that prevent cleavage by the FACE1 endoprotease.
- 60 -
Investigating Dystroglycan Deficiency at the Cellular Level -

A Role in Cytokinesis (37)
Higginson Jennifer and Winder S.J.

University of Glasgow and University of Sheffield, Sheffield, UK
Dystroglycan is a component of the dystrophin-glycoprotein complex, first identified

in skeletal muscle, which links the actin cytoskeleton to the extracellular matrix.
Dystroglycan has since been identified in tissues other than muscle in association
with the dystrophin homologue utrophin. Dystroglycan is encoded by a single gene,
DAG1, which is expressed as a single polypeptide and post-translationally cleaved
into - and -subunits. Mice deficient in dystroglycan are embryonic lethal, due to
their failure to produce an early basement membrane and chimaeric mice produced
using DAG-/- embryonic stem cells have a muscular dystrophy phenotype. In this
study, we have investigated dystroglycan deficiency at the cellular level and
identified a possible role for dystroglycan in cytokinesis. Dystroglycan was found to
localise to the cleavage furrow of dividing Swiss 3T3 fibroblasts overexpressing
dystroglycan-GFP. In dividing Ref52 cells, dystroglycan co-localised with the ERM
protein, ezrin at the cleavage furrow and midbody. Swiss 3T3 fibroblasts were
depleted of dystroglycan using retroviral infection of an siRNA vector and were
characterised by both morphological and biochemical techniques. These findings into
dystroglycan deficiency will provide greater understanding of the increasingly
complex roles for this protein and may provide insight into diseases such as
Duchenne muscular dystrophy.
- 61 -
Fibroblast Contraction Is Coordinated By

Intercellular Mechanical Coupling (38)
Follonnier L., Pittet P., Schaub S., Meister J.J. and Hinz Boris
Laboratory of Cell Biophysics (LCB), Ecole Polytechnique Fédérale de Lausanne (EPFL),
Lausanne, Switzerland
During tissue repair and fibrosis, fibroblasts differentiate into myofibroblasts by de

novo expressing alpha-smooth muscle actin, rendering them specialized for matrix
contraction. Correlating with the acquisition of a contractile phenotype, fibroblasts
couple stress fibers intercellularly at sites of adherens junctions in vivo and in vitro.
The aim of this study was to elucidate the role of adherens junctions in coordinating
myofibroblast contraction. Fibroblast contraction was previously shown to be
regulated by variations of the intracellular calcium concentration [Ca2+]i (flashing).
We here show that both, rat subcutaneous fibroblasts (SCF) and myofibroblasts
(SCM) exhibit spontaneous and highly periodic [Ca2+]i flashing when grown in 2D
monolayer, on microcontact printed lines and in 3D collagen culture. Spontaneous
flashing is synchronized between contacting SCM but random between SCF. [Ca2+]i
flashing in SCM was randomized upon inhibition of adherens junction but not by
chemical uncoupling of gap junctions. Inhibition of myofibroblast contraction and
actin depolymerization abolished coordinated [Ca2+]i flashing. In contrast, SCF
cultures that have been synchronized with the use of smooth muscle cell contraction
agonists, loose coordination of [Ca2+]i flashing only after gap junction uncoupling.
Hence, adherens junctions appear to coordinate myofibroblast communication
mechanically, whereas fibroblasts communicate predominantly electrochemically via
gap junctions. Second, to assess the relation between intracellular propagation of
[Ca2+]i flashing and single cell contraction we analyzed these events simultaneously
in 3D collagen culture, using [Ca2+]i imaging, confocal reflection microscopy and
texture tracking image analysis. Our results suggest that single cell contraction
mechanically stimulates a [Ca2+]i increase and subsequent contraction in the
adjacent cell via adherens junctions.
- 62 -
The Tobacco LIM Protein NtWLIM1 Stabilises the Actin Cytoskeleton

against Depolymerisation by Latrunculin B (39)
Hoffmann Céline1, Thomas C. 1 and Steinmetz A.1,2

1
2
NtWLIM1 is one of six LIM domain proteins identified in tobacco. Transcripts of the
gene have been detected in all organs of the tobacco plant, but not in pollen grains
and BY2 cell suspension cultures. Initial attempts to express the protein in fusion
with GFP and under the control of the cauliflower mosaic virus 35S promoter failed to
generate transgenic cell lines as well as tobacco plants, suggesting that an
overproduction of the protein interferes with an essential cellular function. Therefore,
in order to study the subcellular localisation of the protein, we have placed the fusion
construct under the control of an inducible promoter in the plant transformation
vector pTA7002 [Aoyama and Chua (1997) Plant J. 11: 605-612]. The recombinant
plasmid was then introduced into tobacco BY2 cells using Agrobacterium-mediated
transformation, and recombinant cell lines were selected on appropriate medium. We
observed that NtWLIM1 exhibits a dual nuclear and cytoplasmic distribution. In the
cytoplasm it was predominantly associated with a filamentous network identified as
the actin cytoskeleton by co-labeling experiments as well as by treatment of the cells
with latrunculin B, a drug that specifically disrupts actin filament assembly.
Importantly, we noticed that the ratio of actin-associated NtWLIM1 relative to the
unbound protein varied with the culture conditions: for instance, a reduction of cell
oxygenation significantly enhanced the binding of NtWLIM1 to microfilaments. In
addition, when these cells were treated with latrunculin B and subsequently analysed
at different times after addition of the drug, the actin cytoskeleton was still clearly
visible after 40 minutes while in control cells not expressing the protein the
filamentous structures had completely disappeared. These data indicate that binding
of the NtWLIM1 protein to the actin cytoskeleton stabilizes the latter against
depolymerisation by latrunculin B.
- 63 -
Two Distinct Assembly Mechanisms are Utilized in Generation of the

Actin Stress Fiber Network (40)
Hotulainen Pirta and Lappalainen P.

Institute of Biotechnology, University of Helsinki, Finland
The actin cytoskeleton is organized to a variety of structures in non-muscle cells.

These include three dimensional networks found in lamellipodia, bundles of unipolar
actin filaments in filopodia, and mixed polarity actin filament bundles called stress
fibers. In contrast to relatively well analyzed lamellipodia and filopodia, the
mechanisms of stress fiber formation and turnover are poorly understood. To
examine stress fiber assembly, known stress fiber components and a focal adhesion
marker were tagged with GFP spectral variants and the localization and dynamics of
these proteins were examined by real-time multicolour widefield epifluorescence
microscopy and by FRAP analysis. In U2OS cells, stress fibers form a polygonal
network consisting of two major types of actin bundles: 1) longitudinal actin bundles,
which are attached to focal adhesions, and 2) transverse actin bundles, which
typically do not directly associate with focal adhesions. Although the two types of
actin bundles assemble into a continuous network, our studies show that they are
generated by distinct assembly mechanisms. Longitudinal bundles elongate through
rapid actin polymerization at focal adhesions, whereas transverse actin bundles
assemble from short alpha-actinin cross-linked structures derived from the actin
network at the leading edge of a motile cell. These short actin arrays assemble with
myosin II bundles to generate periodic mixed polarity transverse actin filament
bundles. They then flow rearward accompanied by shortening of the widths of
myosin II and alpha-actinin bands, apparently due to contraction of the filament
system. In contrast, the focal adhesion polymerized filaments first form a longitudinal
alpha-actinin cross-linked bundle. Myosin II is then incorporated into this relatively
long bundle resulting in the formation of a periodic alpha-actinin:myosin II structure
and probably subsequent mixed polarity arrangement of the actin filaments.
- 64 -
Abi1 Regulates the Activity of N-WASP and WAVE in Distinct

Actin-Based Processes (41)
Innocenti Metello1, Gerboth S.1, Rottner K.2, Carlier M. F.3 and Scita G.1
1
IFOM, Istituto FIRC di Oncologia Molecolare, Milan, Italy
2
Cytoskeleton Dynamics Group, Signalling and Motility Group, German Research Centre for
Biotechnology (GBF), Braunschweig, Germany
3
Dynamique du Cytosquelette, Laboratoire d'Enzymologie et Biochimie Structurales,
C.N.R.S., Gif-sur-Yvette, France
N-WASP and WAVE are members of a family of proteins that use Arp2/3 complex to
stimulate actin assembly in actin-based motile processes. By entering into distinct
macromolecular complexes, they act as convergent nodes of different signaling
pathways. The role of WAVE in generating lamellipodial protrusion during cell
migration is well established. Conversely, the precise cellular functions of N-WASP
have remained, by large, elusive. Here, we report that Abi1, an essential component
of the WAVE protein complex, also plays a critical role in regulating N-WASP-
dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and
potently induces, cooperating with Cdc42, N-WASP activity in vitro. Molecular genetic
approaches demonstrated that Abi1 and WAVE, but not N-WASP, are essential for
Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-
WASP, but not WAVE, regulate actin-based vesicular trafficking, EGFR receptor
endocytosis, EGFR and Transferrin Receptor (TfR) cell-surface distribution. Thus,
Abi1 is a dual regulator of WAVE and N-WASP activities in specific actin dynamic-
dependent processes.
- 65 -
Phosphorylation of L-Plastin on Serine 5 Increases its F-Actin-

Bundling Activity and Promotes its Targeting to Sites of
Actin Assembly in Cells (42)
Giganti A.1*, Janji Bassam1*, De Corte V.2, Catillon M.1, Lentz D.1, Bruyneel E.3,
Plastino J.4, Gettemans J.2 and Friederich E.1
* These authors contributed equally to this work
1
Laboratoire de Biologie Moléculaire, d’Analyse Génique et de Modélisation. Centre de
Recherche Public-Santé (CRP-Santé), 42, rue du Laboratoire, L-1911 Luxembourg
2
Department of Biochemistry, Faculty of Medicine and Health Sciences, Gent University,
Albert Baertsoenkaai 3, B-9000 Ghent, Belgium and Flanders Interuniversity Institute for
Biotechnology (V.I.B.), Belgium
3
Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear
Medicine, Ghent University Hospital (1P7), De Pintelaan 185, B-9000 Gent, Belgium
4
Laboratoire Physicochimie “Curie”, UMR168 CNRS/Institut Curie, 11, rue Pierre et Marie
Curie, 75231 Paris cedex 05, France
Plastins/fimbrins are actin cross-linking proteins harbouring two actin-binding

domains and an amino-terminal regulatory headpiece domain comprising two EF
hand Ca2+-binding sites. Among plastin isoforms, L-plastin, a cell transformation
associated protein, is the only one to be phosphorylated. Although it was suggested
that phosphorylation of L-plastin on serine-5 might regulate its F-actin-binding and
cross-linking properties and thus its biological function, experimental support for
such a mechanism of regulation is still lacking. Here, we combined cell-based and
biochemical assays to address this issue. Vero cells that do not express endogenous
L-plastin, were transfected with constructs encoding wild type L-plastin (WT) or L-
plastin phosphorylation variants (S5/A-L-plastin, S5/E-L-plastin). Immuno-staining of
transfected cells producing WT-L-plastin with a Ser5-P specific antibody, revealed
that the phosphorylated protein localised to actin-rich structures, including focal
adhesions, microspikes and the leading edge. S5/E-L-plastin also localised to actin-
rich regions and, in addition, promoted the formation of spike-like F-actin structures
in two cell lines. Conversely, S5/A substitution highly reduced the capacity of L-
plastin to localise with actin in cells. In-vitro assays revealed that S/E-L-plastin had
markedly increased F-actin bundling activity when compared to non-phosphorylated-
L-plastin. To investigate the impact of phosphorylated L-plastin on cell migration,
phosphorylation variants were tested in a collagen gel invasion assay. Expression of
WT- or S/E-L-plastin conferred invasive properties to non-invasive HEK-293T cells,
while S/A-L-plastin had no effect. Based on these results we propose that
phosphorylation of L-plastin on ser-5 switches the protein from a low to a high
activity state, increasing its bundling activity and its avidity for F-actin in cells. Thus,
L-plastin might act as an integrator of signals controlling the assembly of the actin
cytoskeleton and cell motility.
- 66 -
Regulation of Actin Dynamics and Actin-Based Movement by the

Actin Filament Cross-Linking Protein T-Fimbrin/Plastin (43)
Giganti A.1, Plastino J.2§, Janji Bassam1§, Van Troys M.3, Lentz D.1, Catillon M.1,
Hoffmann C.4, Ampe C.3, Sykes C.* and Friederich E.1
§
These authors contributed equally to this work
1
Laboratoire de Biologie Moléculaire, d’Analyse Génique et de Modélisation, Centre de
Recherche Public-Santé, 42, rue du Laboratoire, L-1911, Luxembourg
2
Laboratoire Physicochimie “Curie”, UMR168 CNRS/Institut Curie, 11, rue Pierre et Marie
Curie, 75231 Paris cedex 05, France
3
Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University,
and Medical Protein Research, Flanders Interuniversity Institute for Biotechnology (VIB09)
Belgium
4
Increasing evidence suggests that actin cross-linking or bundling proteins may not
only structure the cortical actin cytoskeleton but also control actin dynamics. Here,
we analysed the effects of T-fimbrin/T-plastin, a representative member of an
important actin filament cross-linking protein in by combining a quantitative
biomimetic motility assay with biochemical and cell-based approaches. Beads coated
with the VCA domain of WASp (Wiskott Aldrich Syndrom protein) recruit the actin
nucleating Arp2/3 complex, polymerise actin at their surface and undergo movement
when placed in cell-free extracts. T-fimbrin increased the velocity of VCA beads by
1.5-fold, stabilised actin comets and concomitantly displaced cofilin, an actin-
depolymerising protein. T-fimbrin decreased F-actin disassembly rate and inhibited
cofilin-mediated depolymerisation of actin filaments, in vitro . Importantly, a
bundling-incompetent variant comprising the first actin-binding domain (ABD1) had
similar effects. In cells, this domain induced the formation of long actin cables to
which other actin-regulating proteins were recruited. Analysis of the intracellular
distribution of GFP-T-fimbrin in living cells revealed that the protein rapidly
redistributed to ruffling membranes where actin assembly occurs. Altogether, these
results favor a mechanism in which binding of ABD1 controls actin turn over,
independent of cross-link formation. In cells, this activity may contribute to the
assembly and maintenance of the actin cytoskeleton of plasma membrane
protrusions.
- 67 -
Characterization of Nuclear Myosin I (44)
Kahle Michal, Dzijak R. and Hozák P.

