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

Publications de l’équipe


  • N. Essawy, C. Samson, A. Petitalot, S. Moog, A. Bigot, I. Herrada, A. Marcelot, A. - A. Arteni, C. Coirault, et S. Zinn-Justin, « An Emerin LEM-Domain Mutation Impairs Cell Response to Mechanical Stress », Cells, vol. 8, nᵒ 6, juin 2019.
    Résumé : Emerin is a nuclear envelope protein that contributes to genome organization and cell mechanics. Through its N-terminal LAP2-emerin-MAN1 (LEM)-domain, emerin interacts with the DNA-binding protein barrier-to-autointegration (BAF). Emerin also binds to members of the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Mutations in the gene encoding emerin are responsible for the majority of cases of X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). Most of these mutations lead to an absence of emerin. A few missense and short deletion mutations in the disordered region of emerin are also associated with X-EDMD. More recently, missense and short deletion mutations P22L, ∆K37 and T43I were discovered in emerin LEM-domain, associated with isolated atrial cardiac defects (ACD). Here we reveal which defects, at both the molecular and cellular levels, are elicited by these LEM-domain mutations. Whereas K37 mutation impaired the correct folding of the LEM-domain, P22L and T43I had no impact on the 3D structure of emerin. Surprisingly, all three mutants bound to BAF, albeit with a weaker affinity in the case of K37. In human myofibroblasts derived from a patient's fibroblasts, emerin ∆K37 was correctly localized at the inner nuclear membrane, but was present at a significantly lower level, indicating that this mutant is abnormally degraded. Moreover, SUN2 was reduced, and these cells were defective in producing actin stress fibers when grown on a stiff substrate and after cyclic stretches. Altogether, our data suggest that the main effect of mutation K37 is to perturb emerin function within the LINC complex in response to mechanical stress.
    Mots-clés : actin, atrial cardiac defects, B3S, BAF, CRYOEM, emerin, INTGEN, mechano-transduction, PF.


  • A. Belyy, U. Mechold, L. Renault, et D. Ladant, « ExoY, an actin-activated nucleotidyl cyclase toxin from P. aeruginosa: A minireview », Toxicon: Official Journal of the International Society on Toxinology, vol. 149, p. 65-71, juill. 2018.
    Résumé : ExoY is one of four well-characterized Pseudomonas aeruginosa type 3 secretion system (T3SS) effectors. It is a nucleotidyl cyclase toxin that is inactive inside the bacteria, but becomes potently activated once it is delivered into the eukaryotic target cells. Recently, filamentous actin was identified as the eukaryotic cofactor that stimulates specifically ExoY enzymatic activity by several orders of magnitude. In this review, we discuss recent advances in understanding the biochemistry of nucleotidyl cyclase activity of ExoY and its regulation by interaction with filamentous actin.
    Mots-clés : ACTIN.

  • A. Belyy, I. Santecchia, L. Renault, B. Bourigault, D. Ladant, et U. Mechold, « The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin », Journal of Biological Chemistry, vol. 293, nᵒ 51, p. 19785-19796, déc. 2018.
    Résumé : Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 35-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants.
    Mots-clés : ACTIN, B3S, ExoY, Pseudomonas aeruginosa (P, Pseudomonas aeruginosa (P. aeruginosa).

  • C. Ciobanasu, H. Wang, V. Henriot, C. Mathieu, A. Fente, S. Csillag, C. Vigouroux, B. Faivre, et C. Le Clainche, « Integrin-bound talin head inhibits actin filament barbed end elongation », The Journal of Biological Chemistry, vol. 293, nᵒ 7, p. 2586-2596, 2018.
    Résumé : Focal adhesions (FAs) mechanically couple the extracellular matrix (ECM) to the dynamic actin cytoskeleton, via transmembrane integrins and actin-binding proteins. The molecular mechanisms by which protein machineries control force transmission along this molecular axis, i.e. modulating integrin activation and controlling actin polymerization, remain largely unknown. Talin is a major actin-binding protein that controls both the inside-out activation of integrins and actin-filament anchoring and thus plays a major role in the establishment of the actin-ECM mechanical coupling. Talin contains three actin-binding domains (ABDs). The N-terminal head domain contains both the F3 integrin-activating domain and ABD1, while the C-terminal rod contains the actin-anchoring ABD2 and ABD3. Integrin binding is regulated by an intramolecular interaction between the N-terminal head and a C-terminal five-helix-bundle (R9). Whether talin ABDs regulate actin polymerization in a constitutive or regulated manner has not been fully explored. Here, we combine kinetics assays using fluorescence spectroscopy and single actin filament observation in TIRF microscopy, to examine relevant functions of the three ABDs of talin. We find that the N-terminal ABD1 blocks actin filament barbed end elongation while ABD2 and ABD3 do not show any activity. By mutating residues in ABD1, we find that this activity is mediated by a positively charged surface that is partially masked by its intramolecular interaction with R9. Our results also demonstrate that, once this intramolecular interaction is released, integrin-bound talin head retains the ability to inhibit actin assembly.
    Mots-clés : ACTIN.

