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Accueil > Départements > Biochimie, Biophysique et Biologie Structurale > Christophe LE CLAINCHE & Louis RENAULT : Dynamique du cytosquelette et motilité

Regulation of actin assembly, actomyosin networks architecture and mechanotransduction associated with cell-matrix adhesion

Christophe LE CLAINCHE

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LE CLAINCHE Christophe [Chargé de Recherche - CNRS]
Equipe Le Clainche C./Renault L. - Dynamique du cytosquelette et motilité [Responsable]
01 69 82 34 80 Gif - Bât 34

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Keywords : cell migration, force, cell-matrix adhesion, actin, myosin, talin, vinculin, actin binding proteins, actin polymerization, actomyosin contractility, mechanosensitivity, biochemistry, kinetic assays, single actin filaments, in vitro reconstitution, fluorescence spectroscopy, TIRF microscopy, image analysi

Main research projects

Cell migration is involved in many physiological and pathological processes. Force is produced by the growth and the contraction of the actin cytoskeleton 1. To produce force, these actin networks must be anchored to the extracellular matrix (ECM) by adhesion complexes 1,3,4. These structures contain transmembrane integrins that mechanically couple the ECM to the intracellular actin cytoskeleton via actin binding proteins (ABPs) 3,4. This system acts as a molecular clutch that controls force transmission across adhesion complexes. This molecular clutch is a complex interface made of multiple layers of regulated protein-protein interactions 4. The multiple activities of the ABPs present in these structures play a critical role in the dynamics of this interface. In addition to the control of actin filament binding and polymerization 1-4, these proteins sense and respond to the force applied by the actomyosin cytoskeleton to adjust the anchoring strength 4,6. Finally, some of these ABPs are involved in the activation of integrins (Figure 1) 4.

In the short term, our project aims at understanding :
1) the coordinated regulation of actin binding and assembly by the numerous ABPs present in adhesion complexes ; 2) the resulting architecture and dynamics of the polymerizing and contractile actomyosin networks ; 3) the mechanisms by which ABPs sense and respond to the force applied by contractile actomyosin networks ; 4) the relative contribution of the various ABPs to integrin activation (Figure 1).

Recently, we studied the activities and the mechanosensitive properties of the two interacting ABPs talin and vinculin. We first described the mechanism by which vinculin regulates actin polymerization (Figure 2 A-D) 3. We also developed a new microscopy assay with pure proteins in which the force, generated by actomyosin cables, stretches talin, immobilized on a micropatterned surface, to expose vinculin binding sites (Figure 2 E-G) 5,6. Altogether, our results reveal the mechanism by which the mechanosensitive talin-vinculin machinery controls actin anchoring to the adhesion complexes5.

In the long term, we are also interested in studying the interplay between the actin-associated machineries present in cell-matrix adhesion complexes, cell-cell adhesion complexes and at the leading edge of the lamellipodium.

cell-matrix adhesion complexes and actin cytoskeleton
Figure 1. Mechanical coupling between cell-matrix adhesion complexes and the actin cytoskeleton during cell migration. (A) Force is produced by the growth (red arrows) and the contraction (blue arrows) of the actin cytoskeleton. To produce force, these actin networks must be anchored to the extracellular matrix (ECM, grey) by adhesion complexes (dark blue). Figure adapted from 1. (B) Adhesion complexes contain transmembrane integrins that couple the extracellular matrix to the intracellular actin cytoskeleton via actin binding proteins (ABPs, ex : talin and vinculin). This system acts as a molecular clutch that controls force transmission across adhesion complexes. ABPs present in these structures control actin filament binding and polymerization (1), which dictates the dynamics and the ultrastructure of the contractile actomyosin networks (2). These proteins also sense and respond to the force applied by the actomyosin cytoskeleton to adjust the anchoring strength (3). Finally, some of these ABPs are involved in the activation of integrins (4).