Institute of Experimental Medicine, Academy of Sciences of The Czech Republic, Prague,
Czech Republic
Nuclear myosin I (NMI) is the only molecular motor described in the nucleus so far.
It is closely related to cytoplasmic myosin MYO1C from which it differs only by 16
amino acid N-terminal extension. It was shown that NMI is required for transcription
by both RNA polymerase I and II. By immunofluorescence microscopy, we found
NMI to be expressed in all tested mouse tissues. We observed generally higher levels
of NMI in cells with more intensive metabolism. On the level of mRNA, the
expression of NMI varied among different tissues with the highest expression in
spleen and testis. We measured the level of NMI in cells brought to quiescence by
serum deprivation and subsequently activated by addition of the serum. The amount
of NMI reacts relatively slowly to changed proliferation rate of the cells. To test the
pace of degradation of NMI, we observed its level after inhibition of protein synthesis
by cycloheximide. We found NMI to be stable with no significant decrease after 16
hours. While the presence of NMI is required for transcription, the slow turnover of
NMI argues against its being a transcription regulator of RNA polymerase I. This
work was supported by Grant Agency of the Czech Republic (Reg. No.
2004/04/0108), Grant Agency of the Academy of Sciences of the Czech Republic
(Reg. No. IAA5039202), and by the Institutional Grant No. AV0Z5039906.
- 68 -
Internal Fragments of ZASP Isoforms Co-Localize with

-Actinin (45)
Klaavuniemi Tuula1 and Ylänne J.1,2

1
Biocenter Oulu and Department of Biochemistry, University of Oulu, Oulu, Finland
2
Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä,
Finland
The cytoskeleton of muscle cell is organized into sarcomeres. -Actinins crosslink

antiparallel actin filaments at the Z-discs. A group of PDZ-LIM proteins interact with
-actinin at the muscle Z-lines. Of these proteins, ALP, CLP36 and ZASP have a
conserved ZASP-like motif (ZM) motif in the internal region between PDZ and LIM
domains. This motif is composed of 26 conserved residues. We have shown that ALP
and CLP36 have two interaction sites with -actinin (Klaavuniemi et al. 2004). The
PDZ domain recognizes the C-terminus of -actinin and the internal part interacts
with the rod region. The ZM-motif is important in the internal interaction. In ALP and
ZASP, the ZM-motif is either spliced from exon 4 or from exon 6. ZASP has one N-
terminal PDZ domain and three LIM domains at the C-terminus. ZASP/Cypher
deficient mice die with severe disruption of sarcomeres (Zhu et al. 2001). The PDZ
domain of ZASP recognizes the C-terminus of -actinin. Our first aim was to study
whether the internal interaction site with -actinin exists also in ZASP. We found that
the internal fragments of ZASP exon 4 and exon 6 ZM-variants co-localized with -
actinin on actin fibers in cultured cells. ZASP variant 1, which does not have LIM
domains, inhibited the binding of the -actinin rod region to ALP internal fragment in
surface plasmon resonance (SPR)-measurement. Based on these results, we propose
that also ZASP has an internal interaction site with -actinin. Our second aim was to
study the effect of nonmuscle myosin light chain kinase inhibitor (ML-7) on ZASP
localization in cultured cells. This inhibitor destroys stress fibers by preventing the
polymerization of nonmuscle myosin. We detected two distinct pools of -actinin in
relation to ZASP, one co-localizing with ZASP on actin filaments, and one separate
pool of -actinin accumulating in the cell periphery in the presence of ML-7. We
suggest that ZASP is bound to -actinin mainly when actinin is linked to filamentous
actin. This supports our hypothesis of the two interaction sites in regulating the
capacity of -actinin to crosslink filamentous actin in stress fibers and myofibrils.
- 69 -
Host Cell Invasion of Campylobacter jejuni Involves

Rho GTPases Rac1 and Cdc42 (46)
Krause Malgorzata, König W. and Backert S.

Otto von Guericke University Magdeburg, Department of Medical Microbiology, Magdeburg,
Germany
Campylobacter jejuni is the leading bacterial cause of food-borne illness worldwide

and a major cause of Guillain-Barre paralysis. Invasion of host cells is one of the
primary mechanisms of tissue damage caused by C. jejuni. Recent studies have
provided evidence for an microtubule-dependent (actin-filament-independent) and/or
actin-filament-dependent mechanism by which C. jejuni invaded gut tissue cells.
Here, we describe the identification of a novel set of intracellular signals involved in
the internalization of clinical C. jejuni strains into cultured human intestinal epithelial
cells as investigated by gentamycin protection assays, confocal microscopy and live-
cell imaging. To characterize the sequence of events in the host cell entry in more
detail, we investigated the role of small Rho GTPases which are well-known
regulators of both actin and microtubule polymerization and organization.
Inactivation of the small Rho GTPases using clostridial toxin B or mevastatin
significantly reduced the uptake of C. jejuni. Conversely, cytotoxic necrotizing factor
CNF-1 from Escherichia coli, known to activate the small GTPase members Rho, Rac,
and Cdc42, increased the internalization of C. jejuni. Furthermore, expression of
constitutively-active forms of Cdc42 and Rac1, but not RhoA, promoted the uptake of
C. jejuni, whereas expression of their dominant-negative variants hampered host cell
entry. Collectively, our results suggest that the small Rho family GTPase members
Cdc42 and Rac1 play a crucial role in the host cell invasion of C. jejuni.
- 70 -
Evaluating the Mechanics of Single IFs In Vitro - Towards

Understanding the Mechanical Role of IFs In Situ (47)
Kreplak Laurent, Mücke N., Bär H., Leterrier J.F., Herrmann H. and Aebi U.
M.E. Müller Institute, Biozentrum, University of Basel, Switzerland
Intermediate filaments (IFs) are generally considered as rigid structures providing

cells with some resilience to mechanical stresses. This notion stems from dramatic
phenotypes observed in mechanically stressed tissues such as skin or muscle, where
due to mutations IFs were deficient. Apart from these indirect evidences there are
very few data available on the mechanical properties of Ifs in vitro or in situ. In this
communication, we will present our recent findings concerning the mechanics of
single IFs in vitro using an scanning force microscopy (SFM) approach. The elastic
behaviour of IFs was investigated by measuring the persistence length of vimentin
IFs adsorbed to different supports and a value of 1 micrometer was derived. The
extensibility and toughness of IFs was studied by an online nanomanipulation
approach where the SFM tip was used to move or stretch single IFs directly on the
support where they were adsorbed. By this method, IFs were stretched severalfold
and clear morphological changes were observed, especially a large reduction of the
apparent diameter. All these data will be compared to earlier mechanical
measurements on macroscopic, IF containing fibres such as, for example, Hagfish
slime threads, and we will discuss the possibility that in situ IFs may function as
mechanical shock absorbers.
- 71 -
Identifying New Binding Partners of SWAP-70 (48)
Kühnl Jochen, Oberbanscheidt P. and Bähler M.

Institut für Allgemeine Zoologie und Genetik, Münster, Germany
Functionally different subsets of actin filament arrays controlled by different signaling

pathways contribute to cellular organization and motility. The protein SWAP-70 has
been shown to localize to a previously unrecognized array of loose actin filaments
localized behind protruding lamellipodia. Since the subset of actin filaments marked
by SWAP-70 lacks myosin II and SWAP-70 is diffusely distributed in the cytosol of
non-motile cells, it has been suggested that SWAP-70 marks filaments which are in
transition between generation in the lamellipodium and incorporation into contractile
bundles. So far, no direct interaction between F-actin and SWAP-70 could be
demonstrated by in vitro sedimentation analysis. In addition, SWAP-70 was also
found to associate with macropinosomes, association which is independent of F-actin
filaments. To identify potential binding partners of SWAP-70 that control its different
subcellular localization, we carried out bacterial two-hybrid screens. Several potential
candidates have been identified and are in the process of being analysed.
- 72 -
Activation of the Lysosome-Associated p61Hck Isoform Triggers the

Biogenesis of Podosomes (49)
Labrousse Arnaud, Cougoule C., Carreno S., Castandet J., Astarie-Dequeker C.,
Poincloux R., Le Cabec V. and Maridonneau-Parini I.
IPBS - CNRS UMR 5089, Department of Molecular Mechanisms of Mycobacterial Infections,
Toulouse, France
Podosomes are actin-rich membrane structures which participate in cell migration via
adhesion and extracellular matrix proteolysis. Podosomes are present only in cells
which migrate through anatomic boundaries such as osteoclasts, phagocytes and
invasive tumor cells. In human macrophages, we observed that Hck, a Src family
tyrosine kinase expressed as two isoforms, is present at lysosomes and podosomes.
Ectopic expression of the lysosome-associated isoform p61Hck triggered the de novo
formation of podosomes. It required its kinase activity, its SH2 and SH3 adaptor
function, the integrity of microfilament and microtubule networks and concerted
action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished
when p61Hckca was re-addressed from lysosomes to the cytosol or triggered when
p59Hckca was re-localized to lysosomes. Lysosomal markers were present at
podosome rosettes. By stimulating exocytosis of p61Hckca-lysosomes with a calcium
ionophore, the formation of podosome rosettes was enhanced. Interestingly, we
confirm that, in human macrophages, Hck and lysosomal markers were present at
podosomes which were spatially re-organized as clusters, a foregoing step to form
rosettes, upon expression of p61Hckca. We propose that lysosomes, under the
control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon
in the migration of phagocytes.
- 73 -
Tumoral Phenotype Reversion by Pharmacologic Modulation of Actin

Cytoskeleton Structure (50)
Le Moigne R.1, Polard V.2, Maksimenko A.2, Subra F.1, Auclair C1.
1
Laboratoire de Biotechnologie et Pharmacologie Génétique Appliquée, CNRS UMR 8113, 61
avenue du président Wilson, 94230 Cachan, France
2
Bioalliance Pharma, 59 avenue du général Martial Valin, 75015 Paris, France
Tumoral cells commonly present major phenotypic modifications. An increasing

number of works show that some of these morphological and phenotypical changes
are due to variations of actin network structure caused by deterioration of its
polymerization or reshaping [Pawlak and Helfman (2001) Curr. Opin. Genet. Dev.
11: 41-47]. It is notably the case of the fibroblastic model NIH3T3 expressing the
EWS-Fli1 (E/F) fusion oncoprotein (responsible of Ewing sarcoma) we developed in
our laboratory. While NIH3T3 cells are non-tumorigenic and show a highly organized
cytoskeleton, E/F cells are tumorigenic and their actin cytoskeleton is completely
disrupted. Previous investigations have shown that E/F cell under-expressed the
zyxin protein, involved in the architecturation of actin cytoskeleton. The zyxin re-
expression at a normal level in E/F cells induces the tumoral phenotype reversion
(Amsellem et al., 2005). Based on these results, we hypothesized that
pharmacological agents capable of restructuring malignant cell actin cytoskeleton
would induce the phenotypic reversion as well.
In order to identify new anticancerous agents, we developed a fluorescence
anisotropy based assay. First, this assay allowed us to show the weakest capacity of
tumoral cells to polymerize actin with regard to their non tumoral equivalents. Then,
we made the primary screening of a bank containing numerous natural product
extracts. A plant extract (Peganum harmala) induces tumoral phenotype reversion of
E/F cells by restructuring actin cytoskeleton and restoring all associated structures.
The reversion phenomenon occurs at non cytotoxic extract concentration and is
independent of a direct actin interaction. In vivo, P. harmala nude mouse treatment
induces 60 % inhibition of E/F tumor growth. After purification, the BA512, molecule
responsible for the pharmacological activity was able to be characterized and is at
present optimized for future clinical trials.
Anticancerous treatment aiming actin cytoskeleton is feasible and we are undertaking
research on how the tumoral phenotype reversion mechanism occurs (BA512 target,
involved pathways).
- 74 -
Actomyosin Contraction Controls B Lymphocyte Polarization and

BCR-Driven Ag Presentation (51)
Vascostto F.1, Lankar D.1, Faure-André G.1, Raposo G.2, Boes M.3, Bonnerot C.1,
Manoury B.1 and Lennon-Duménil Ana-Maria1
1
U653, Institut Curie, Paris
2
UMR144, Institut Curie, Paris
3
Harvard Medical School, Boston (USA)
B lymphocytes capture exogenous antigens (Ag) through their BcR (B Cell Receptor),
which includes a specific membrane immunoglobulin (Ig) and a signalling module.
Interaction of multivalent antigens with the BcR initiates a cascade of events that
concomitantly leads to B cell activation and induces Ag internalization, processing
and presentation to CD4 T cells by MHC class II molecules. Activated T cells will, in
turn, modify the response of the B cells by regulating their capacity to produce
antibodies against this specifically captured Ag, a process referred to as T-B cell
cooperation. We used freshly purified B cells from MHC II-GFP (I-Abb-GFP) knock-in
mice to analyze the dynamics of antigen processing upon BcR stimulation. We found
that, in resting B lymphocytes, MHC class II molecules concentrate essentially at the
plasma membrane and in the ER. Drastic differences in MHC class II distribution are,
however, observed in BCR-activated cells: although surface MHC class II levels do
not change, most intracellular MHC II molecules are found endosomal compartments
full of internal membranes that cluster at the centre of the cell in close apposition
with the centrosome. Redistribution of MHC II molecules appears to be part of a
global re-polarization program, as highlighted by MTOC and nucleus re-orientation.
Time-lapse experiments suggest that this re-polarization event is coupled to
important modifications in the contractile activity of the B cell, a pathway known to
be control by Myosin II. In agreement with this result, we observe that: (1) MLC, the
regulatory subunit of Myosin II becomes phosphorylated and relocalizes upon BCR
stimulation, (2) inhibitors of ROCK and MLC-kinase, two enzymes capable of
phosphorylating MLC, prevents clustering of MHC II-containing endosomes,
positioning of the MTOC at the cell centre, and (3) presentation of BCR-internalized
Ag to specific T cells. These are the first results implicating the Actomyosin
contractile activity in Ag trafficking, processing and presentation and suggest that, by
triggering such global transformation of antigen-stimulated B cells, the ROCK/Myosin
II pathway may have an important role in regulating the antibody response.
- 75 -
The Novel Jamip Family in T Lymphocytes: a Role in the

Immune Synapse ? (52)
Libri Valentina, Pizza G. and Pellegrini S.

Unité de Signalisation des Cytokines, Institut Pasteur, Paris, France
A two hybrid screen performed with the FERM-homology domain of Tyk-2 led to the
identification of a novel protein called Jamip1, an acronym for Jak and microtubule
interacting protein. Jamip1 is one of a three-member family of closely related genes.
These family proteins lack known conserved domains, but contain predicted coiled
coils. Jamip1 is expressed uniquely in neuronal cells and in lymphocytes.
Immunolocalization of the endogenous Jamip1 in Jurkat T cells revealed organized
filamentous structures that colocalize, in confocal microscopy images, with alfa-
tubulin. Strong staining of the MTOC is also detected. We are presently addressing
the involvement of Jamip1 in cytoskeletal rearrangements occurring during antigen-
induced lymphocyte activation. To this end, we perform functional studies of Jamip
proteins in human CD4+ T lymphocytes to study their role in cytoskeletal
rearrangements and polarization processes. Confocal analysis of T cell-APC
conjugates shows that the endogenous Jamip1 undergoes a TCR-dependent
reorientation toward the immunological synapse together with the MTOC. This
observation was confirmed by studying the activation of a Jurkat stable clone
expressing EGFP-Jamip1 fusion protein. Using a siRNA approach, we recently studied
the effect of silencing Jamip1 and Jamip2 on TCR-induced events in Jurkat and
HuT78 T cell lines. Silencing of endogenous proteins is very reproducible and
efficient (>80). Cell imaging studies of activated cells depleted for Jamip1 and
Jamip2 seems to be a promising tool to understand the role of Jamip proteins in the
reorientation of MTOC and the redistribution of key molecules at the immunological
synapse.
- 76 -
Subcellular Distribution of Cytoplasmic Linker Proteins in Migrating

Epithelial Carcinoma Cells (53)
Long Heather, Boczonadi V. and Maatta A.