  • D. Georgess, P. Spuul, C. Le Clainche, D. Le Nihouannen, I. Fremaux, T. Dakhli, D. M. Delannoy López, D. Deffieux, P. Jurdic, S. Quideau, et E. Génot, « Anti-osteoclastic effects of C-glucosidic ellagitannins mediated by actin perturbation », European Journal of Cell Biology, sept. 2018.
    Résumé : Actin subunits assemble into actin filaments whose dynamics and three-dimensional architectures are further regulated by a variety of cellular factors to establish the functional actin cytoskeleton. The C-glucosidic ellagitannin vescalagin and its simpler analogue vescalin, affect both the dynamics and the ultrastructure of the actin cytoskeleton by directly binding to F-actin. Herein, we show that in vitro, the two compounds induce the formation of distinct F-actin networks characterized by different superstructures and dynamics. In living mature osteoclasts, highly specialized bone-degrading cells that constantly remodel their cytoskeleton, vescalagin and vescalin alter actin dynamics at podosomes and compromise the integrity of the podosome belt that forms the bone-degrading apparatus. Both compounds target the bone-resorbing activity at concentrations that preserve osteoclastic maturation and survival and with no detectable impact on the behaviour of bone-forming osteoblastic cells. This anti-osteoclastic activity of vescalagin and vescalin reveals the potential of targeting actin dynamics as a new therapeutic opportunity and, in this case, as a plausible approach for the local treatment of osteoporosis.
    Mots-clés : ACTIN, Actin dynamics, B3S, Cytoskeleton, Medicinal biology, Osteoclast biology, Podosomes, Polyphenol sciences.

  • A. Mahuzier, A. Shihavuddin, C. Fournier, P. Lansade, M. Faucourt, N. Menezes, A. Meunier, M. Garfa-Traoré, M. - F. Carlier, R. Voituriez, A. Genovesio, N. Spassky, et N. Delgehyr, « Ependymal cilia beating induces an actin network to protect centrioles against shear stress », Nature Communications, vol. 9, nᵒ 1, p. 2279, juin 2018.
    Résumé : Multiciliated ependymal cells line all brain cavities. The beating of their motile cilia contributes to the flow of cerebrospinal fluid, which is required for brain homoeostasis and functions. Motile cilia, nucleated from centrioles, persist once formed and withstand the forces produced by the external fluid flow and by their own cilia beating. Here, we show that a dense actin network around the centrioles is induced by cilia beating, as shown by the disorganisation of the actin network upon impairment of cilia motility. Moreover, disruption of the actin network, or specifically of the apical actin network, causes motile cilia and their centrioles to detach from the apical surface of ependymal cell. In conclusion, cilia beating controls the apical actin network around centrioles; the mechanical resistance of this actin network contributes, in turn, to centriole stability.
    Mots-clés : ACTIN, B3S.

  • D. Raoux-Barbot, A. Belyy, L. Worpenberg, S. Montluc, C. Deville, V. Henriot, C. Velours, D. Ladant, L. Renault, et U. Mechold, « Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin », PloS One, vol. 13, nᵒ 11, p. e0206133, 2018.
    Résumé : Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.
    Mots-clés : ACTIN, adenylate-cyclase, B3S, calmodulin, cytoskeleton disruption, domains, edema factor, exotoxin, PF, PIM, pseudomonas-aeruginosa exoy, structural basis, toxin, yersinia protein-kinase.