Approaches

To develop this project we use the following techniques/approaches :
1) Proteins and complexes are purified from tissues or expressed in baculovirus/insect cells and E. Coli systems.
2) Protein complexes containing actin, ABPs and regulatory factors are studied by fluorescence spectroscopy and a variety of biochemical techniques.
3) The effects of ABPs on the kinetics of actin polymerization are studied by fluorescence spectroscopy (Figure 2 A,B)
4) The mechanism by which ABPs regulate actin polymerization is also studied by the direct observation of single actin filament elongation in Total Internal Reflection Fluorescence (TIRF) microscopy (Figure 2 C,D)
5) Systems of increased gomplgxyty are reconstituted in vitro with multiple purified proteins to study :
- the regulation of actin polymerization
- the architecture of actin networks (Figure 2 E,F)
- the dynamics of actomyosin-dependent mechanosensitive protein complexes (Figure 2 E,G)

Actin polymerization
Figure 2. Approaches used and recent results obtained in this project. (A) Kinetics of actin polymerization in the presence of increasing concentrations of the actin filament-binding domain of vinculin (Vt). Actin polymerization is measured by the increase of fluorescence of pyrenyl-labeled actin 2. (B) The quantification of the rate of actin polymerization as a function of vinculin concentrations indicates that Vt inhibits barbed end elongation specifically 2. (C) Time-lapses showing the direct real-time observation of single actin filament elongation in TIRF microscopy in the absence (top) and in the presence (bottom) of the actin filament-binding domain of vinculin (Vt). Actin filaments are visualized by the fluorescence of Alexa Fluor 488–labeled actin 2. (D) Kymographs corresponding to actin filaments presented in panel C. In the presence of Vt, pauses correspond to barbed end capping events 2. (E) Basic principle of the assay that we developed to study mechanosensitive protein complexes. In this assay, talin is immobilized in disc-shaped islands micropatterned on a glass surface. In the absence of mechanical stimulus, vinculin-binding sites are buried in talin rod domain. In response to the force applied by a contractile actomyosin network, talin is stretched. This mechanical stretching of talin reveals cryptic vinculin-binding sites. EGFP-vinculin fluorescence reveals talin stretching. Actomyosin structures are visualized by the fluorescence of Alexa Fluor 594–labeled actin 5,6. (F) Self-organization of actomyosin cable{, crksSlinked by α-actinin, between talin-coated discs on a micro-patterned surface. The actomyosin cables are revealed by the fluorescence of mCherry-α-actinin. (G) The attachment of contractile actomyosin cables, made of actin, myosin and α-actinin, to talin-coated discs, induces vinculin binding to stretched talin. The detachment of these cables allows talin re-folding and vinculin dissociation. In this time-lapse, EGFP-vinculin is in green and Alexa Fluor 594–labeled actin is in red 5.

Open positions

We are interested in recruiting students (M1/M2 internships and PhD), post-docs and confirmed researchers for new projects. Candidates are very welcome to contact us to discuss projects and funding.

Publications

Selected recent publications (related to the current project)

1 Christophe Le Clainche , and Marie-France Carlier
Regulation of actin assembly associated with protrusion and adhesion in cell migration.
Physiological Reviews (2008) Apr ;88(2):489-513.

2 Christophe Le Clainche , Satya P Dwivedi, Dominique Didry, Marie-France Carlier
Vinculin is a dually regulated actin filament barbed-end capping and side-binding protein.
Journal of Biological Chemistry (2010) Jul 23 ;285(30):23420-32.

3 Corina Ciobanasu, Bruno Faivre, Christophe Le Clainche
Actin dynamics associated with focal adhesions.
International Journal of Cell Biology (2012) 2012:941292.

4 Corina Ciobanasu, Bruno Faivre, Christophe Le Clainche
Integrating actin dynamics, mechanotransduction and integrin activation : The multiple functions of actin binding proteins in focal adhesions.
European Journal of Cell Biology (2013) (92) 339-348.

5 Corina Ciobanasu, Bruno Faivre, Christophe Le Clainche
Actomyosin dependent formation of the mechanosensitive talin-vinculin complex reinforces actin anchoring.
Nature Communications (2014) 5:3095 DOI : 10.1038/ncomms4095

6 Corina Ciobanasu, Bruno Faivre, Christophe Le Clainche
Reconstituting actomyosin-dependent mechanosensitive protein complexes in vitro.
Nature Protocols (2015), Jan ;10(1):75-89. doi : 10.1038/nprot.2014.200

Publications related to past projects

1. Research articles

- Marie-France Carlier, Pierre Nioche, Isabelle Broutin-L’Hermite, Rajaa Boujemaa, Christophe Le Clainche, Coumaran Egile, Christiane Garbay, Arnaud Ducruix, Philippe Sansonetti, Dominique Pantaloni.
Grb2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASp) with actin-related protein (Arp2/3) complex.
Journal of Biological Chemistry (2000) Jul 21 ;275(29):21946-52.