School of Biological and Biomedical Sciences, University of Durham, Durham, UK
Plakins are large multidomain proteins that connect intermediate filaments to cellular
junctions and other cytoskeletal networks. Plakins have been implicated in both
maintenance of tissue integrity and orchestration of cytoskeletal organisation in
development and in cell migration.
To investigate the biological functions of cytoskeletal linker proteins in cell migration,
we have studied periplakin and desmoplakin in MCF7 mammary carcinoma cells.
Upon in vitro wounding of confluent epithelial sheets, periplakin, unlike desmoplakin,
disappears from cell borders, becomes polarised and is partially co-localised with
thick keratin 8/18 bundles forming at the ‘wound’ edges. Changes in periplakin
distribution are closely associated with changes in -catenin distribution. Using
okadaic acid and SB203580 we have examined the effect of disrupting the
phosphorylation states on the redistribution of periplakin in MCF7 cells and the
subsequent effects on keratin filament organisation. Inhibition of p38 MAPK disrupts
the interaction of periplakin with keratin filaments and the addition of okadaic acid
causes co-collapse of periplakin with the keratin into granular aggregates. -catenin
is unaffected by the addition of okadaic acid and SB203580, suggesting that
periplakin has at least two separate roles in MCF7 cells.
Transfections with NT and CT domains of periplakin show the different roles of these
domains. The NT domain is localised to the membrane and the CT domain is
associated with the keratin network. The addition of okadaic acid to CT transfected
cells causes collapse of both filaments and periplakin into aggregates, however the
NT domain is relatively unaffected by the addition of this drug. Using RNAi, we have
been examining the effect of knockdown of keratin 8 and periplakin on cell migration
in MCF7 cells.
We have, thus, established that the subcellular distribution of plakins and
intermediate filaments is dynamic in cell migration.
- 77 -
The Src-Cortactin Pathway Regulates Podosome Dynamics during

Osteoclast Polarization (54)
Luxenburg Chen1, Addadi L2. and Geiger B.1

1
Department of Molecular Cell Biology and
2
Department of Structural Biology, The Weizmann Institute of Science, Rehovot, Israel
Osteoclasts are giant, multinucleated, bone-degrading cells, whose biological activity

is central to the development and maintenance of the skeleton. During osteoclast
polarization, podosomes, which are unique adhesion structures, develop into a
peripheral ring-like structure at the cell periphery - the sealing zone. Osteoclasts
display four sequential patterns of podosomal organization. These include individual
podosomes, podosome clusters, developing rings and finally a sealing-zone-like
structure at the cell periphery. Focusing on the dynamic features of podosomes in
their different states of organization we show that podosome clusters, throughout
the ventral cell surface, turn over within 3 min, while those forming the developing
rings and the sealing-zone-like structure are considerably more dynamic. Src catalytic
activity is essential for osteoclast polarization. Here we show that it is crucial for
podosome maturation and dynamics as well. Osteoclasts expressing constitutively-
active src are polarized and possess ectopic ring-like structures with very high
turnover. Cells expressing dominant negative src fail to polarize, and their
podosomes are highly stable and fail to mature. The differences in podosome
dynamics during polarization are likely attributable to the phosphorylation of
cortactin. Cortacin is an actin nucleating protein whose activity is regulated by src-
mediated phosphorylation. We show here that this protein is tyrosine-phosphorylated
in podosome clusters but not in the developing rings or the mature sealing-zone-like
structure. Furthermore, we demonstrate that phosphorylation of cortactin regulates
the rate of podosome turnover, but not osteoclast polarization.
- 78 -
Modulation of ADF/Cofilin Stimulated F-Actin ATPase by

Actin Binding Proteins (55)
Dansranjavin T., Gremm D., Strotkamp D., Wegner A. and Mannherz Hans Georg
Department of Anatomy and Cell Biology and Physiological Chemistry, Ruhr University,
Bochum, Germany
The rate of ATP-hydrolysis catalysed by F-actin correlates with e-ADP release and
thus with actin treadmilling. This rate was demonstrated to increase by a factor of 10
in the presence of ADF or cofilin. Maximal stimulation of the actin-ATPase or of e-
ADP release by ADF or cofilin was attained at an actin to ADF/cofilin ratio of 2. At
higher ratios the ATPase decreased most probably due to F-actin depolymerisation
and formation of stable actin:ADF/cofilin complexes. In the presence of both ADF
and cofilin the actin-ATPase was stimulated in an additive manner. We determined
the effect of thymosin ß4, gelsolin and tropomyosin on the ADF or cofilin stimulated
actin-ATPase. Both thymosin ß4 and tropomyosin decrease the ADF or cofilin
stimulated actin-ATPase. Thymosin ß4 is shown to support the depolymerising
activity of ADF/cofilin. In contrast, the effect of gelsolin was more complex in that it
depended on its ratio to actin. At a ratio of 1/500, when all actin filaments were
assumed to be capped by gelsolin, the effect of cofilin or ADF on the actin-ATPase
was minimal, whereas at lower or higher ratios a further stimulation of the cofilin or
ADF increased actin-ATPase was observed. Furthermore, we determined the rate of
actin monomer dissociation from the pointed-ends of gelsolin capped F-actin by
addition of increasing concentrations of cofilin. Cofilin increased the rate of monomer
dissociation as expected, however, this rate continuously increased with further
addition of cofilin suggesting that the increasing decoration of F-actin by cofilin
further stimulated the rate of monomer dissociation. Only after complete decoration
of the F-actin by tropomyosin was a maximal rate obtained with increasing cofilin
concentration. The data obtained also indicated that cofilin only affected the rate of
actin monomer dissociation but not of monomer association at the pointed-ends.
- 79 -
Regulation of Cytoskeletal Dynamics and Cell Migration

by Plectin (56)
Marshall Lorna and Maatta A.

Integrative Cell Biology Laboratory, Centre for Stem Cell Biology and Regenerative Medicine,
School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE
Plectin is a large cytoskeletal linker protein that can connect different cytoskeletal
networks to each another and to cell-cell and cell-matrix junctions. Mutations in
plectin disrupt tissue integrity in skin, skeletal and heart muscle. The versatility of
plectin as a linker protein is further enhanced by complex alternative splicing that
results in a large number of plectin isoforms. We are investigating the role of plectin
in the cytoskeletal organisation and invasiveness of epithelial carcinomas. Using
published information on mouse gene structure we have cloned seven alternative
first exons of human plectin gene and show that alternative plectin N-terminal
domains comprising the first exon and the following calponin-homology actin binding
domain target EGFP constructs to different subcellular localisations. Thus, the first
exon appears to determine where in the cell plectin can form actin – intermediate
filament cross-bridges. Using quantitative real-time PCR we have found that grade
3/4 colon cancer cell line SW480 that is highly motile on collagen has altered
expression profile of plectin N-terminal isoforms compared to less motile grade 1 or 2
cell lines. The altered expression pattern is reflected by differences in the subcellular
distribution of plectin. Notably, plectin is absent from lamellipodia of SW480 cells but
can be localised in circular dorsal ruffles. We propose that a change in plectin
isoform expression can lead to altered cytoskeletal and junctional organisation and
can contribute to invasive properties of carcinoma cells.
- 80 -
Analysis of MIM and ABBA1, Two Homologous Regulators of the

Actin Cytoskeleton (57)
Mattila Pieta1, Saarikangas J.1, Varjosalo M.2, Taipale J.2, Salminen M.1 and
Lappalainen P.1
1
Institute of Biotechnology, University of Helsinki, Helsinki, Finland
2
Molecular/Cancer Biology Program, University of Helsinki, Helsinki, Finland
The diverse essential functions of the actin cytoskeleton are regulated by a large
number of actin-binding proteins. Two novel regulators of the actin cytoskeleton,
MIM (missing in metastasis) and ABBA1, were recently identified from mammals.
They are approximately 55% identical to each other and are composed of an N-
terminal actin filament bundling IMD domain and a C-terminal actin monomer-
binding WH2 domain. Interestingly, MIM was recently also identified as an enhancer
of Gli-dependent transcription of the Shh signalling pathway [Callahan et al. (2004)
Genes Dev. 18: 2724-2729]. Our Northern blot and in situ hybridization analyses
revealed that MIM and ABBA1 show distinct expression patterns. In adult mouse MIM
is especially strongly expressed in liver, kidney and Purkinje cells, whereas ABBA1 is
mainly expressed in spine and molecular layer of the cerebellum. Both proteins are
also strongly expressed during the development. Over-expression of MIM and ABBA1
in various cell types resulted in the loss of stress fibers and appearance of abnormal
actin structures and microspikes. To reveal the biological roles of these proteins, we
have generated MIM knockout mice and are currently generating ABBA1 knockout
mice. Mice lacking MIM are viable and do not display any gross abnormalities. The
lack of obvious developmental defects in MIM -/- mice suggests that this protein
does not play a central role in the Shh pathway during embryogenesis. To further
investigate the role of MIM in Shh signalling, we carried out an in vitro Gli-luciferase
reporter assay in NIH-3T3 cells. Expression of MIM and ABBA1 had no effect on Hh
signalling or on transcriptional activity with Gli1/2 in these cells. Together, these
studies suggest that MIM and ABBA1 regulate certain specialized actin-dependent
processes in mammals but do not significantly contribute to Shh signalling as
previously suggested.
- 81 -
Behaviour of Mutant Cardiac Actins after Transfection into

Various Cell Lines (58)
Masur Antonina1, Fister S. 2, Milting H. 2 and Mannherz H.G.1

1
Department of Anatomy and Embryology, and
2
Herz- und Diabeteszentrum, Bad Oeynhausen, Ruhr-University Bochum, Germany
Point mutations in -cardiac actin have been made responsible for some cases of
hypertrophic cardiomyopathy which result in heart failure. So far we have
investigated two point mutations found in ACTC gene of patients with hypertrophy,
Tyr168Cys and Met305Leu. Fibroblastic (NRK and NIH3T3) and cardiomyocyte-like
(HL-1) cell lines were transfected with plasmid pcDNA3.1/NT-GFP-TOPO containing
the GFP gene fused to the 5’-end (N-terminus) of either wild type human -cardiac
actin cDNA or to its mutant forms. Transfected cells were stained with
rhodamine–phalloidin or antibodies directed against actin or cytoskeletal proteins
interacting with actin and analyzed by confocal microscopy. Wild type GFP--cardiac
actin was found integrated into pre-existing stress fibers or sarcomeric structures
without obviously disturbing their supramolecular organisation. In contrast,
transfection with Met305Leu and Tyr168Cys mutants resulted in changes in cell
morphology and visible alterations of stress fiber organisation and sarcomer
structure. The Tyr168Cys mutant was found unable to integrate in pre-existing stress
fibers. Substitution of Tyr168 with Cys must have resulted in a significant effect on
the actin monomer structure with marked effects on its ability to form filamentous
actin. We have also observed after transfection that GFP-actin products often formed
cytoplasmic aggregates that were more frequently observed for the mutants than for
the wild type cardiac -actin. Work is presently in progress investigating the nature
and cause of these aggregates.
- 82 -
The Formin Homology 1 Domain Modulates the Actin Nucleation and

Bundling Activity of Arabidopsis FORMIN1 (AFH1) (59)
Michelot Alphée1, Guérin C1., Huang S.2, Ingouff M.1, Rodiuc N.1, Staiger C.J.2 and
Blanchoin L.1
1
Laboratoire de Physiologie Cellulaire Végétale, CEA/CNRS/INRA/UJF, UMR 5168, Grenoble,
France
2
Department of Biological Sciences and the Bindley Bioscience Center, Purdue University,
West Lafayette, IN 47907-2064, USA
The organization of actin filaments into large ordered structures is a tightly controlled
feature of many cellular processes. However, the mechanisms by which actin
filament polymerization is initiated from the available pool of profilin-bound actin
monomers remain unknown in plants. Since the spontaneous polymerization of actin
monomers bound to profilin is fully inhibited, the intervention of an actin promoting
factor is required for efficient actin polymerization. Two such factors have been
characterized from yeasts and metazoans, the Arp2/3 complex, a complex of seven
highly-conserved subunits including two actin-related proteins (ARP2 and ARP3), and
the formin family of proteins. The recent finding that Arabidopsis plants lacking a
functional Arp2/3 complex exhibit rather modest morphological defects leads us to
consider whether the large FORMIN family plays a central role in the regulation of
actin polymerization. Here we have characterized the mechanism of action of
Arabidopsis FORMIN1 (AFH1). Overexpression of AFH1 in pollen tubes has been
shown previously to induce abnormal actin cable formation. We demonstrate that
AFH1 has a unique behavior when compared with non-plant formins. The activity of
the formin homology domain 2 (FH2), containing the actin binding activity, is
modulated by the formin homology domain 1 (FH1). Indeed, the presence of the FH1
domain switches the FH2 domain from a tight capper (Kd ~ 3.7 nM) able to nucleate
actin filaments that grow only in the pointed ends direction to a leaky capper that
allows barbed ends elongation and efficient nucleation of actin filaments from actin
monomers bound to profilin. Another very exciting feature of AFH1 is its ability to
bind and bundle actin filaments. For the first time we identified an actin nucleator
able to organize actin filaments directly into unbranched actin filament bundles. We
suggest that AFH1 plays a central role in the initiation and organization of actin
cables from the pool of actin monomers bound to profilin.
- 83 -
A Two-Amino Acid Mutation in the Talin Rod Domain Inactivates the

Rod Integrin Binding Site (60)
Moes Michèle, Salsmann A., Falsetti S., Tremuth L., Melchior C., Schaffner-
Reckinger E. and Kieffer N.
Laboratoire LBPI (CNRS/GDRE-ITI), Université du Luxembourg, Luxembourg
Talin is a cytoskeletal protein that links integrins to actin filaments and plays a major
role in integrin-dependent bidirectional signal transduction. Two distinct integrin
binding sites in talin have been identified, one present in the 47 kDa talin head
domain, and a second in the talin rod domain. We have previously delimitated the
second integrin binding site to a 130 residue sequence within the rod domain
[Tremuth et al. (2004) J. Biol. Chem. 279: 22258-22266]. Here we have used a
bioinformatics as well as a molecular biology approach to further map the rod
integrin binding site. Sequence identity analysis of all cloned talin molecules revealed
an overall moderate identity of 39 % between human and drosophila talin except for
known functional domains, such as the talin rod actin binding domain (60%
sequence identity). To tentatively localize the integrin binding sequence within the
130 residue fragment, we searched for sequence stretches having at least 60%
sequence identity. This in silico approach was then validated by expressing 4
overlapping myc-tagged fragments of about 40 residues each (J1 – J4) in CHO cells
stably expressing an autofluorescent aIIbb3GFP integrin. Only one of the transfected
fragments, J4, colocalized with b3GFP in focal adhesions. An in vitro peptide overlay
assay using immobilized GST-b3 and 3 biotinylated overlapping peptides covering the
40 residue sequence allowed to narrow down the binding site to 8 residues. Finally,
all conserved residues within this octamer were mutated in fragment J by PCR
mutagenesis and expression of these mutant J fragments in CHO cells allowed to
determine that a two amino-acid mutation in the talin rod domain is sufficient to
inactivate the integrin binding site. Work is now in progress to test the effect of this
2 amino-acid mutation on recombinant full length talin function in talin-/- cells.
- 84 -
Microtubule Array Rearrangement during

Epithelial Cell Apoptosis (61)
Moss David and Lane J.D.