  • M. Zaghouani, O. Gayraud, V. Jactel, S. Prévost, A. Dezaire, M. Sabbah, A. Escargueil, T. - L. Lai, C. Le Clainche, N. Rocques, S. Romero, A. Gautreau, F. Blanchard, G. Frison, et B. Nay, « Multifaceted study on a cytochalasin scaffold: lessons on reactivity, multidentate catalysis and anticancer properties », Chemistry (Weinheim an Der Bergstrasse, Germany), vol. 24, nᵒ 62, p. 16686-16691, août 2018.
    Résumé : We report an intramolecular Diels-Alder reaction efficiently accelerated by Schreiner's thiourea, to build a functionalized cytochalasin scaffold (periconiasin series) amenable to biological purpose. DFT calculation highlighted a unique multidentate cooperative hydrogen bonding in this catalysis. The deprotection end-game afforded a collection of diverse structures and showed the peculiar reactivity of the Diels-Alder cycloadducts upon functionalization. Biological studies revealed strong cytotoxicity of a few compounds on breast cancer cell lines, while preserving actin polymerization.
    Mots-clés : acids, ACTIN, B3S, Cytochalasins Thiourea multidentate catalysis Diels-Alder reaction.


  • M. - F. Carlier et S. Shekhar, « Global treadmilling coordinates actin turnover and controls the size of actin networks », Nature Reviews. Molecular Cell Biology, vol. 18, nᵒ 6, p. 389-401, 2017.
    Résumé : Various cellular processes (including cell motility) are driven by the regulated, polarized assembly of actin filaments into distinct force-producing arrays of defined size and architecture. Branched, linear, contractile and cytosolic arrays coexist in vivo, and cells intricately control the number, length and assembly rate of filaments in these arrays. Recent in vitro and in vivo studies have revealed novel molecular mechanisms that regulate the number of filament barbed and pointed ends and their respective assembly and disassembly rates, thus defining classes of dynamically different filaments, which coexist in the same cell. We propose that a global treadmilling process, in which a steady-state amount of polymerizable actin monomers is established by the dynamics of each network, is responsible for defining the size and turnover of coexisting actin networks. Furthermore, signal-induced changes in the partitioning of actin to distinct arrays (mediated by RHO GTPases) result in the establishment of various steady-state concentrations of polymerizable monomers, thereby globally influencing the growth rate of actin filaments.
    Mots-clés : ACTIN.

  • G. Dimchev, A. Steffen, F. Kage, V. Dimchev, J. Pernier, M. - F. Carlier, et K. Rottner, « Efficiency of lamellipodia protrusion is determined by the extent of cytosolic actin assembly », Molecular Biology of the Cell, vol. 28, nᵒ 10, p. 1311-1325, mai 2017.
    Résumé : Cell migration and cell-cell communication involve the protrusion of actin-rich cell surface projections such as lamellipodia and filopodia. Lamellipodia are networks of actin filaments generated and turned over by filament branching through the Arp2/3 complex. Inhibition of branching is commonly agreed to eliminate formation and maintenance of lamellipodial actin networks, but the regulation of nucleation or elongation of Arp2/3-independent filament populations within the network by, for example, formins or Ena/VASP family members and its influence on the effectiveness of protrusion have been unclear. Here we analyzed the effects of a set of distinct formin fragments and VASP on site-specific, lamellipodial versus cytosolic actin assembly and resulting consequences on protrusion. Surprisingly, expression of formin variants but not VASP reduced lamellipodial protrusion in B16-F1 cells, albeit to variable extents. The rates of actin network polymerization followed a similar trend. Unexpectedly, the degree of inhibition of both parameters depended on the extent of cytosolic but not lamellipodial actin assembly. Indeed, excess cytosolic actin assembly prevented actin monomer from rapid translocation to and efficient incorporation into lamellipodia. Thus, as opposed to sole regulation by actin polymerases operating at their tips, the protrusion efficiency of lamellipodia is determined by a finely tuned balance between lamellipodial and cytosolic actin assembly.
    Mots-clés : ACTIN.

  • F. Kage, M. Winterhoff, V. Dimchev, J. Mueller, T. Thalheim, A. Freise, S. Brühmann, J. Kollasser, J. Block, G. Dimchev, M. Geyer, H. - J. Schnittler, C. Brakebusch, T. E. B. Stradal, M. - F. Carlier, M. Sixt, J. Käs, J. Faix, et K. Rottner, « FMNL formins boost lamellipodial force generation », Nature Communications, vol. 8, p. 14832, mars 2017.
    Résumé : Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.
    Mots-clés : ACTIN.