- Rajaa Boujemaa, Edith Gouin, Guido Hansen, Stanislav Samarin, Christophe Le Clainche, Dominique Didry, Pierre Dehoux, Pascale Cossart, Christine Kocks, Marie-France Carlier and Dominique Pantaloni.
The Listeria protein ActA mimics WASP family proteins : it activates filament branching by Arp2/3 complex.
Biochemistry (2001) Sep 25 ;40(38):11390-404.

- Flavia Castellano, Christophe Le Clainche, Delphine Patin, Marie-France Carlier, and Philippe Chavrier. A WASp/VASP complex regulates actin polymerisation at the plasma membrane.
EMBO Journal (2001) Oct 15 ;20(20):5603-14.

- Christophe Le Clainche, Dominique Didry, Marie-France Carlier and Dominique Pantaloni.
Activation of Arp2/3 Complex by Wiskott-Aldrich Syndrome Protein Is Linked to Enhanced Binding of ATP to Arp2.
Journal of Biological Chemistry (2001) Dec 14 ;276(50):46689-92.

- Christophe Le Clainche, Dominique Pantaloni and Marie-France Carlier.
ATP hydrolysis on Arp2/3 complex causes debranching of dendritic actin arrays.
Proceedings of the National Academy of Sciences of USA (2003) May 27 ;100(11):6337-42.

- Stéphane Romero, Christophe Le Clainche, Dominique Didry, Coumaran Egile, Dominique Pantaloni, Marie-France Carlier.
Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis.
Cell (2004) Oct 29 ;119(3):419-29.

- Carole Gautier-Courteille, Christophe Le Clainche, Carine Barreau, Yann Audic, Antoine Graindorge, Dominique Maniey, H. Beverley Osborne and Luc Paillard.
EDEN-BP-dependent post-transcriptional regulation of gene expression in Xenopus somitic segmentation.
Development (2004) 131 (24) : 6107-6117.

- Christophe Le Clainche #, Dominik Schlaepfer #, Aldo Ferrari #, Mirko Klingauf, Katarina Grohmanova, Alexey Veligodskiy, Dominique Didry, Diep Le, Coumaran Egile, Marie-France Carlier, Ruth Kroschewski. IQGAP1 stimulates actin assembly through the N-WASP-Arp2/3 pathway.
Journal of Biological Chemistry (2007) Jan 5 ;282(1):426-35. # Equal contribution

- Nalini Ramarao, Christophe Le Clainche, Tina Izard, Raphaëlle Bourdet-Sicard, Elisabeth Ageron, Philippe J Sansonetti, Marie-France Carlier, Guy Tran Van Nhieu.
Capping of actin filaments by vinculin activated by the Shigella IpaA carboxyl-terminal domain.
FEBS Letter (2007) Mar 6 ;581(5):853-7.

- Christophe Le Clainche, Barbara S Pauly, Claire X Zhang, Åsa E Engqvist-Goldstein, Kimberley Cunningham, David G Drubin.
A Hip1R-cortactin complex negatively regulates actin assembly associated with endocytosis.
EMBO Journal (2007) Mar 7 ;26(5):1199-210.

- Claire Vincent, Isabelle Maridonneau-Parini, Christophe Le Clainche, Pierre Gounon, Arnaud Labrousse. Activation of p61Hck triggers WASp- and Arp2/3-dependent actin-comet tail biogenesis and accelerates lysosomes.
Journal of Biological Chemistry (2007) Jul 6 ;282(27):19565-74.

- Elodie Lewkowicz, Floriane Herit, Christophe Le Clainche, Pierre Bourdoncle, Franck Perez, Florence Niedergang.
The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis.
Journal of Cell Biology (2008) Dec 29 ;183(7):1287-98.