School of Medical Sciences, University Walk, Bristol, UK
Apoptotic cellular dynamics are thought to be driven predominantly by the

actomyosin cytoskeleton. Here we demonstrate that microtubules play an essential
role during the apoptotic execution phase. Flow cytometry and cell fragmentation
assays show that microtubules are responsible for apoptotic body formation in the
A431 cell line. Fluorescence and electron microscopy reveal that numerous extra-
centrosomal bundles of densely packed microtubules assemble in the cytoplasm of
apoptotic cells, often in the vicinity of ribonucleoprotein granules and condensed
chromatin. Live-cell imaging demonstrates that interphase microtubules rapidly
depolymerise at the start of the execution phase. Subsequently, rigid bundles of
microtubules assemble, forming long spikes that extend through the cell cortex,
driving the dispersal of chromatin-containing cell fragments. Although apoptotic
spikes contain microtubules and F-actin, only microtubules are essential for spike
formation, with actin stabilising these structures.
- 85 -
Targeted Deletion of Myotilin and Palladin Genes in Mice (62)
Moza Monica1, Wang H.-V.2, Moser M.2, Fässler R.2 and Carpén O.1,3
1
Neuroscience Program, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
2
Max Planck Institute of Biochemistry, Department of Molecular Medicine, Martinsried,
Germany
3
Department of Pathology, University of Turku and Turku University Hospital, Turku, Finland
The newly characterized myotilin, palladin and myopalladin form a small subfamily of
cytoskeletal Ig-like containing proteins. Myotilin and myopalladin show high
expression levels in the heart tissue, with highest myotilin expression observed in the
skeletal muscle cells. Both myotilin and myopalladin in heart and skeletal muscle cells
are known to localize to Z-discs, the sarcomeric region where opposite actin
filaments are cross-linked together. Palladin expression is widespread and found as
many isoforms in the embryonic and adult mouse tissues. Current updates of the
genomic DNA databases and generation of novel antibodies against palladin were
used to identify a 200 kDa new palladin isoform as component of the striated muscle
that could play important roles in maintaining the highly organized structure of
sarcomeres. In our previous work, to functionally investigate in vivo myotilin's roles,
we generated a mouse with targeted inactivation of myotilin gene. Characterized by
the protein absence in skeletal muscle and heart cells, the myotilin knockout mouse
did not develop a dystrophic phenotype. However, a well-documented role of
myotilin in cross-linking of actin filaments in concert with the major component of
the Z-disc, alpha-actinin, could point towards compensatory roles of other proteins
for an apparent normal structural and functional Z-disc in the myotilin knockout
mice. Similarly, the mouse 200 kDa palladin was ablated and phenotypic
investigation showed no gross morphologic change of the skeletal and cardiac
sarcomeric structure. To address the question of redundancy among the members of
this small subfamily of proteins, we are currently generating a mouse with targeted
disruption of both myotilin and palladin gene. The crossing of the myotilin knockout
strain with palladin knockout, produced viable double knockout mice, and the
phenotyping results will be presented at the meeting. Our aim is to unveil the
common and distinct roles of this particular palladin isoform and myotilin in the adult
skeletal muscle cells and cardiomyocytes.
- 86 -
Sequence and comparative genomics analysis of

Actin Related Proteins (63)
Muller Jean1,2, Oma Y.3, Vallar L.1, Muller A.2, Friederich E.1, Poch O.2 and
Winsor B.3
1
Laboratoire de Biologie et Génomique Structurales, Institut de Génétique et de Biologie
Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch cedex, France
2
Laboratoire de Biologie Moléculaire, d'Analyse Génique et de Modélisation, Centre de
Recherche Public-Santé, 42, rue du Laboratoire, L-1911, Luxembourg, Luxembourg
3
UMR 7156 Département “Génétique Moléculaire et Cellulaire“ Institut de Physiologie et de
Chimie Biologique, 21, rue René Descartes, 67084 Strasbourg cedex, France
Actin Related Proteins (ARPs) are key players in cytoskeleton activities, the ARP2/3
complex in actin dynamics and ARP1 and ARP11 in microtubule-based vesicle
trafficking, while ARP4-ARP9 are components of many chromatin modulating
complexes. Conventional actins and ARPs co-define a large family of homologous
proteins, the actin superfamily, with a tertiary structure known as the actin fold.
Since ARPs and actin share high sequence conservation, clear family definition
requires distinct features to easily and systematically identify each subfamily.
Numerous new ARP sequences in protein databases motivated us to perform an in
depth sequence and comparative genomic analysis of ARP subfamilies. Based on
high quality Multiple Alignment of Complete Sequences of ~700 proteins homologous
to actin including 148 ARP sequences, we extended the ARP family classification to
new organisms. Sequence alignments revealed conserved residue, motif and inserted
sequence signatures to define each ARP subfamily. We developed ARPAnno
(http://bips.u-strasbg.fr/ARPAnno and mirrored at www.bioinfomatics.lu.) a freely
available web server dedicated to the annotation of ARP sequences. Analyses of
sequence conservation highlight part of the actin fold and suggest perspectives of
interactions between ARPs and Actin Binding Proteins. Finally, genomic sequence
exploration allowed us to define the distribution of ARPs among eukaryotic phyla,
emphasizing the central importance of nuclear ARPs, particularly the multifunctional
ARP4.
- 87 -
Involvement of Motility-and Adhesion-Related Genes in

Tumor Metastasis (64)
Naffar-Abu-Amara Suha and Geiger B.

Molecular Cell Biology Department, The Weizmann Institute of Science, Rehovot, Israel
The objective of our work is to identify and characterize new specific tumor-
associated genes that are specifically involved in regulating the metastatic properties
of cancer cells, by affecting their migratory or adhesive activity. Our screen based on
the classical 2D motility phagokinetic track assay. We develop a high throughput
screening protocol based on the use of 96 wells plate covered with latex-beads. Cells
are incubated at 37°C for 5-8 hours, then fixed with 3% paraformaldehyde. The
migrating cells ingest or push the beads located along their migratory pathway and
leave free area as a permanent record of their movements. The phagokinetic tracks
were recorded using a home-built cell-screening digital microscope system and laser
based auto-focusing device. For our application images are taken at adjacent fields
so we can fuse them to view a montage of the entire well. Analysis program was
developed also to analyze and calculate cell shape as well as velocity and persistence
of cell migration. This system gives a reliable detection and quantitative analysis of
cell motility changes as a result of large scale of perturbations, gene overexpression,
siRNA, and drugs that may be applied to the cells. Our screen is done on MCF-7
cells, poorly metastatic and non migrating mammary tumor cell line infected with
cDNAs library packed in retroviral vector (pEYK) of MDA-MB-231, highly migrating
and metastatic breast carcinoma cell line. Infected MCF7 cells are plated on the
beads covered 96 wells plate. MCF-7 cells with increased migration velocity can be
detected and the migration promoting genes will be cloned, identified and
characterized.
- 88 -
Biochemical, Cell Biological, and Structural Analysis of

Mouse Coactosin (65)
Naumanen Perttu1, Hellman M.2, Paavilainen V.O.1, Annila A.2, Permi P.2 and
Lappalainen P.1
1
Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Finland
2
Program in Structural Biology and Biophysics, Institute of Biotechnology, University of
Helsinki, Finland
Coactosin is a small evolutionarily conserved actin filament binding protein. It shows

remote sequence homology to ADF-H family of actin regulatory proteins, including
ADF/cofilins, twinfilins, and Abp1/drebrins. However, the biochemical activity of
coactosin as well as its role in actin dynamics have been poorly understood. By
Northern blot and in situ hybridization studies, we revealed that coactosin is widely
expressed in non-muscle tissues of developing and adult mouse. Interestingly,
coactosin is strongly expressed in certain highly polar epithelial and neuronal cells
including the gastric epithelial cells, cells of the renal cortex and the cerebellar
Purkinje cell layer. Immunofluorescence studies showed that in cultured mammalian
cells coactosin localizes to specific actin filament structures, including actin stress
fibers. Furthermore, these studies also identified a putative coactosin binding
partner, which may regulate coactosin’s localization to actin stress fibers. To
elucidate the mechanisms by which coactosin regulates actin dynamics, we
determined the structure of mouse coactosin by NMR. Coactosin shows structural
homology to cofilin and the amino terminal ADF-H domain of twinfilin, but the
proteins also have certain key differences that might explain their different
biochemical activities. Structural comparison also revealed that residues critical for
actin filament binding in yeast cofilin are structurally conserved in coactosin. Site-
directed mutagenesis of these residues diminished the actin binding activity of
coactosin in vitro and altered its subcellular localization in vivo. Together, these data
show that coactosin and cofilin bind actin through an evolutionarily conserved
molecular interface, but that these proteins play distinct roles in regulation of actin
dynamics in cells.
- 89 -
Regulation of Mitochondria Form and Distribution

by Fibronectin (66)
Nekrasova Oksana E.1, Minin An.A.1, Kulik A.V.3 and Minin A.A.2
1
N.K. Koltsov's Institute of Developmental Biology, 119991 Moscow, Vavilova 26, Russia
2
Institute of Protein Research RAS, 119334 Moscow, Vavilova 34, Russia
3
Moscow Institute of Physics and Technology, 141700 Moscow region, Dolgoprudny,
Institutsky pereulok 9, Russia
This study deals with distribution and morphology of mitochondria in cultured cells
that depend on cytoskeleton structures and are controlled by external factors.
Fibronectin is one of the main components of extracellular matrix (ECM) that
interacts with the receptors on the cell surface and determines the shape of the
cellular cytoskeleton. It was shown earlier that different parts of fibronectin molecule
bind to different receptors which cause rearrangements in cytoskeleton. Thus it was
demonstrated that while cell-binding domain of fibronectin interacting with integrins
was important for cell attachment, stress fibers and focal contacts were induced in
cells by a heparin-binding domain. We suggest that some of these rearrangements
play role in mitochondria shape and distribution. To investigate the possible role of
fibronectin in these processes we have developed a model based on covalent binding
of this protein or its fragments to activated glass and cell cultivation on such
modified surfaces in serum free medium. Cells with fluorescently tagged
mitochondria spread on glass coverslips covered with fibronectin or its 120 kD
fragment that contained cell-binding domain but were unable to do so on its 40 kD
fragment containing heparin-binding domain. However, mitochondria in these cells
were localized in perinuclear region and had round grain-like morphology. We have
found that addition of serum, fibronectin or 40 kD fragment solution induced the fast
distribution of mitochondria to the periphery and caused the elongation of these
organelles. At the same time 120 kD fragment of fibronectin had no such an effect
on mitochondria. These data indicate that interaction of heparin-binding domain of
fibronectin with receptors on the cellular surface induce cytoskeleton rearrangements
indispensable for mitochondria distribution.
- 90 -
Cas Proteins and the Integration of Signal Transduction with

Cell Morphology (67)
Bargon S.D.1, Cowell L.1, Bac C.1, Zhong J.1, Andrew S.1, Gunning P.W.1 and O'Neill
Geraldine2
1
Oncology Research Unit, The Children's Hospital at Westmead, NSW 2145 Australia
2
The Discipline of Paediatrics and Child Health, University of Sydney, NSW 2006
It is clear that integrin-based adhesion sites contribute to the co-ordination of both

signalling to the nucleus and cellular morphology. Complex networks of signalling
molecules are found to localize to adhesion sites, yet how cells achieve spatially and
temporally distinct signalling through these sites is not clear. The Cas family of
proteins are a family of docking molecules that contain multiple protein-protein
interaction domains and can localize to focal adhesions. Based on their structure and
sub-cellular localization it is likely that these molecules are at least one component
that integrates cellular morphology with signals transduced through adhesion sites.
We have been studying the Cas family protein, HEF1, to determine whether this
molecule integrates signals at adhesion sites. To understand the role of HEF1, we
have been examining the sub-cellular localization of HEF1 under different adhesion
conditions, the affect of HEF1 on cell shape and the post-translational regulation of
HEF1 phosphorylation. Using cultured breast cancer and neuronal cells, we find that
HEF1 associates with multiple adhesion structures and can promote differing
morphologies, dependent on cell context. We further find that the regulation of
HEF1 phosphorylation correlates with altered cellular morphologies and propose that
altered phosphorylation of HEF1 contributes to the function of this molecule. We are
currently testing the role of HEF1 phosphorylation in signalling and shape changes
down-stream of this molecule.
- 91 -
Mutational Analysis of C. elegans AIP1 (UNC-78) in Disassembly

of ADF/Cofilin-Bound Actin Filaments In Vitro and
Actin Cytoskeletal Organization In Vivo (68)
Mohri K., Ono K., Yu R. and Ono Shoichiro

Department of Pathology, Emory University, Atlanta, GA 30322, USA
Actin-interacting protein 1 (AIP1) is a conserved WD-repeat protein that enhances

actin filament disassembly in the presence of ADF/cofilin. Involvement of AIP1 in
actin reorganization has been demonstrated in several different model systems. In C.
elegans, AIP1 is encoded by the unc-78 gene and is required for organized assembly
of actin filaments in muscle cells. Recent biochemical studies on AIP1 have shown
that AIP1 caps the barbed ends and also actively disassembles ADF/cofilin-bound
actin filaments. Crystal structure of AIP1 revealed two seven-bladed beta-propeller
domains, and we identified five functional surface residues in the N-terminal
propeller that are involved in disassembly of ADF/cofilin-bound actin filaments but
not in barbed-end capping [Mohri et al. (2004), J. Biol. Chem. 279: 31697-31707].
However, cellular functions of capping and disassembly activities of AIP1 are
unknown. We found a novel missense mutation in the unc-78 gene from a C. elegans
mutant that was isolated by Zengel and Epstein [(1980), Cell Motil. 1: 73-97]. This
mutation changes a surface residue in the N-terminal propeller, and the mutant
worms exhibited severe disorganization of the actin filaments. Biochemical analysis
of this mutant UNC-78 protein showed that the activity to disassemble ADF/cofilin-
bound filaments was significantly reduced, and the capping activity was impaired.
This is the only mutant with impaired capping activity and suggests the location of
critical surface for capping. Furthermore, we found that expression of GFP-tagged
UNC-78 in the unc-78-null mutant could rescue the mutant phenotype and restore
nearly normal actin organization. Using this system, we are currently testing in vivo
functions of mutant forms of UNC-78 as GFP-fusion proteins.
- 92 -
Actin Cytoskeleton Dynamics via SH3 Domain Protein Interactions:

Development of a New Caged Fluorophore for
Spatio-Temporal Studies (69)
Orange Clélia1,2, Specht A.1, Sakr E.1, Goeldner M.1 and Winsor B.2
1
UMR7514/CNRS 74 route du Rhin, 67400 Illkirch, France
2
FRE2375/CNRS 21 rue René Descartes, 67000 Strasbourg, France
The actin cytoskeleton dynamic is crucial for multiple inter-related cellular

phenomena such as motility, endocytosis, polarized morphogenesis and cytokinesis.
Signals coming from Rho/Rac GTPases implicate multiples proteins including WASP
(Wiskott-Aldrich Syndrome Protein) and the Arp2/3 complex to polymerize actin. SH3
(Src homology 3) domain containing proteins interact with proline-rich proteins, such
as Las17p, yeast WASP homologue, verprolin and the type myosins. Our goal is to
synthesize and develop a new fluorescent probe : a caged coumarin able to interact
with histidine tags on proteins of interest. After light activation in a limited area of
the cell, the tagged protein could be tracked and followed by time lapse fluorescence
microscopy. This new system could be used to study in time and space the SH3
domain proteins interacting with the actin polymerization activation complex. The
synthesis and initial testing of the caged compound will be presented.
- 93 -
Palladin Binds Directly to F-Actin and Cross-Links