  • A. Le Dur, T. L. Laï, M. - G. Stinnakre, A. Laisné, N. Chenais, S. Rakotobe, B. Passet, F. Reine, S. Soulier, L. Herzog, G. Tilly, H. Rézaei, V. Béringue, J. - L. Vilotte, et H. Laude, « Divergent prion strain evolution driven by PrP(C) expression level in transgenic mice », Nature Communications, vol. 8, p. 14170, janv. 2017.
    Résumé : Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrP(C) into PrP(Sc). Structurally distinct PrP(Sc) conformers can give rise to multiple prion strains. Constrained interactions between PrP(C) and different PrP(Sc) strains can in turn lead to certain PrP(Sc) (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrP(C). Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrP(C). While breeding true in mice expressing PrP(C) at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrP(C) overexpression. Our results support the view that PrP(C) gene dosage can influence prion evolution on homotypic transmission.
    Mots-clés : ACTIN.

  • T. Motomura, M. Suga, R. Hienerwadel, A. Nakagawa, T. - L. Lai, W. Nitschke, T. Kuma, M. Sugiura, A. Boussac, et J. - R. Shen, « Crystal structure and redox properties of a novel cyanobacterial heme-protein with a His/Cys heme axial ligation and a per-arnt-sim (PAS)-like domain », The Journal of Biological Chemistry, avr. 2017.
    Résumé : Photosystem II (PSII) catalyzes the light-induced water oxidation leading to the generation of dioxygen indispensable for sustaining aerobic life on Earth. The PSII reaction center is composed of D1 and D2 proteins encoded by the psbA and psbD genes, respectively. In cyanobacteria, different psbA genes are present in the genome. The thermophilic cyanobacterium Thermosynechococcus elongatus contains 3 psbA genes, psbA1, psbA2 and psbA3 and a new c-type heme protein, Tll0287, was found to be expressed in a strain expressing the psbA2 gene only, but the structure and function of Tll0287 are unknown. Here we solved the crystal structure of Tll0287 at a 2.0 Å resolution. The overall structure of Tll0287 was found to be similar to some kinases and sensor proteins with a per-arnt-sim (PAS)-like domain, rather than to other c-type cytochromes. The 5(th) and 6(th) axial ligands for the heme were Cys and His, instead of the His/Met or His/His ligand pairs observed for most of the c-type hemes. The redox potential, E1/2, of Tll0287 was -255 ± 20 mV versus normal hydrogen electrode at pH values above 7.5. Below this pH value, the E1/2 increased by ≈57 mV/pH unit at 15°C, suggesting the involvement of a protonatable group with a pKred = 7.2 ± 0.3. Possible functions of Tll0287 as a redox sensor under micro-aerobic conditions or a cytochrome subunit of an H2S-oxidising system, are discussed in view of the environmental conditions in which psbA2 is expressed as well as phylogenetic analysis, structural and sequence homologies.
    Mots-clés : ACTIN.

  • S. Shekhar et M. - F. Carlier, « Enhanced Depolymerization of Actin Filaments by ADF/Cofilin and Monomer Funneling by Capping Protein Cooperate to Accelerate Barbed-End Growth », Current biology: CB, juin 2017.
    Résumé : A living cell's ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers, which feed polarized filament growth [1, 2]. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like actin depolymerizing factor (ADF)/cofilin and filament cappers like capping protein (CP) are essential agonists of motility [3-8], but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades [9-12]. Whereas filament fragmentation by ADF/cofilin has long been demonstrated by total internal reflection fluorescence (TIRF) [13, 14], filament depolymerization was only inferred from bulk solution assays [15]. Using microfluidics-assisted TIRF microscopy, we provide the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. Using a conceptually novel assay to directly visualize ADF's effect on a population of pre-assembled filaments, we demonstrate how ADF's enhanced pointed-end depolymerization causes an increase in polymerizable actin monomers, thus promoting faster barbed-end growth. We further reveal that ADF-enhanced depolymerization synergizes with CP's long-predicted "monomer funneling" [16] and leads to skyrocketing of filament growth rates, close to estimated lamellipodial rates. The "funneling model" hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few non-capped filaments transiently grow much faster [15], an effect proposed to be very important for motility. We provide the first direct microscopic evidence of monomer funneling at the scale of individual filaments. These results significantly enhance our understanding of the turnover of cellular actin networks.
    Mots-clés : ACTIN.