- Franck Bazile, Aude Pascal, Isabelle Arnal, Christophe Le Clainche, Franck Chesnel, Jacek Z Kubiak.
Complex relationship between TCTP, microtubules and actin microfilaments regulates cell shape in normal and cancer cells.
Carcinogenesis (2009) Apr ;30(4):555-65.

- Maud Hertzog, Francesca Milanesi, Larnell Hazelwood, Andrea Disanza, HongJun Liu, Emilie Perlade, Maria Grazia Malabarba, Sebastiano Pasqualato, Alessio Maiolica, Stefano Confalonieri, Christophe Le Clainche, Nina Offenhauser, Jennifer Block, Klemens Rottner, Pier Paolo Di Fiore, Marie-France Carlier, Niels Volkmann, Dorit Hanein, Giorgio Scita.
Molecular basis for the dual function of Eps8 on actin dynamics : bundling and capping.
PLOS Biology (2010) Jun 1 ;8(6)

- Sabrina Marion, Eik Hoffmann, Daniela Holzer, Christophe Le Clainche, Marianne Martin, Martin Sachse, Iva Ganeva, Paul Mangeat, Gareth Griffiths.
Ezrin Promotes Actin Assembly at the Phagosome Membrane and Regulates Phago-Lysosomal Fusion.
Traffic (2011) Apr ;12(4):421-37

- Andrea Pelikan-Conchaudron, Christophe Le Clainche, Dominique Didry, Marie-France Carlier.
The IQGAP1 protein is a calmodulin-regulated barbed end capper of actin filaments : possible implications in its function in cell migration.
Journal of Biological Chemistry (2011) Oct 7 ;286(40):35119-28.

- Dominique Didry, François Xavier Cantrelle, Clotilde Husson, Pierre Roblin, Ana M Moorthy, Javier Perez, Christophe Le Clainche, Maud Hertzog, Eric Guittet, Marie-France Carlier, Carine van Heijenoort, Louis Renault.
How a single residue in individual β-thymosin/WH2 domains controls their functions in actin assembly.
EMBO Journal (2012) Feb 15 ;31(4):1000-13

- Jan Müller, Julia Pfanzelter, Christoph Winkler, Akihiro Narita, Christophe Le Clainche, Maria Nemethova, Marie France Carlier, Yuichiro Maeda, Matthew D. Welch, Taro Ohkawa, Christian Schmeiser, Guenter P. Resch and J. Victor Small.
Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of propulsion.
PLOS Biology (2014) Jan 14 ; 12(1) : e1001765. DOI:10.1371/journal.pbio.1001765

2. Reviews and chapters

- Dominique Pantaloni, Christophe Le Clainche and Marie-France Carlier.
Mechanism of actin-based motility.
Science (2001) May 25 ;292(5521):1502-6.

- Marie-France Carlier, Sebastian Wiesner, Christophe Le Clainche, Dominique Pantaloni.
Actin-based motility as a self-organized system : mechanism and reconstitution in vitro.
Comptes Rendus Biologies (2003) Feb ; 326(2):161-70.

- Marie-France Carlier, Christophe Le Clainche, Sebastian Wiesner, Dominique Pantaloni.
Advances in actin-based motility : from Arp2/3 complex to biomimetics.
Bioessays (2003) 25(4):336-45.

- Christophe Le Clainche and Marie-France Carlier.
Actin-based motility assay.
Current Protocols in Cell Biology (2004) Oct ;Chapter 12:Unit12.7.

- Christophe Le Clainche and David G. Drubin.
Actin lessons from pathogens.
Molecular Cell (2004) Feb 27 ;13(4):453-4.

- Marie-France Carlier, Dominique Pantaloni, Stéphane Romero and Christophe Le Clainche.
How actin assembly is modulated at filament barbed ends in motile processes ?
Landes Bioscience (2006) Edited by Pekka Lappalainen. Eurekah.com. Book chapter.

- Beata Bugyi, Christophe Le Clainche, Guillaume Romet-Lemonne, Marie-France Carlier.
How do in vitro reconstituted actin-based motility assays provide insight into in vivo behavior ?
FEBS Letter (2008) Jun 18 ;582(14):2086-92.

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