Actin Filaments (70)
Rachlin A.S.1 and Otey Carol A.1,2

1
Department of Cell and Molecular Physiology
2
Neuroscience Center, University of North Carolina, Chapel Hill, NC 27599-7545, USA
Palladin is a multi-isoform protein that localizes to actin-rich structures, including

stress fibers, focal adhesions, cell-cell junctions and the Z-line of cardiac myocytes.
Palladin's role as a potent cytoskeletal scaffold is supported by an emerging list of
cytoskeletal proteins with which it interacts. This group includes the f-actin
crosslinking protein alpha-actinin, proteins with well-defined roles in actin
polymerization, such as VASP and profilin, and the protein Lasp-1, which plays an
important role in cellular motility. All of these proteins form direct associations with
filamentous and/or monomeric actin. The overexpression of palladin in a variety of
cultured cells has been shown to induce the formation of unusually large, hyper-
bundled stress fibers or star-like structures. We set out to understand if these
bundles are generated indirectly, as the result of palladin’s interactions with its
binding partners, or if palladin also has direct effects on actin organization. To
address this question, baculovirus expression technology was used to generate
purified palladin, and differential centrifugation was used to determine if palladin
binds directly to F-actin and co-fractionates with actin pellets. The results showed
that when palladin was mixed with actin in a 10:1 molar ratio (actin:palladin),
virtually all of the palladin pelleted with F-actin. In addition, purified palladin was
found to crosslink individual actin filaments into higher order bundles, as determined
both by fluorescence microscopy and differential centrifugation. These results
suggest that palladin's overexpression phenotype of actin bundling may be due in
part to its own independent crosslinking activity. Taken together, the evidence to
date indicates that palladin is both an actin-binding protein and a scaffold for the
recruitment of multiple other actin-binding proteins of widely varying activities,
suggesting that this one molecule may occupy an unusual functional niche in
regulating actin assembly in cells.
- 94 -
Artificial Lipid Vesicles Accumulate in a Cell Plate Region (71)
Ozdoba Agnieszka, Emons A.M.C. and van Lammeren A.M.M.

Laboratory of Plant Cell Biology, Wageningen University, The Netherlands
For cytokinesis in plant cells, Golgi vesicles fuse into a net-like structure and then
into a more or less rigid plate consisting of vesicle-derived membrane at the outside,
and vesicle content, matrix cell wall material, at the inside. It is generally thought
that these vesicles are released from Golgi bodies and are transported to the forming
cell plate, aided by the cytoskeleton. We study which properties the vesicles need to
have in order to be transported to and to fuse together in the cell plate. We
developed a bioassay to microinject fluorescently labelled synthetic ‘vesicles’ of which
we can change parameters like size, number, content, composition, coating etc., into
dividing cells of Tradescantia virginiana stamen hairs. We observed that
microinjected polystyrene beads with diameter of 40 nm did distribute through the
cytoplasm and accumulated in the phragmoplast cytoskeleton but did not accumulate
in the cell plate region. On the contrary, synthetic lipid vesicles made of
dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) with
diameters of 45-150 nm did distribute and accumulate in the region of the cell plate.
Whether fusion of those vesicles occurs, is under investigation. The difference in the
behaviour of polystyrene beads and lipid vesicles can be caused by their nature. Both
lipid vesicles and polystyrene beads are taken up into cytoplasmic streaming, after
injection. Our explanation is that vesicles and polystyrene beads are coated with
cytoplasmic motor proteins and transported to the cell plate area, or that
hydrodynamic flow plays a role in their transport.
- 95 -
The Rho Family GTPase Rif Induces Filopodia Through mDia2 (72)
Pellegrin Stéphanie and Mellor H.

Department of Biochemistry, University of Bristol, Bristol, UK
Rho family GTPases are critical regulators of the actin cytoskeleton and individual
Rho GTPases activate distinct signalling pathways, leading to specific actin
reorganisation. Of the 23 mammalian Rho GTPases identified so far, three have been
well characterised: RhoA, Rac1 and Cdc42. We have isolated a novel Rho-family
GTPase, Rif (Rho in filopodia), which is present in chordates including ascidians.
When transiently expressed in mammalian cells, Rif is localised at the plasma
membrane and induces long actin-rich filopodia. The Rho GTPase Cdc42 is a key
mediator of filopodia formation. Cdc42 may generate filopodia by regulating CRIB
motif-containing effectors including WASP proteins, IRSp53 and mDia2. Rif induces
filopodia independently from Cdc42 and does not bind CRIB motifs. However, Rif
interacts with the formin mDia2 and mDia2 is required for Rif-induced filopodia
formation. The CRIB motif of mDia2 is not required for its interaction with Rif. Also,
the filopodia generated by Cdc42 are morphologically different to those induced by
Rif. Thus Rif and Cdc42 represent two distinct routes to the induction of filopodia,
producing structures with both shared and unique properties.
- 96 -
CEACAM3-Mediated Phagocytosis of Human Pathogens Requires

Cytoskeletal Dynamics and Rac Activation (73)
Schmitter T., Pils Stefan and Hauck C.R.

Research Center for Infectious Diseases, University Würzburg, Würzburg, Germany
Several human pathogens utilize carcinoembryonic antigen-related cell adhesion

molecules (CEACAMs) such as CEA, CEACAM1, or CEACAM6 to interact with the
apical surface of mucosal epithelial cells of their host. Interestingly, these CEACAM-
binding pathogens of the genera Neisseria , Moraxella and Haemophilus can be
recognized and phagocytosed by cells of the innate immune system in the absence
of opsonizing antibodies or complement factors. In line with this notion, we recently
identified the granulocyte-specific isoform CEACAM3 as an efficient, opsonin-
independent phagocytic receptor for CEACAM-binding microorganisms. In contrast to
CEACAM1 or CEACAM6, bacterial engagement of CEACAM3 triggers membrane
recruitment and increased GTP-loading of the small GTPase Rac. Stimulation of Rac
depends on the integrity of a cytoplasmic ITAM motif in CEACAM3 and requires Src
family protein tyrosine kinase activity. Transduction of isolated primary human
granulocytes with a TAT-fusion protein of dominant-negative Rac (RacN17), but not
a dominant-negative version of the closely related GTPase Cdc42, severely interfered
with phagocytosis of CEACAM-binding pathogens. Furthermore, blockage of
CEACAM3, but not CEACAM6, by monoclonal antibodies reduced uptake and killing
by primary granulocytes. Current investigations into the upstream regulators and
downstream effectors of GTP-bound Rac critical for CEACAM3-mediated phagocytosis
of human pathogens will be discussed. Together, our data assign an important
function to the orphan receptor CEACAM3 in the control of human-specific pathogens
by the innate immune system.
- 97 -
The Actin Binding Protein ABP1 Interacts With N-WASP and this
Complex May Link Actin Dynamics to Endocytosis (74)
Pinyol Roser, Koch A., Qualmann B. and Kessels M.M.

Leibniz-Institute for Neurobiology, Magdeburg, Germany
We have identified and characterized several proteins at the functional interface

between endocytosis and the actin cytoskeleton. Among those is mammalian Abp1, a
signal-responsive F-actin-binding protein that is involved in membrane trafficking
processes by an association with the GTPase dynamin controlling vesicle formation.
Our most recent data show that Abp1 does not only associate with polymerized actin
fibers, but furthermore suggest a role for Abp1 in controlling actin filament
polymerization via an interaction with N-WASP, a potent stimulator of the Arp2/3
complex actin nucleation machinery. Affinity purifications demonstrate that Abp1
binds to the proline-rich domain of N-WASP via its SH3 domain. The mutation of two
residues in the SH3 domain abolished the association of Abp1 with N-WASP.
Coimmunoprecipitation studies and reconstitution of the N-WASP/Abp1 complex at
intracellular membranes demonstrate the in vivo relevance of this interaction.
Immunofluorescence microscopy studies in primary hippocampal neurons showed a
colocalization of Abp1 and N-WASP at actin-rich sites. In vitro reconstitution assays
indicate that Abp1 is capable to induce actin polymerization by regulating the activity
of N-WASP in an SH3 domain-dependent manner. The Abp1 SH3 domain coated to
the surface of beads was sufficient to promote actin nucleation and polymerization in
homogenates while the mutant N-WASP binding domain did not. Furthermore, the
Abp1 SH3 domain acts synergistically with the GTPase Cdc42 to stimulate N-WASP-
dependent activation of Arp2/3 complex-mediated actin filament nucleation and
polymerization, as we demonstrate in fluorimetric analyses of actin polymerization
using purified components. We propose that the binding of Abp1 promotes the open
conformation of N-WASP, allowing N-WASP to activate the Arp2/3 complex and
thereby promoting actin polymerization. These findings, together with our previous
characterization of N-WASP as an important endocytic component, suggest that actin
polymerization during endocytosis is an Arp2/3 complex-based mechanism controlled
by proteins such as Abp1.
- 98 -
The Adhesion Strength between Fibroblasts and Myofibroblasts (75)
Pittet Philippe, Meister J.J. and Hinz B.

Laboratory of Cell Biophysics, Swiss Federal Institute of Technology, Lausanne, Switzerland
Fibroblast to myofibroblast modulation is characterized by expression of -smooth

muscle actin (-SMA) and represents a crucial step in granulation tissue contraction
during wound healing. We have recently demonstrated that myofibroblasts develop
cadherin-type cell-cell adherens junctions (AJs), which join stress fibers over the cell
membrane. Cadherin expression changes from N-cadherin to OB-cadherin upon TGF-
induced myofibroblast differentiation in culture and coincides with the enlargement
of AJs. We here show that myofibroblast AJs exhibit a higher mechanical resistance
compared with that of -SMA-negative fibroblasts by measuring the binding strength
between two cells. 1) By separating two suspended cells placed in contact with laser
tweezers, we revealed two types of Ca2+-dependent adhesion bonds with different
strength. The first bond exhibited a breaking force of 5.5 ± 1.5 nN and existed in
both, fibroblasts and myofibroblasts. The strength of the second bond differed
between the two cells, breaking at 18.2 ± 0.8 nN in fibroblasts and at 23.0 ± 1.5 nN
in myofibroblasts. Measuring the adhesion force between two plated cells by atomic
force microscopy after short contact grossly confirmed these observations. Since no
cyoskeletal reinforcement was involved in these experimental conditions, we suggest
that these differences represent the different cadherins involved in (myo)fibroblast
adhesion. 2) Subjecting suspended cells that were attached to plated cells to
hydrodynamic forces in a flow chamber demonstrated ~18% higher adhesion of
myofibroblasts over fibroblasts at shear forces of 4 Nm-2. This higher adhesion was
reduced to the level of fibroblast attachment by a peptide that inhibits -SMA-
mediated contraction, indicating AJ reinforcement by -SMA–positive stress fibers.
To conclude, high cell-cell adhesion of myofibroblasts appears to be determined by
their specific cadherin pattern and by -SMA-mediated mechanical reinforcement of
AJs.
- 99 -
Characterising the Role of WASP/WAVE Proteins in

Cell Migration In Vivo (76)
Pocha Shirin, Stramer B., Martin P. and Cory G.

Department of Biochemistry, University of Bristol, UK
The actin cytoskeleton is crucial for eukaryotic cell motility and morphology. In vitro
studies have shown that numerous protein are involved in regulating actin dynamics,
amongst which are members of the WASP/WAVE family. Tissue culture studies have
implicated WASP/WAVE proteins as key regulators of cell migration, however their
precise physiological relevance has not been elucidated. To investigate the
physiological importance of these proteins, we are using Drosophila as a genetically
tractable model system. Previous research into the roles of WASP and SCAR,
(Drosophila WAVE homologue) carried out in Drosophila, identified defects in bristle
formation and axon morphology respectively, indicating an actin-related defect. In
order to directly assess the physiological role of WASP/WAVE proteins in cell
migration, we are using live imaging of the inflammatory response in Drosophila
embryos. This technique, pioneered to investigate the roles of small GTPases during
cell migration, enables us to visualise the ability of WASP/SCAR mutant hemocytes
( Drosophila macrophages) to migrate to the site of a highly reproducible laser
wound. Using this technique, we plan to look at many other cytoskeletal regulators.
- 100 -
Separation of Hepatoma Morris 5123 Cells with

Higher Migration Ability (77)
Popow Agnieszka, Kozera L., Nowak D. and Malicka-Blaszkiewicz M.

Department of Cell Pathology, Institute of Biochemistry and Molecular Biology, Wroclaw,
Poland
Tumor cells must mobilize a complex network of signals associated with extracellular
matrix digestion and cytoskeleton architecture dynamic remodeling, in order to get
through the barrier of the vessel walls in the process of intravasion. In addition to
this, the correlation between metastatic potential of the cancer cells and the
subcellular localization of certain proteins has been widely studied, however it is not
entirely characterized yet. It has been suggested lately that there may exist a
relationship between the overexpression of beta actin and it’s membrane crosslinker
ezrin, and metastatic potential of tumor cells. Our research has been focused on the
isolation of the highly motile cells fraction from the hepatoma Morris 5123
population. Cells which underwent several migration cycles through the Matrigel -
coated filters were successfully cultured. The invasion factor was determined by
means of Matrigel invasion assay. We have noticed a statistically significant increase
in the values of invasion factors for the independent fractions isolated in successive
separation steps. The invasion factor was the lowest for the parental population. The
considerable changes in the cell shape were accompanied by the reorganization of
the actin cytoskeleton structure - including noticable, subcortical congestion in the
distribution of ezrin and actin. The visualization of these two proteins in tumor cells
was performed by immunofluorescence staining and fluorescent confocal microscopy.
- 101 -
Effect of Gadolinium Chloride, an Inhibitor of Stretch-Activated

Channels, on Focal Adhesions and the Associated
Actin Cytoskeleton (78)
Prager-Khoutorsky Masha, Bershadsky A. and Geiger B.

Department of Molecular Cell Biology, Weizmann Institute, Rehovot, Israel
Cell adhesion to the extracellular matrix plays a critical role in a variety of

fundamental biological processes, such as cell migration, embryonic development,
and wound healing, and in pathological processes such as cancer invasion and
metastasis. Cells adhere to the extracellular matrix (ECM) via dynamic molecular
complexes known as focal adhesions that physically connect ECM molecules with the
cytoskeleton. In addition, focal adhesions operate as signal transduction organelles
“informing” cells about the chemical composition and mechanical properties of the
ECM. Recent studies revealed that integrin-containing focal complexes and focal
adhesions behave as “mechanosensors” responding by directional assembly to local
force. While a great deal of information, concerning the molecular composition and
molecular assembly of focal adhesions is available; little is known about the cascade
of events that links the mechanical tension and the assembly process. We began to
examine the hypothesis that stretch-activated calcium channels are involved in the
regulation of focal complexes. Using NIH3T3 fibroblasts, we investigated the effect of
gadolinium chloride, a blocker of stretch-activated channels, on focal adhesions and
actin cytoskeleton. We found that focal adhesion components such as vinculin,
paxillin and zyxin are removed from the focal adhesion as result of gadolinium
application in a time- and concentration-dependent manner. In contrast, a-actinin
accumulates in the periphery of the cell following gadolinium application. Actin
cytoskeleton is less affected by gadolinium, and the organization of stress fibers is
preserved even in cells that completely loss vinculin, paxillin and zyxin in focal
adhesions. Our results suggest that stretch-activated channels are involved in the
regulation of focal adhesion assembly and maintenance.
- 102 -
Effect of Src Kinase on Localisation of Actin and Cadherin in

MDCK Cells Grown in Three-Dimensional Environment (79)
Rahikkala Mira, Vuoristo J. and Eskelinen S.