  • J. V. G. Abella, C. Galloni, J. Pernier, D. J. Barry, S. Kjær, M. - F. Carlier, et M. Way, « Isoform diversity in the Arp2/3 complex determines actin filament dynamics », Nature Cell Biology, vol. 18, nᵒ 1, p. 76-86, janv. 2016.
    Résumé : The Arp2/3 complex consists of seven evolutionarily conserved subunits (Arp2, Arp3 and ARPC1-5) and plays an essential role in generating branched actin filament networks during many different cellular processes. In mammals, however, the ARPC1 and ARPC5 subunits are each encoded by two isoforms that are 67% identical. This raises the possibility that Arp2/3 complexes with different properties may exist.  We found that Arp2/3 complexes containing ARPC1B and ARPC5L are significantly better at promoting actin assembly than those with ARPC1A and ARPC5, both in cells and in vitro. Branched actin networks induced by complexes containing ARPC1B or ARPC5L are also disassembled ∼2-fold slower than those formed by their counterparts. This difference reflects the ability of cortactin to stabilize ARPC1B- and ARPC5L- but not ARPC1A- and ARPC5-containing complexes against coronin-mediated disassembly. Our observations demonstrate that the Arp2/3 complex in higher eukaryotes is actually a family of complexes with different properties.
    Mots-clés : ACTIN.

  • A. Belyy, D. Raoux-Barbot, C. Saveanu, A. Namane, V. Ogryzko, L. Worpenberg, V. David, V. Henriot, S. Fellous, C. Merrifield, E. Assayag, D. Ladant, L. Renault, et U. Mechold, « Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins », Nature Communications, vol. 7, p. 13582, déc. 2016.

  • C. Deville, C. Girard-Blanc, N. Assrir, N. Nhiri, E. Jacquet, F. Bontems, L. Renault, S. Petres, et C. van Heijenoort, « Mutations in actin used for structural studies partially disrupt β-thymosin/WH2 domains interaction », FEBS Letters, vol. 590, nᵒ 20, p. 3690-3699, 2016.

  • P. Montaville, S. Kühn, C. Compper, et M. - F. Carlier, « Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments », The Journal of Biological Chemistry, vol. 291, nᵒ 7, p. 3302-3318, févr. 2016.
    Résumé : Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end.
    Mots-clés : ACTIN.

  • J. Pernier, S. Shekhar, A. Jegou, B. Guichard, et M. - F. Carlier, « Profilin Interaction with Actin Filament Barbed End Controls Dynamic Instability, Capping, Branching, and Motility », Developmental Cell, vol. 36, nᵒ 2, p. 201-214, janv. 2016.
    Résumé : Cell motility and actin homeostasis depend on the control of polarized growth of actin filaments. Profilin, an abundant regulator of actin dynamics, supports filament assembly at barbed ends by binding G-actin. Here, we demonstrate how, by binding and destabilizing filament barbed ends at physiological concentrations, profilin also controls motility, cell migration, and actin homeostasis. Profilin enhances filament length fluctuations. Profilin competes with Capping Protein at barbed ends, which generates a lower amount of profilin-actin than expected if barbed ends were tightly capped. Profilin competes with barbed end polymerases, such as formins and VopF, and inhibits filament branching by WASP-Arp2/3 complex by competition for filament barbed ends, accounting for its as-yet-unknown effects on motility and metastatic cell migration observed in this concentration range. In conclusion, profilin is a major coordinator of polarized growth of actin filaments, controlled by competition between barbed end cappers, trackers, destabilizers, and filament branching machineries.
    Mots-clés : ACTIN.

  • L. Renault, « Intrinsic, Functional, and Structural Properties of β-Thymosins and β-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling », Vitamins and Hormones, vol. 102, p. 25-54, 2016.
    Résumé : β-Thymosins are a family of heat-stable multifunctional polypeptides that are expressed as small proteins of about 5kDa (~45 amino acids) almost exclusively in multicellular animals. They were first isolated from the thymus. As full-length or truncated polypeptides, they appear to stimulate a broad range of extracellular activities in various signaling pathways, including tissue repair and regeneration, inflammation, cell migration, and immune defense. However, their cell surface receptors and structural mechanisms of regulations in these multiple pathways remain still poorly understood. Besides their extracellular activities, they belong to a larger family of small, intrinsically disordered actin-binding domains called WH2/β-thymosin domains that have been identified in more than 1800 multidomain proteins found in different taxonomic domains of life and involved in various actin-based motile processes including cell morphogenesis, motility, adhesions, tissue development, intracellular trafficking, or pathogen infections. This review briefly surveys the main recent findings to understand how these small, intrinsically disordered but functional domains can interact with many unrelated partners and can thus integrate and coordinate various intracellular activities in actin self-assembly dynamics and cell signaling pathways linked to their cytoskeleton remodeling.
    Mots-clés : ACTIN.

