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INVITED SPEAKERS
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Karsenti Eric
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Mooseker Mark
Department of Molecular Cellular Developmental Biology, Yale University, New Haven,
Connecticut, USA
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Carlier Marie-France
Laboratoire d'Enzymologie et Biochimie Structurale (LEBS), CNRS, Gif-sur-Yvette, France
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Pepperkok Rainer
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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).
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Beckerle Mary
Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84103 USA
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Arpin Monique
Institut Curie, Biology Division, Paris, France
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Dogterom Marileen
FOM Institute for Atomic and Molecular Physics (AMOLF), Amsterdam, The Netherlands
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
POSTER ABSTRACTS
are in alphabetical order with respect to the presenting author (underlined)
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Colombelli Julien, Reynaud E.G., Rietdorf J., Pepperkok R. and Stelzer E.H.K.
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
de Rooij Johan, Kerstens A., Danuser G., Schwartz M.A. and Waterman-Storer C.M.
The Netherlands Cancer Institute (NKI), Amsterdam, The Netherlands
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
A New Role for the Axial Budding Protein Bud3 in Regulating Septins
Network for Cytokinesis in Saccharomyces cerevisiae (26)
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Gatti Sabrina1, Shen W.H.2, Grausem B.1, Dieterle M.1, 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
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Ghosh Indraneel
The Weizmann Institute of Science, Rehovot, Israel
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
- 56 -
FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
- 60 -
FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
- 61 -
FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
- 64 -
FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
Laboratory of Plant Molecular Biology, CRP-Santé, Luxembourg
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Kreplak Laurent, Mücke N., Bär H., Leterrier J.F., Herrmann H. and Aebi U.
M.E. Müller Institute, Biozentrum, University of Basel, Switzerland
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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
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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
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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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
The Rho Family GTPase Rif Induces Filopodia Through mDia2 (72)
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
The Actin Binding Protein ABP1 Interacts With N-WASP and this
Complex May Link Actin Dynamics to Endocytosis (74)
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
Reynaud Emmanuel, Colombelli J., Rietdorf J., Stelzer E.H.K. and Pepperkok R.
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
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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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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Role of Emerin in the Association of the MTOC With the Nucleus (85)
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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.
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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FEBS/ESF Workshop on Integrated Approaches in Cytoskeleton Research
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