Biocenter Oulu and the Department of Pathology, University of Oulu, University of Oulu, Oulu,
Finland
The effects of Src tyrosine kinase was analysed using temperature sensitive (ts)-Src-
transformed Madin-Darby canine kidney (MDCK) cells grown within a gel of growth
factor reduced Matrigel. The cells were cultivated for 5 days, fixed and stained for
actin and cadherin. The morphology of cell cysts and the protein distribution were
visualised using an Olympus confocal microscope. At permissive temperature (35°C)
the cells formed clusters without any orientation or signs of polarisation. Actin and
cadherin delineated all cell walls. The process of polarisation was analysed by
shifting the cells grown in 3D gel at permissive temperature for 5 days to non-
permissive (40.5°C) temperature for 1 to 4 hours. Only 1 h after the shift to non-
permissive temperature, the formation of small lumina could be seen within the cell
cluster. Actin began to accumulate towards the apical side of the lumina. Cadherin
delineated the lateral walls of the cells facing matrix. The effect of temperature shift
was compared to treatment of the cells with specific Src-inhibitor pp2 at permissive
temperature. After one hour treatment there was no lumen formation, and the cells
remained an irregular cluster. It seems that shutting down the Src kinase by
temperature shift was beneficial to the cells and improved the cell polarity, whereas
the Src inhibitor impaired the cell morphology. Hence, the factors behind the
mesenchymal – epithelial transition are more complex than simply inhibiting the
phosphorylation activity of Src kinase. At the moment we are investigating the
changes in gene expression in Src-transformed cells during mesenchymal-epithelial
transition with RNA-microarray technique.
- 103 -
Study of Formin Homology Domains FH1 and FH2 of mDia1 (80)
Renault Louis, Romero S., Didry D., Pantaloni D. and Carlier M.F.
Dynamique du Cytosquelette et Motilité Cellulaire, Laboratoire d’Enzymologie et Biochimie
Structurale, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France
Formins are conserved multidomain proteins that nucleate actin filaments and bind
to the filament barbed end (Zigmond SH. (2004) Curr Opin Cell Biol. 16, 99-105).
They lead to the formation of unbranched filaments that are bundled into structures
found in yeast actin cables, actin stress fibers and the cytokinetic ring. Formins share
an N-terminal Rho-GTPase binding domain (GBD), conserved formin homology
domains FH1 (Proline rich domain) and FH2 (actin binding domain) which initiate
actin assembly, and a C-terminus which interacts with the GBD in the absence of
activated Rho GTP-binding proteins and thus auto-inhibits the capacity to initiate new
filaments. It has recently been shown that, in association with profilin and actin, FH1
and FH2 domains form together a processive motor that catalyzes and steers
filament assembly at the barbed end, remaining bound to the rapidly growing
filament [Romero et al. (2004), Cell 119: 419-429). FH1 and FH2 domains for their
processive walk accelerate ATP hydrolysis on actin during filament assembly. We are
investigating the mechanism of this processive machinery by structural studies.
Preliminary results will be shown on the expression and characterization in solution
of fragments of mDia1 containing FH1 and FH2 domains.
- 104 -
What is the Role of Villin in Cell Dynamics ? (81)
Revenu Céline, Louvard D. and Robine S.

Institut Curie, Paris, France
Villin is an actin-binding protein (ABP) of the gelsolin family mainly expressed in the
epithelial cells of the intestine and the kidney proximal tubule where it associates
with the actin bundles supporting the microvilli. Among all ABPs, villin is the only one
to present in vitro bundling, capping, nucleating and severing activities towards actin
filaments depending on the calcium concentration. A structural role for villin in the
formation and maintenance of microvilli has been demonstrated ex vivo and is linked
to its bundling property. The knock-out of the gene encoding the villin protein in
mice did not affect the organisation of the microvilli. However, by using different
stresses that increase intracellular calcium concentration, we revealed an essential
function for villin in tissue plasticity and repair. We hypothesised that villin could be
essential in cytoskeleton remodelling of intestine epithelial cells. Indeed, they have
large amount of apically concentrated actin bundles that need to be rapidly broken
down in order for the cell to depolarise and rebuild its cytoskeleton towards a motile
morphology. This could be achieved through villin severing property. In order to test
the importance of this activity, we designed a mutant of villin based on sequence
comparison with the CapG protein. This set of mutations strongly affects the
severing activity as tested by pyrene-actin assay but do not decrease the ability to
nucleate, cap and bundle filaments. The impact of this loss of function will be tested
in vivo by trying to rescue the lack of plasticity of the villin knock-out mice. Whereas
wild type villin should be able to rescue the phenotype, the severing mutant should
not.
- 105 -
Inducing Viscoelastic Retraction of F-Actin Stress Fibers In Vivo (82)
Reynaud Emmanuel, Colombelli J., Rietdorf J., Stelzer E.H.K. and Pepperkok R.
Intracellular tension is considered an important determinant of cell shape, motility,

and cellular responses to biochemical and biophysical signals. However, many details
about cytoskeletal tension remain poorly understood because these forces cannot be
directly measured in living cells. We report on the manipulation of actin stress fibers
using a nanosurgery system based on a short pulsed UV laser optimized for the
localized severing of biological polymers. Laser nanosurgery allows severing of
selected actin stress fibers without affecting cell viability. The induced fiber motion
can be analyzed and fitted to viscoelastic models. This points out a double
exponential retraction phenomenon. We discuss the existence of a frictional
coefficient characterizing the hydrodynamic coupling of the filaments to the
surrounding network. Furthermore, using a fringe FRAP (Fluorescence Recovery After
Photobleaching) approach, we can measure the entire fiber dynamic of retraction to
estimate viscoelastic parameters. We propose this method as a new and regional
way to quantify actin forces and dynamics.
- 106 -
Formin is a Processive Motor that Requires Profilin to Accelerate

Actin Assembly and Associated ATP Hydrolysis (83)
Romero Stéphane1, Le Clainche C.1, Didry D.1, Egile C.2, Pantaloni D.1 and
Carlier M.F.1
1
Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France
2
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, U.S.A.
Formins are initiators of actin assembly that are thought to remain bound to the
barbed ends of growing filaments by an unknown mechanism. Using the conserved
FH2 and FH1-FH2 domains of formin mDia1 as models, we show that FH1-FH2, but
not FH2, drives processive polymerization of profilin-actin. Profilin is required for and
takes part in the processive motor function. Formin increases 15-fold (above the
diffusion limit) the rate constant for association of profilin-actin to barbed ends,
accelerates the hydrolysis of ATP coupled to profilin-actin polymerization, and uses
the derived free energy for processive polymerization. Single filaments grow at least
10 m long (several thousand subunits) from formin-bound beads without detaching.
Propulsion of formin-coated beads is reconstituted in solutions of actin, ADF and
profilin that support fast treadmilling, at the rapid rates displayed by formin-driven
processes in vivo. Transitory formin-associated processes are generated by poisoning
of the processive cycle by capping proteins.
- 107 -
Characterization of Alpha-Skeletal Muscle Actin Mutants Causing

Nemaline Myopathy: Expression in Cultured Skeletal Muscle
Cells Causes Various Aberrant Phenotypes (84)
Rommelaere Heidi1, Constantin B.2, Waterschoot D.1, Vandekerckhove J.1 and

Ampe C.1
1
Department of Medical Protein Research, Flanders Interuniversity Institute for
Biotechnology (VIB) and Department of Biochemistry, Faculty of Medicine and Health
Sciences, Ghent University, Ghent, Belgium
2
Institut de Physiologie et Biologie Cellulaire, CNRS, UMR-6187, Pôle Biologie Santé,
Universtité de Poitiers, Poitiers, France
Mutations in the gene encoding alpha-skeletal muscle actin, ACTA1, cause congenital
myopathies of various phenotypes. We conducted a biochemical analysis on 35
nemaline myopathy-causing point mutants (1, and unpublished results) and found
various defects in actin. A few mutants have folding defects, and display increased
binding to the chaperones prefoldin and CCT (cytosolic chaperonin containing TCP-
1), of which the latter is strictly required for the proper folding of actin (2). Some
other mutants, altered in or near the ATP-binding pocket, are unstable resulting in
an increased binding to the cyclase associated protein, CAP (3). Finally some
mutants display polymerisation defects, whereas others behave normally. These
observations were complemented by analysing the incorporation of 18 of these actin
mutants into endogenous cytoskeletal structures of fibroblasts (1). In some cases
disease associated cellular phenotypes, e.g. rod formation in muscle cells were
mimicked. Recently, we also expressed these mutants in skeletal muscle cells in
culture and analysed the incorporation and/or the morphological effects of the actin
mutants in differentiated myotubes. For a few mutants we observed apparently
normal incorporation into developing myofilaments. The majority of the mutants,
however, displayed different defects ranging from altered morphology of myotubes,
deformation of the myotube wall (blebbing) to formation of unusual myotube tips. At
the subcellular level some of the mutants cause aggregation of myofilament
structures and/or accumulation of actin rods in the cytoplasm or in nuclei.
References
1. Costa C.F., Rommelaere H., Waterschoot D., Sethi K.K., Nowak K.J., Laing N.G.,
Ampe C. and Machesky L.M. (2004) J Cell Sci. 117: 3367-3377.
2. Rommelaere H., Van Troys M., Gao Y., Melki R., Cowan N.J.,Vandekerckhove J. and
Ampe C. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 11975-11979.
3. Rommelaere H., Waterschoot D., Neirynck K., Vandekerckhove J. and Ampe C.
(2003) Structure 11: 1279-1289.
- 108 -
Role of Emerin in the Association of the MTOC With the Nucleus (85)
Salpingidou Georgia, Rzepecki R., Simon B. and Hutchison C.

Department of Biological Sciences, University of Durham, UK
The function of emerin was investigated by the use of Xenopus cell-free extracts.
Four deletion mutants of human emerin consisting of residues 1-70, 1-176, 1-220
and 73-180 were created and inoculated into Xenopus egg extracts in order to
investigate their effects on the assembly of sperm pronuclei. Experiments showed
that emerin peptides containing the LEM domain were able to inhibit sperm
decondensation and nuclear envelope assembly while peptides without the LEM
domain had no effect on nuclear assembly. Emerin mutants were subsequently used
in pull-down experiments in an attempt to identify interacting proteins in the
Xenopus cytosol. Mass spectrometric analysis identified beta-tubulin as an emerin
binding protein. To further investigate the role of the emerin-tubulin interaction
immunofluorescence analysis on normal Human Dermal Fibroblasts (HDF) and
emerin-null HDF derived from X-EDMD patients with a beta-tubulin specific antibody
was performed. Experiments revealed a localisation of the MTOC away from the the
nucleus in X-EDMD cells suggesting a role for emerin in the anchorage of the MTOC
to the nuclear envelope.
- 109 -
A New Functional Role of the Fibrinogen RGD Motif as the Molecular

Switch that Selectively Triggers Integrin aIIbb3-Dependent RhoA
Activation during Cell Spreading (86)
Salsmann Alex, Schaffner-Reckinger E. and Kieffer N.

Laboratoire de Biologie et Physiologie Intégrée (CNRS/GDRE-ITI), Université du Luxembourg,
Luxembourg
A number of RGD-type integrins rely on a synergistic site in addition to the canonical

RGD site for ligand binding and signaling, although it is still unclear whether these
two recognition sites function independently, synergistically or competitively.
Experimental evidence has suggested that fibrinogen binding to the RGD-type
integrin aIIbb3 occurs exclusively through the synergistic g400-411 sequence, thus
questioning the functional role of the RGD recognition site. Here we have
investigated the respective role of the fibrinogen g400-411 sequence and the RGD
motif in the molecular events leading to ligand-induced aIIbb3-dependent CHO cell
or platelet spreading, by using intact fibrinogen and well characterized plasmin-
generated fibrinogen fragments containing either the RGD motif (fragment C) or the
g400-411 sequence (fragment D), and CHO cells expressing resting wild type
(aIIbb3wt), constitutively active (aIIbb3T562N) or non-functional (aIIbb3D119Y)
receptors. Our data provide evidence that the g400-411 site by itself is able to
initiate aIIbb3 clustering and recruitement of intracellular proteins to early focal
complexes, mediating cell attachment, FAK phosphorylation and Rac1 activation,
while the RGD motif subsequently acts as a molecular switch on the b3 subunit to
trigger cell spreading. More importantly, we show for the first time that the premier
functional role of the RGD site is not to reinforce cell attachment, but rather to
imprint a conformational change on the b3 subunit leading to maximal RhoA
activation and actin cytoskeleton organization in CHO cells as well as in platelets.
Finally, aIIbb3-dependent RhoA stimulation and cell spreading, but not cell
attachment, are Src-dependent and PI3 kinase-independent and are inhibited by the
Src antagonist PP2.
- 110 -
Mapping the Movement and Assembly of Filamentous Actin and

Myosin II in Migrating Fish Epidermal Keratocytes (87)
Bohnet S.1, Schaub Sébastien1, Laurent V.1,2, Meister J.J.1 and Verkhovsky A.B1
1
Laboratory of Cell Biophysics, Swiss Federal Institute of Technology, Lausanne, Switzerland
2
University Paris 5, Paris, France
To understand the mechanism of cell migration one needs to know how the parts of
the motile machinery of the cell are assembled and move with respect to each other.
We use fluorescence speckle and conventional fluorescence microscopy, image
analysis and computer tracking techniques to quantitatively characterize the
assembly and movement of actin and myosin II, in a simple model system of
persistently migrating cells (fish keratocytes). Filamentous actin exhibited a slow
retrograde flow in the leading lamellipodium and faster forward motion in the trailing
cell body. Transition zone localized in the middle of the lamellipodium. Myosin II also
moved forward in the cell body, but no motion with respect to the substratum was
detected at the leading edge. Forward motion in the cell body was slower at its
bottom and faster at its top, consistent with cell body rotation. At the cell sides, actin
and myosin II exhibited motion towards the center with the velocities increasing with
the distance from the center. These patterns of motion were consistent with
longitudinal and transverse contraction along the lamellipodium/cell body junction.
Simultaneous tracking of actin and myosin II in the same cell demonstrated a slow
forward motion of myosin relative to actin, suggesting a mechanism of anisotropic
network contraction via sliding of myosin II assemblies along divergent actin
filaments. Net assembly of actin occurred locally at the leading edge of the
lamellipodium. The organization of actin in the rest of the cell was accounted for by
its relative motion away from the leading edge, distributed disassembly and local
contraction. In contrast, myosin II assembled in a distributed manner in the
lamellipodium and disassembled in the cell body. Thus, velocity and assembly maps
reflected mechanisms of polarization and force generation in crawling cells.
Supported by SNSF 31-61589.
- 111 -
Modulation of Cell Migration by Integrin-Filamin Linkage (88)
Shevchenko Galina
University of Oulu, Department of Biochemistry, Oulu, Finland
The process of cell migration is crucial for many normal and pathological processes
occurring during organism development (e.g. embryo formation, wound healing,
tumor metastasis etc.). Migration can be divided into several consecutive steps: cell
polarization and formation of protrusions, focal adhesions and cell retraction. The
precise mechanisms by which focal adhesions are formed in migrating cells is a
major challenge for modern molecular biology. It is known that this process largely
depends upon the contact of extracellular matrix and the cytoskeleton through the
integrins, the major family of migration-promoting receptors. Our aim is to
investigate how actin-binding protein filamin A contributes into integrin signal
transduction during cell migration. Filamins contain 24 immunoglobulin-like domains
of which 19-21 have been shown to interact with the cytoplasmic tails of integrin
beta subunits. Recently, a splice variant of filamin domain 19-21 variant 1 has been
found which was shown to enhance the binding of filamin to integrins. Our aim is to
study if overexpression of filamin domains 19-21 affect cell morphology and integrin-
mediated migration. We have found that morphology of CHO cells transfected by
pEGFP-FLN19-21 var1 differs from that one of pEGFP-FLN19-21 by appearance of
numerous protrusions. Therefore, it is assumed that splice variants could somehow
impact some stages of cell migration. Currently, we are investigating how
transfection with pEGFP-FLN19-21 var1 influences the formation of protrusions and
cell spreading. Above research could help to establish the role of filamin variants in
the properties of migrating cells.
- 112 -
Functional and Biochemical Characterization of EFA6, a Key Player in

Cytoskeletal Remodeling (89)
Sironi Cristian Lynne, Matafora V., Bachi A. and Talarico D.