  • S. Shekhar et M. - F. Carlier, « Single-filament kinetic studies provide novel insights into regulation of actin-based motility », Molecular Biology of the Cell, vol. 27, nᵒ 1, p. 1-6, janv. 2016.
    Résumé : Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles that drive actin remodeling have provided a mechanistic and quantitative understanding of such processes. We specifically consider key barbed-end regulators such as capping protein and formins as illustrative examples. We compare and contrast different kinetic approaches, such as the traditional pyrene-polymerization bulk assays, as well as more recently developed single-filament and single-molecule imaging approaches. Recent development of novel biophysical methods for sensing and applying forces will in future allow us to address the very important relationship between mechanical stimulus and kinetics of actin-based motility.
    Mots-clés : ACTIN.

  • S. Shekhar, J. Pernier, et M. - F. Carlier, « Regulators of actin filament barbed ends at a glance », Journal of Cell Science, vol. 129, nᵒ 6, p. 1085-1091, mars 2016.
    Résumé : Cells respond to external stimuli by rapidly remodeling their actin cytoskeleton. At the heart of this function lies the intricately controlled regulation of individual filaments. The barbed end of an actin filament is the hotspot for the majority of the biochemical reactions that control filament assembly. Assays performed in bulk solution and with single filaments have enabled characterization of a plethora of barbed-end-regulating proteins. Interestingly, many of these regulators work in tandem with other proteins, which increase or decrease their affinity for the barbed end in a spatially and temporally controlled manner, often through simultaneous binding of two regulators at the barbed ends, in addition to standard mutually exclusive binding schemes. In this Cell Science at a Glance and the accompanying poster, we discuss key barbed-end-interacting proteins and the kinetic mechanisms by which they regulate actin filament assembly. We take F-actin capping protein, gelsolin, profilin and barbed-end-tracking polymerases, including formins and WH2-domain-containing proteins, as examples, and illustrate how their activity and competition for the barbed end regulate filament dynamics.
    Mots-clés : ACTIN.


  • B. S. Avvaru, J. Pernier, et M. - F. Carlier, « Dimeric WH2 repeats of VopF sequester actin monomers into non-nucleating linear string conformations: An X-ray scattering study », Journal of Structural Biology, vol. 190, nᵒ 2, p. 192-199, mai 2015.
    Résumé : VopF and VopL are highly similar virulence-factors of Vibrio cholerae and Vibrio parahaemolyticus respectively that disrupt the host's actin cytoskeleton, using a unique organization in dimerized WH2 repeats. Association of dimerized WH2 domains with the barbed face of actin confers multifunctional activities to VopF in vitro, including G-actin sequestration and filament nucleation, barbed end tracking and uncapping. Here, small angle X-ray scattering (SAXS) measurements of complexes of VopF with actin and structural modeling reveal that VopF stabilizes linear actin-strings that differ from canonical actin filament architectures but represent non-polymerizable sequestered forms of actin. The results exclude that VopL binds the pointed end of actin filaments in the template filament nucleation mechanism derived from crystallographic studies.
    Mots-clés : ACTIN.

  • M. - F. Carlier, J. Pernier, P. Montaville, S. Shekhar, S. Kühn, et Cytoskeleton Dynamics and Motility group, « Control of polarized assembly of actin filaments in cell motility », Cellular and molecular life sciences: CMLS, vol. 72, nᵒ 16, p. 3051-3067, août 2015.
    Résumé : Actin cytoskeleton remodeling, which drives changes in cell shape and motility, is orchestrated by a coordinated control of polarized assembly of actin filaments. Signal responsive, membrane-bound protein machineries initiate and regulate polarized growth of actin filaments by mediating transient links with their barbed ends, which elongate from polymerizable actin monomers. The barbed end of an actin filament thus stands out as a hotspot of regulation of filament assembly. It is the target of both soluble and membrane-bound agonists as well as antagonists of filament assembly. Here, we review the molecular mechanisms by which various regulators of actin dynamics bind, synergize or compete at filament barbed ends. Two proteins can compete for the barbed end via a mutually exclusive binding scheme. Alternatively, two regulators acting individually at barbed ends may be bound together transiently to terminal actin subunits at barbed ends, leading to the displacement of one by the other. The kinetics of these reactions is a key in understanding how filament length and membrane-filament linkage are controlled. It is also essential for understanding how force is produced to shape membranes by mechano-sensitive, processive barbed end tracking machineries like formins and by WASP-Arp2/3 branched filament arrays. A combination of biochemical and biophysical approaches, including bulk solution assembly measurements using pyrenyl-actin fluorescence, single filament dynamics, single molecule fluorescence imaging and reconstituted self-organized filament assemblies, have provided mechanistic insight into the role of actin polymerization in motile processes.
    Mots-clés : ACTIN.