DIBIT, H.S. Raffaele, via Olgettina 58, 20132 Milano, Italy
EFA6 is preferentially expressed in brain tissue and is believed to be involved in actin

cytoskeleton remodelling and neurite outgrowth. Through its Sec7 domain, it is able
to activate the small GTPase ARF6, known to play an important role in endosomal-
plasma membrane recycling and in cytoskeleton dynamics; moreover, overexpression
of EFA6 in neuronal cells induces neurite outgrowth, while in HeLa cells it induces
membrane ruffling and protrusions. The C-terminal of EFA6 appears to be required
for the full biological activity of the protein. The PH domain is important for its
localization to the membrane; the presence of two putative coiled coil motifs might
suggest a possible interaction with other proteins, likely involved in the cytoskeletal
remodelling signalling pathway. To further characterize the COOH terminal of EFA6,
we have prepared tagged constructs that encompass different portions of the PH
domain and of the coiled coil motifs. We show that overexpression of an entire PH-
coiled coil construct in neuronal cells induces neurite branching, while in HeLa cells it
induces cell elongation and formation of dorsal protrusions, where it colocalizes with
F-actin. Actin colocalization is even more striking when HeLa cells are transfected
with a truncated construct missing the second coiled coil motif. The importance of
the coiled coil motifs in the protein activity is also being analysed by site-directed
mutagenesis. Moreover we are currently investigating whether EFA6 undergoes any
post-translational modification, as this might represent a mechanism to modulate
interaction with effector proteins. With these approaches, we would like to elucidate
the components of the EFA6 pathway, and to better understand its role in the
cytoskeleton dynamics.
- 113 -
Actin Polymerization and the Role of Different

Interacting Proteins (90)
Sokoll Andrea1, Shimada A.2 and Mannherz H.G.1

1
Department of Anatomy and Embryology, Ruhr-University Bochum, Bochum, Germany
2
Max-Planck Institut fur Molekulare Physiologie, Dortmund, Germany
There are numerous actin binding proteins, but under all these proteins there are
only a few that preferentially bind to (G)-actin. Thymosin beta 4 (Tß4) is a prototype
of this kind of proteins. It binds to (G)-actin and inhibits the polymerisation of (G)-
actin to (F)-actin. The members of the ADF/Cofilin family also abolish the actin
polymerisation. Recently a new group of proteins called Diaphanous-related formins
(Drf) was shown to interact with actin and to promote the nucleation step of actin
polymerisation. They are activated by Rho GTPases and induce polymerisation of
unbranched actin filaments. MDia is a member of this group. The Drfs contain three
formin homology domains called FH1/FH2. In the present study we measured the
polymerisation of actin in an in vitro assay in the presence of different proteins. We
show that Tß4 abrogates the polymerisation of actin. On the other side we
demonstrate that the actin polymerisation is accelerated in the presence of the FH2-
domain of MDia. To test whether this domain was able to abrogate the
depolymerisation effect of Tß4 we incubated both proteins in the in vitro assays. We
show that the FH2-domain of MDia was able to neutralize the depolymerisation effect
of Tß4. Furthermore we demonstrated that the members of the ADF/Cofilin family
react differently in the presence of MDia. ADF abrogates the polymerisation of actin,
like Cofilin. We can show that the FH2-domain of MDia was able to neutralize the
depolymerisation effect of Cofilin but not of ADF. In summary our data proof that the
FH2-domain of MDia, a member of the Drfs, is an enhancer of actin polymerisation.
- 114 -
The Arabidopsis AtWLIM1 protein associates with actin cytoskeleton

and modifies its organisation (91)
Thomas Clément1, Dieterle M.1, Hoffmann C.1 and Steinmetz A.1,2

1
2
The Arabidopsis genome encodes six CRP-related LIM domain proteins three of
which are specifically and abundantly expressed in mature pollen (AtPLIM1, AtPLIM2
and AtPLIM3), while the other three (AtWLIM1, AtWLIM2 and AtWLIM3) are
expressed in the vegetative tissues. We are investigating the function of AtWLIM1
(At1g10200) in various tissues of the Arabidopsis plant. We have analysed the
subcellular localisation of the protein following inducible expression of the GFP-fusion
protein in transgenic Arabidopsis seedlings. Laser scanning confocal microscopy
observations showed that the fusion protein associates with filamentous structures in
several cell types. Following incubation of the seedlings with Latrunculin B the
cortical cytoskeletal structures disappeared while the more internal structures
appeared to be more resistant. This sensibility of the LIM-GFP-labeled cables to Lat B
identified these structures as actin filaments. Interestingly also, while in root hairs
the cytoskeletal organisation was identical to that observed for fimbrin-GFP-
expressing cells, in another root cell type marked differences in cytoskeletal structure
were observed : most notably, the AtWLIM1-GFP expressing cells showed thick,
more randomly organized actin cables, whereas in fimbrin-GFP-expressing cells the
cables were much thinner and longitudinally oriented. These data suggest that plant
LIM proteins represent new actin cytoskeleton organisers.
- 115 -
Modelling the Dynamics of the Microtubule Cytoskeleton :

a Top-Down Approach (92)
Tindemans Simon and Mulder B.M.

FOM Institute for Atomic and Molecular Physics, Amsterdam, The Netherlands
The cytoskeleton within living cells exhibits rich pattern forming behavior in the
various stages of the cell cycle. Similar patterns have also been observed in in vivo
experiments using only microtubules and multimeric motors. We attempt to obtain a
theoretical understanding of pattern formation in the cytoskeleton, concentrating
only on microtubules. Microtubules and their associated proteins have a large
repertoire of interactions, of which the exact nature and relevance to pattern
formation is often not known. For this reason, instead of constructing a model based
on microscopic interactions, we propose a top-down approach to modelling. We
introduce a mean field model that describes coarse grained average properties of
microtubules around any given point. These properties are represented by three
independent fields: density, polarization and nematic order. We derive generic
dynamical equations for these fields, using only symmetry considerations and
assuming that the total number of microtubules is conserved. The resulting model
has a large number of undetermined parameters. We wish to identify those regions
in the parameter space where a given initial pattern becomes unstable and gives way
to another stable pattern. Specifically, we will investigate the formation of the
transverse cortical array and the pre-prophase band in plant cells. To efficiently
identify the corresponding regions within the high-dimensional parameter space, we
will make use of an evolutionary algorithm.
- 116 -
Design and Evaluation of a Thematic Microarray for Expression

Profiling of Actin Cytoskeleton-Related Genes (ACTIchip) (93)
Vallar Laurent1, Muller J.1,2, Vetter G.1, Mehlen A.1, Poch O.2 and Friederich E.1
1
Laboratoire de Biologie Moléculaire, d’Analyse Génique et de Modélisation, 84 Val Fleuri, L-
1526 Luxembourg
2
Laboratoire de Biologie et de Génomique Structurales, IGBMC, UPR 9004 du CNRS, 1 rue
Laurent Fries, F-67404 Illkirch, France
The actin cytoskeleton is a highly dynamic meshwork that plays a critical role in the
regulation of many aspects of cell behavior, including morphology, adhesion,
migration, cell growth, and apoptosis. In cancer cells, structural and functional
alterations of the actin cytoskeleton correlate with higher proliferation rates and
uncontrolled movement. Understanding these alterations can form the basis for the
development of novel diagnostic, prognostic and therapeutic approaches that might
facilitate control over cancer diseases.
We developped a thematic oligonucleotide microarray for the analysis of actin
cytoskeleton-associated gene expression. The 367 genes represented on the chip
were selected based on bibliography and gene ontology searches. The corresponding
genomic data and sequence analysis features were retrieved from GenBank and
stored in an integrative database (Actinome). From these data, 60-mer probe
sequences with homogeneous pre-defined properties were designed using a home-
made program (CADO4MI) through a multistep protocol allowing the automatic
selection of one optimized probe per target gene. The oligonucleotide probes were
spotted in quadruplicate together with 45 positive controls, 30 negative controls, and
a quality and normalisation probe set (Wang et al., Genome Biology 2003).
Using samples harbouring well-defined expression profiles, series of gene profiling
experiments were performed with ACTIchip and with commercial DNA- and short
oligonucleotide microarrays. Comparative data will be presented that demonstrate
the good quality of ACTIchip in terms of reactivity, specificity and reproducibility,
allowing the use of the chip in routine for the analysis of clinical samples.
- 117 -
Regulation of the Nucleocytoplasmic Transport of the

SRF Cofactor MAL by Actin (94)
Vartiainen Maria, Sebastian Guettler and Richard Treisman

Cancer Research UK, London Research Institute, Lincoln’s Inn Fields Laboratories, London
WC2A 3PX, UK
Serum response factor (SRF) is a transcription factor that controls the expression of
both growth factor-regulated and muscle–specific genes. Two main signalling
pathways regulate SRF activity in cells. MAPK pathway uses TCFs as cofactors,
whereas MRTFs are regulated by Rho GTPase signalling. The MRTF cofactors
MAL/MKL1 and MAL16/MKL2 are related to the constitutively active muscle-specific
SRF coactivator myocardin. While myocardin is constitutively nuclear, MAL
redistributes from the cytoplasm to the nucleus upon Rho signalling. The depletion of
the G-actin pool seems to induce the nuclear accumulation of MAL, but the
mechanisms underlying this process have remained unclear.
To study the nucleocytoplasmic transport of MAL in living cells we have fused the
full-length MAL or its various mutant and deletion constructs to GFP,
photoactivatable GFP and photoswitchable CFP. Using bleaching and photoactivation
techniques we show that MAL rapidly shuttles between nucleus and cytoplasm even
in unstimulated cells. Leptomycin B, an inhibitor of the export receptor Crm1, causes
rapid accumulation of MAL in the nucleus. The effect of leptomycin B can be inhibited
by expression of the Rho inhibitor C3 or unpolymerizable actin mutant R62D, or by
treatment with the actin-binding drug latrunculin B, suggesting that basal import of
MAL requires actin dynamics.
Experiments with photoactivatable GFP show that in unstimulated cells the export
rates of MAL are extremely fast. In contrast, FLIP and photoactivation experiments
show that inhibition of the actin-MAL complex formation by cytochalasin D treatment
or serum stimulation result in substantially reduced rates of MAL export. Consistent
with this, MAL mutants that are unable to bind actin are not efficiently exported from
the nucleus even in the absence of Rho signalling.
- 118 -
Role of Actin Bundling Protein Fascin in Cancer Metastasis (95)
Vignjevic Danijela, Bousquet G., Rosty C., Robine S. and Louvard D.

Equipe de Morphogenèse et Signalisation Cellulaires, Institut Curie, Paris, France
Cancer cells become metastatic by acquiring a motile and invasive phenotype. This
step requires remodelling of the actin cytoskeleton and the expression of exploratory,
sensory organelles known as filopodia. Because of their structure, filopodia can easily
infiltrate between cells and by identifying appropriate targets for adhesion, generate
traction forces to move the cell body. Previously, we tested whether efficient
bundling of actin filaments within filopodia, is critical for filopodia formation. Fascin
was the only actin bundling protein found to be localized along the whole length of
all filopodia. Targeted depletion of fascin by siRNA lead to the substantially reduced
number of filopodia. Further, by participating in the formation of filopodia, fascin
may promote metastasis. Using qRT-PCR and immunohistochemistry, we found that
fascin expression was significantly increased in mouse intestinal tumors compared
with normal epithelium, in stage depended manner (normal 0%, adenoma 41%, SIC
71% and invasive carcinoma 89%, n=48). Next, we tested whether fascin can
promote invasion and migration of human adenocarcinoma cells HT29: 1) in vitro, in
trans-filter assays and 2) in vivo, by tail vein injection of cells into SCID mice. Fascin
expressing cells had higher migrational potential in vitro; and in vivo led to more
frequent distant metastasis in the lungs. Moreover, mice injected with fascin positive
cells developed severe paralysis. There were no bone metastases present. Paralysis
was caused by development of tumors along the spine, which invaded a back
muscle. Those tumors were positive for villin (enterocytes marker) and fascin, which
confirmed that they derived from the injected cells. We propose that up-regulation of
fascin, by promoting the formation of filopodia, could be a significant component in
the acquisition of invasive phenotype in carcinomas.
- 119 -
Measuring and Modeling Microtubule Organisation in Interphase

Tobacco BY-2 Cells (96)
Vos Jan1,2, Cosentino Lagomarsino M.2,3, Tanase C.2,4, Emons A.M.C.1,2, Mulder
B.M.2,1 and Dogterom M.2
1
Wageningen University, Laboratory of Plant Cell Biology, Wageningen, The Netherlands
2
FOM Institute for Atomic and Molecular Physics (AMOLF), Amsterdam, The Netherlands
3
Present address: Institut Curie, Section de Recherche, Paris, France
4
Present address: Physics Department, Utrecht University, Utrecht, The Netherlands
How cortical microtubules become organized in growing interphase plant cells, i.e.
change from a random criss-cross network to a transverse oriented array of parallel
bundles, is a very interesting topic of research as this organization plays a major role
in the growth direction of the cell. We have investigated if physical properties of the
microtubules, such as density, length, bending rigidity and confinement, can explain
this organization. Comparing in vivo and in vitro measurements of microtubule
density and organization with mathematical modeling showed that a nematic
ordering model could partly explain the alignment of microtubules in the cortex of
interphase cells, but not their orientation perpendicular to the cell elongation axis. A
second model, based on the dynamic spring hypothesis, which explains the
transverse arrangement of microtubules in the cortex of interphase plant cells, was
tested quantitatively through a combination of analytical calculations and in vitro
experiments in which microtubules were polymerized from nucleation seeds in micro-
fabricated chambers. We show that a dynamic spring model that only takes bending
elasticity and cellular confinement into account can also not account for the
transverse alignment of microtubules. We conclude that there is a correlation
between microtubule density and ordering in the plant cell but that the nematic
ordering and dynamic spring hypotheses most likely do not explain the organization.
We therefore suggest that an additional active, force-generating process is necessary
to create an aligned configuration perpendicular to the growth direction of the cell.
- 120 -
Molecular, Structural and In Vivo Analysis of the

Dominant Plectin Mutation EBS-Ogna (97)
Vukasinovic Nevena, Castañón M.J., Fuchs P. and Wiche G.

Department of Molecular Cell Biology, Max F. Perutz Laboratories, University of Vienna,
Vienna, Austria
Plectin is a versatile cytoskeletal linker protein widely expressed in mammalian

tissues. It associates with all three types of cytoskeletal filament systems
(intermediate filaments, microfilaments and microtubules) and a variety of junctional
complexes (focal adhesion contacts, desmosomes and hemidesmosomes). Several
mutations in the plectin gene have been identified that lead to the recessive disease
epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). In addition, an
autosomal dominant disorder, EBS-Ogna, has been documented that is characterized
by severe epidermal fragility, but absence of muscular symptoms. The EBS-Ogna
phenotype is due to a C-to-T transition in exon 31 of the plectin gene which results
in an arginine to a tryptophan substitution within the -helical coiled coil rod domain
of plectin. Since plectin functions as a dimer, we have analyzed the effect of Ogna
plectin on dimerization in vitro by using recombinant plectin proteins. Mutated plectin
did not prevent dimerization per se. Possible effects of the EBS-Ogna mutation on
dimer stability still have to be analyzed by mass spectrometry. The mutation in the
rod of plectin might influence its folding so that plectin can not fulfill its function as a
dimer any more. Binding sites even out of the rod domain could be grossly disturbed.
Beside loss of function, this mutation could also cause a gain of function, such as
binding to other proteins or structures. This issue is being currently analyzed by
overexpression studies of Ogna plectin in cultured cells.
- 121 -
The Mechanism and Dynamics of Avb3 Integrin Clustering in

Living Cells (98)
Cluzel C., Saltel F., Lussi J., Paulhe F., Imhof B.A. and Wehrle-Haller Bernhard
Department of Cellular Physiology and Metabolism, Centre Médical Universitaire, Geneva,
Switzerland
During cell migration, the physical link between the extracellular substrate and the
actin cytoskeleton mediated by receptors of the integrin family is constantly
modified. Here we analyzed the mechanisms that regulate the clustering and
incorporation of activated avb3 integrins into focal adhesions in living cells. Mn2+ or
mutational activation of integrins resulted in the formation of de novo, F-actin-
independent integrin clusters. These integrin clusters recruited talin but not other
focal adhesion adapters such as paxillin or vinculin. The head domain of talin was
sufficient to mediate integrin clustering, which however required immobilized ligand
and was prevented by the sequestration of phosphoinositol-4,5-bis-phosphate. FRAP
analysis of activated EGFP-tagged integrin mutants revealed that the association-
dissociation rate of clustered integrins is controlled by its extracellular ligand affinity,
but not by activating mutations altering its cytoplasmic tail domain. Furthermore, the
half-maximal rate of F-actin-independent integrin clustering is 45 seconds,
representing the physical limits for the formation of new focal complexes at the front
of migrating cells and determines the upper limits of sliding induced remodeling of
focal adhesions at the cell rear.
- 122 -
Mechanical Stress Induces Immediate TGF- Activation in

Myofibroblast Culture (99)
Wipff Pierre-Jean, Braunecker J., Meister J.-J. and Hinz B.

Swiss Institute of Technology Lausanne, Switzerland
Differentiation of fibroblasts into contractile, -smooth muscle actin (-SMA)

expressing myofibroblasts depends on the action of the cytokine TGF- in
conjunction with mechanical tension. We here investigated whether mechanical
stress plays a role in directly activating TGF-. Myofibroblasts secrete TGF-1 as a
large latent complex (LLC), consisting of the latency associated protein, the latent
TGF-1 binding protein and TGF-1; this complex binds to components of the
extracellular matrix (ECM). To test whether myofibroblasts may activate TGF-1 from
this local stock by exerting mechanical tension, cells first produced a LLC-rich matrix
on flexible silicone membranes for 4d. Such membranes were then subjected to
unique stretch and the time course of TGF-1 activation was quantified using a
luciferase reporter system. Maximum TGF- activation occurred as early as one
minute after stretch and was independent from membrane-bound proteases, as
shown by cell extraction with Triton X-100. To test the importance of intracellular
tension in physiological TGF-1 activation, myofibroblast on rigid substrates were
treated with agonists and antagonists of actomyosin contraction. The level of
activated TGF-1 always positively correlated with the level of contractile activity.
Finally, to test whether stretch-activated TGF-1 was functional we analyzed
phosphorylation levels of SMAD2/3 and autocrine induction of TGF-1 mRNA; both
were increased. Our results suggest that mechanical stress leads to the rapid release
of TGF-1 from ECM stores, which is sufficient to trigger a TGF-1 autocrine loop in
myofibroblast cultures.
- 123 -
Cofilin/ADF Function in the Mouse (100)
Gurniak C., Bellenchi G., Perlas E. and Witke Walter

European Molecular Biology Laboratory (EMBL), Monterotondo, Italy
Cofilin and ADF belong to the family of F-actin depolymerizing factors thought to be
essential for regulating actin polymerization and directed cell migration. Unlike lower
eukaryotes, three cofilin/ADF genes are found in the mouse: non-muscle cofilin (n-
cofilin), muscle cofilin (m-cofilin) and ADF. We have generated mouse models for n-
cofilin and ADF applying conventional as well as conditional mutagenesis. Using
these mouse models as well as cells cultured from the respective mutants we have
been able to distill specific roles of the depolymerizing factors in cell migration,
proliferation and cell cycle control. Recent results on n-cofilin/ADF in different mouse
tissues and cell types using cre-mediated deletion will be discussed.
- 124 -
UNC-87, C. elegans Calponin-Repeat Protein, Inhibits both F-Actin-

Binding and Severing Activities of ADF/Cofilin (101)
Yamashiro Sawako, Woo J. H., Gimona M. and Ono S.

Emory University, Atlanta, GA 30322 (USA)
During assembly and maintenance of myofibrils in muscle cells, actin filament

dynamics must be spatially and temporally regulated, but the regulatory mechanism
is still unclear. It has been shown that, in C. elegans body wall muscle, ADF/cofilin
(UNC-60B) enhances actin filament dynamics and is essential for assembly of
myofibrils. However, myofibrils are highly stable structures after they are assembled,
so it is suggested that there are some factors which inhibit ADF/cofilin to stabilize
actin filaments in myofibrils. Although it has been reported that C. elegans
tropomyosin (CeTM) inhibits ADF/cofilin-dependent actin dynamics in vitro and in
vivo , the inhibitory effect of tropomyosin is relatively weak. Here, we present
evidence that UNC-87, a C. elegans calponin-repeat protein, is a new candidate
negative modulator of ADF/cofilin-dependent actin dynamics. UNC-87 has seven
calponin repeats that are found in the C-terminal region of calponin, but it does not
have a calponin-homology (CH) domain. Previous studies showed that UNC-87 binds
and cross-links F-actin in vitro, is a thin filament protein in body wall muscle, and is
important for maintenance of myofibrils. However, its cellular function is not clearly
understood. We purified UNC-87, CeTM and UNC-60B and examined their effects on
actin filaments by co-sedimentation assays. We found that UNC-87 bound to F-actin
by strong and weak actin-binding sites and inhibited binding of UNC-60B to F-actin.
Whereas UNC-60B was pre-incubated with F-actin, both F-actin-binding and bundling
activities of UNC-87 were strongly inhibited. Therefore, UNC-87 and UNC-60B bind to
F-actin in a mutually exclusive manner. Furthermore, UNC-87 bound to F-actin
independently of CeTM, and UNC-87 and CeTM cooperatively inhibited association of
UNC-60B with F-actin. We also examined the effects of these proteins on actin
filaments by direct observation of fluorescently labeled actin filaments. UNC-87
effectively inhibited F-actin-severing activity of UNC-60B. This inhibitory effect of
UNC-87 was stronger than that of CeTM. These results suggest that UNC-87 is a
strong inhibitor of UNC-60B-mediated actin dynamics and stabilizes actin filaments
together with CeTM in muscle cells.
- 125 -
Tropomyosin in the Lamellipodium of Migrating Cells (102)
Zhao Rathje Li-Sophie, Hillberg L. and Lindberg U.

Department of Cell Biology, The Wenner-Gren Institute, Stockholm, Sweden
Tropomyosin has been shown to be crucial to the formation of actin cables in

budding yeast Sacharomyces cerevisiae. It is also known that tropomyosin is able to
dissociate gelsolin from gelsolin:actin oligomers subsequently facilitating the
annealing of the actin oligomers into long filaments. This suggests the possibility that
tropomyosin is an essential factor in the assembly of actin regardless of the
mechanism of its polymerization. Here we show that high and low molecular weight
tropomyosins are present in significant amounts in the lamellipodia and filopodia of
spreading normal and transformed cells. The presence of tropomyosin in these
locales was ascertained by staining of cells with antibodies reacting with endogenous
tropomyosins as well as through localization of hemaglutinin- and green fluorescent
protein-tagged tropomyosin isoforms. The presented data suggest a refinement of
the current model of tropomyosin being absent from the leading edge.
- 126 -
Expression of Cytoplasmic Actins is Modulated in Transformed

Compared to Normal Cells (103)
Zwaenepoel Ingrid1, Dugina V.2 and Chaponnier C.1

1
Department of Pathology and Immunology, CMU, Geneva, Switzerland
2
Belozersky Institute of Physical and Chemical Biology of Moscow State University, Russia
A major feature of cell transformation during malignancy consists of a reorganization

of the actin cytoskeleton which leads to increased cell motility and invasion.
Using our newly generated monoclonal antibodies against - and -cytoplasmic actin
(CYA) isoforms, we have recently shown that - and -CYA have distinct patterns of
organization: -CYA is organized in parallel filaments such as in stress fibers,
filopodia and circular bundles at cell-cell contacts, suggesting a role for -CYA in cell
attachment and cell contraction. Conversely, -CYA is organized as a meshwork in
cortical and lamellipodial structures, suggesting a role for -CYA in cell motility.
These observations led us to hypothesize that the regulation of - and -CYA
organization may be of major importance in tumoral processes.
We have therefore compared the organization of - and -CYA in normal human lung
fibroblasts WI 38, MRC-5 and in their respective SV40-transformed counterparts
WI38-VA13, MRC-5V2. Transformed cells are mainly characterized by a diminution of
-CYA bundles and an enrichment of -CYA lamellar protrusions. We further
established that the reorganization of - and -CYA in transformed cells is related to
variation of their expression by quantifying protein expression of each isoform by 2D-
gel analyses. In this study, we have shown that the ratio of / isoforms switched
from 2.5 in normal cells to 0.8 in transformed cells. Whether this variation in protein
expression is related to changes in transcriptional or post-transcriptional events will
be addressed in future studies using real-time RT-PCR.
Our results point out for the first time an association between cell transformation and
a reorganization of cytoplasmic actin isoforms which is correlated to modulation of
their expression. Further investigations will be conducted to characterize the
mechanisms through which actin reorganization occurs in malignant transformation.
- 127 -

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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research

Self-Organization Principles and Mitotic Spindle Morphogenesis

The principles invoked to explain cellular morphogenesis are usually restricted to

Dynamic Polymorphism of Actin Molecules in the Filament

References and Notes

Can We Ever Determine the Structure of Intermediate Filaments

Molecular Mechanisms of the Kinesin Motor

Defining the In Vivo Functions for Enterocyte Class I Myosin,

Myo1a is one of 8 class I myosins expressed in vertebrates. Myo1a is a major

Control Mechanisms of Actin Polymerisation

In living cells, actin is assembled at a steady state. In the absence of regulators,

Coordinated Regulation of the Actin Cytoskeleton and Microtubule

Coordination of actin polymerization, myosin-driven contractility and microtubule

Root Hairs: a Model System for Studying Cytoskeletal Dynamics

WASP versus WAVE: Differential Subcellular Distribution,

Cellular actin polymerisation is catalysed by actin filament nucleation or amplification

Studying the Coupling of ER Exit to Microtubules in Living Cells

Coupling Cytoskeletal Dynamics to the Regulation of

Guettler S., Miralles F., Vartiainen M. and Treisman Richard

Lamina Proteins in Nuclear Architecture, Dynamic Chromatin

Dictyostelium, a Model to Assess the Function of

Bacterial Pathogenesis: Signaling to the Actin Cytoskeleton

LIM Proteins: Molecular Scaffolds for Cytoskeletal Regulation

Proteins involved in regulating complex cellular behaviors such as signal

Talin: an Essential Link between Integrins and the Cytoskeleton

Tanentzapf Guy and Brown N.H.

Cellular adhesion and communication are vital during the development of

Plasma Membrane Organization and Cell Growth Signaling by the

Ezrin is a membrane-cytoskeleton linker involved in dynamic functions that occur at

Abi1 Signaling Complexes are Master Regulators of

Biomimetic Systems to Study Actin-Based Movement

Dynamics of Force-Generating Microtubules

We perform in vitro experiments aimed at understanding force generation by the

Mechanical Weakness of Nuclei and Cells in Laminopathies Result

Laminopathies comprise a heterogeneous group of inherited diseases caused by

The Invasion Nature of Mammary Tumors: Insights into

Actin Filament Reorganisation during Early Adhesion Formation in

Alexandrova Antonina1,2, Resch G.P.1 and Small J.V.1

We correlated the dynamics of formation of early substrate adhesions at the leading

The Microtubules Optical Density Analysis Allows to Estimate the

Smurova K.M. and Alieva Irina

In internal cytoplasm the density of microtubules is highest in the centrosome area

Description and Studying the Actin Cytoskeleton-Associated Protein

Amsellem Valérie1, Kryszke M.H.1, Leh H2. and Auclair C.1

ERM-Dependent Regulation of the Actin Cytoskeleton by

Batchelor Clare and Winder S.J.

Dystroglycan is part of an adhesion receptor complex linking the extracellular matrix

Visualization of Unconventional Myosin VIII Isoforms in Plant Cells

Microtubule Plus-End Capture and the Formation of

The classic radial pattern of microtubules focused on a centrally located centrosome

Nuclear Function of Cytoskeletal Proteins: Coordinating Adhesion

Conacci-Sorrell M.1, Gavert N.1, Brabletz T.2 and Ben-Ze’ev Avri1

Aberrant -catenin signaling is prevalent in most colorectal cancer patients. -

In Silico Model for Actin Filament Assembly Based on

Berro Julien and Martiel J.L.

The Role of Vav Family Proteins in Macrophage Migration (9)

Bhavsar Parag and Ridley, A.J.

The ability to migrate is essential to leukocyte function. In response to inflammatory

Identification of Proteins Interacting with the N-Terminal Domain of

Boczonadi Veronica, Long H. and Maatta A.

p120 Catenin Beyond Cell-Cell Junctions: Association with Dynamic

Armadillo family protein p120 catenin (p120) binds juxtamembrane domains of