  • C. Ciobanasu, B. Faivre, et C. Le Clainche, « Reconstituting actomyosin-dependent mechanosensitive protein complexes in vitro », Nature Protocols, vol. 10, nᵒ 1, p. 75-89, janv. 2015.
    Résumé : In many mechanosensitive biological processes, actin-binding proteins (ABPs) sense the force generated by the actomyosin cytoskeleton and respond by recruiting effector proteins. We developed an in vitro assay, with pure proteins, to observe the force-dependent binding of a protein to a cryptic binding site buried in the stretchable domain of an ABP. Here we describe the protocol to study the actomyosin-dependent binding of vinculin to the ABP talin. In this assay, talin is immobilized in 5-μm-diameter disc-shaped islands, which are regularly spaced by 35 μm and micropatterned on a glass coverslip. In response to the force generated by an actomyosin network, talin extension reveals cryptic vinculin-binding sites (VBSs). To follow this reaction, fluorescent proteins are visualized by total internal refection fluorescence (TIRF) microscopy. EGFP-vinculin fluorescence in talin-coated discs reveals the binding of vinculin to stretched talin. Actomyosin structures are visualized by the fluorescence of Alexa Fluor 594-labeled actin. This protocol describes the purification of the proteins, the preparation of the chamber in which talin is coated on a micropatterned surface, and the biochemical conditions to study several kinetic parameters of the actomyosin-dependent binding of vinculin to talin. A stable actomyosin network is used to measure the steady-state dissociation of vinculin from talin under constant force. In the presence of α-actinin-1, actomyosin cables undergo cycles of force application and release, allowing the measurement of vinculin dissociation associated with talin re-folding. Expression and purification of the proteins requires at least 3 weeks. The assay can be completed within 1 d.
    Mots-clés : ACTIN.

  • I. Herrada, C. Samson, C. Velours, L. Renault, C. Östlund, P. Chervy, D. Puchkov, H. J. Worman, B. Buendia, et S. Zinn-Justin, « Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Self-assembly Properties », ACS chemical biology, vol. 10, nᵒ 12, p. 2733-2742, déc. 2015.
    Résumé : More than 100 genetic mutations causing X-linked Emery-Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding emerin variants' loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of β-structures. Analysis of the assembly properties of five EmN variants reveals that del95-99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of emerin-emerin and emerin-lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to emerin aggregation and mislocalization in cells. Our results suggest that emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.
    Mots-clés : ACTIN.

  • S. Shekhar, M. Kerleau, S. Kühn, J. Pernier, G. Romet-Lemonne, A. Jégou, et M. - F. Carlier, « Formin and capping protein together embrace the actin filament in a ménage à trois », Nature Communications, vol. 6, p. 8730, nov. 2015.
    Résumé : Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed.
    Mots-clés : ACTIN.


  • C. Ciobanasu, B. Faivre, et C. Le Clainche, « Actomyosin-dependent formation of the mechanosensitive talin-vinculin complex reinforces actin anchoring », Nature Communications, vol. 5, p. 3095, 2014.
    Résumé : The force generated by the actomyosin cytoskeleton controls focal adhesion dynamics during cell migration. This process is thought to involve the mechanical unfolding of talin to expose cryptic vinculin-binding sites. However, the ability of the actomyosin cytoskeleton to directly control the formation of a talin-vinculin complex and the resulting activity of the complex are not known. Here we develop a microscopy assay with pure proteins in which the self-assembly of actomyosin cables controls the association of vinculin to a talin-micropatterned surface in a reversible manner. Quantifications indicate that talin refolding is limited by vinculin dissociation and modulated by the actomyosin network stability. Finally, we show that the activation of vinculin by stretched talin induces a positive feedback that reinforces the actin-talin-vinculin association. This in vitro reconstitution reveals the mechanism by which a key molecular switch senses and controls the connection between adhesion complexes and the actomyosin cytoskeleton.
    Mots-clés : ACTIN, Actinin, Actins, Actomyosin, Animals, B3S, Feedback, Physiological, Humans, Mechanotransduction, Cellular, Models, Biological, Protein Binding, Protein Folding, Rabbits, Talin, Vinculin.
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Sélection de publications entre 2001-2014

# auteur de correspondance, gras : membres de l’équipe

- Carlier MF, Romet-Lemonne G, Jégou A. (2014) Actin filament dynamics using microfluidics.
Methods Enzymol. 540:3-17 PubMed

-  Mueller J, Pfanzelter J, Winkler C, Narita A, Le Clainche C, Nemethova M, Carlier MF, Maeda Y, Welch MD, Ohkawa T, Schmeiser C, Resch GP, Small JV. (2014) Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.
PLoS Biol. 12(1):e1001765 PubMed

-  Ciobanasu C, Faivre B, Le Clainche C. (2014) Actomyosin-dependent formation of the mechanosensitive talin-vinculin complex reinforces actin anchoring.
Nat Commun. 5:3095 PubMed

- Jiao Y, Walker M, Trinick J, Pernier J, Montaville P, Carlier MF. (2014) Mutagenetic and electron microscopy analysis of actin filament severing by Cordon-Bleu, a WH2 domain protein.
Cytoskeleton (Hoboken).71(3):170-83 PubMed

- Ciobanasu C, Faivre B, Le Clainche C#. (2013) Integrating actin dynamics, mechanotransduction and integrin activation : the multiple functions of actin binding proteins in focal adhesions.
Eur J Cell Biol. 92(10-11), 339-48. Review.

- Renault, L#, Deville, C., van Heijenoort C. (2013) Structural features and interfacial properties of WH2, β-Thymosin domains and other intrinsically disordered domains in the regulation of actin cytoskeleton dynamics.
Cytoskeleton (Hoboken) 70(11):686-705. Review.

- Dias J, Renault L#, Pérez J, Mirande M#. (2013) Small-angle X-ray solution scattering study of the multi-aminoacyl-tRNA synthetase complex reveals an elongated and multi-armed particle.
J Biol Chem. 288(33), 23979-89.

- D. Didry, F.X. Cantrelle, C. Husson, P. Roblin, A. M. Eswara Moorthy, J. Perez, C. Le Clainche, M. Hertzog, E. Guittet, M.F. Carlier, C. van Heijenoort# and L. Renault#. (2012) How a Single Residue in Individual ß-Thymosin/WH2 Domains Controls their Functions in Actin Assembly.
EMBO J. 31(4), 1000-13

- C. Husson, L. Renault, D. Didry, D. Pantaloni, M.F. Carlier #. (2011) Cordon-Bleu uses WH2 domains as multifunctional dynamizers of actin filament assembly.
Molecular Cell 43, 464-77.

- C. Le Clainche#, S. Prakash Dwivedi, D. Didry and M.F. Carlier (2010) Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein.
J. Biol Chem. 285(30),23420-32.

- Le Clainche C#, Carlier M (2008). Regulation of actin assembly associated with protrusion and adhesion in cell migration.
Physiological Reviews. 88(2):489-513. Review.

- Le Clainche C, Pauly BS, Zhang CX, Engqvist-Goldstein AE, Cunningham K, Drubin DG. (2007) A Hip1R-cortactin complex negatively regulates actin assembly associated with endocytosis.
EMBO J. 26(5), 1199-210.

- Bosch M, Le KH, Bugyi B,Correia JJ, Renault L#, Carlier MF#. (2007) Analysis of the Function of Spire in Actin Assembly and Its Synergy with Formin and Profilin.
Molecular Cell 28(4), 555-568.

- Ghosh A., Praefcke G. J. K., Renault L.#, Wittinghofer A.#, Herrmann C. (2006) How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP.
Nature 440, 101-4.

- Romero S, Le Clainche C, Didry D, Egile C, Pantaloni D, Carlier MF# . (2004) Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis.
Cell 119(3), 419-29.

- C. Le Clainche, D. Pantaloni and M.F. Carlier #. (2003) ATP hydrolysis on Arp2/3 complex causes debranching of dendritic actin arrays.
Proceedings of the National Academy of Sciences of USA 100(11), 6337-42.

- Renault L., Guibert B., Cherfils J. # (2003) Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor.
Nature 426, 525-530.

- Renault L., Kuhlmann J., Henkel A. & Wittinghofer A. # (2001) Structural basis for guanine nucleotide exchange on Ran by the Regulator of Chromosome Condensation (RCC1).
Cell 105, 245-255.

- Pantaloni D, Le Clainche C, Carlier MF# (2001). Mechanism of actin-based motility.
Science 292(5521), 1502-6. Review.

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