CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP
2013; Springer Nature; Volume: 32; Issue: 20 Linguagem: Inglês
10.1038/emboj.2013.208
ISSN1460-2075
AutoresAndrea Disanza, Sara Bisi, Moritz Winterhoff, Francesca Milanesi, Dmitry S. Ushakov, David J. Kast, Paola Marighetti, Guillaume Romet‐Lemonne, Hans‐Michael Müller, Walter Nickel, Joern Linkner, Davy Waterschoot, Christophe Ampè, Salvatore Cortellino, Andrea Palamidessi, Roberto Domínguez, Marie‐France Carlier, Jan Faix, Giorgio Scita,
Tópico(s)Cancer-related Molecular Pathways
ResumoArticle27 September 2013free access Source Data CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP Andrea Disanza Andrea Disanza IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Sara Bisi Sara Bisi IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Moritz Winterhoff Moritz Winterhoff Hannover Medical School, Institute for Biophysical Chemistry, Hannover, Germany Search for more papers by this author Francesca Milanesi Francesca Milanesi IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, ItalyPresent address: Center for Genomic Science of [email protected], Istituto Italiano di Tecnologia, at the IFOM-IEO Campus, Milan, Italy Search for more papers by this author Dmitry S Ushakov Dmitry S Ushakov Hannover Medical School, Institute for Biophysical Chemistry, Hannover, GermanyPresent address: Faculty of Biological Sciences, University of Leeds, Leeds, UK Search for more papers by this author David Kast David Kast Department of Physiology, Perelman School of Medicine Philadelphia, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Paola Marighetti Paola Marighetti IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Guillaume Romet-Lemonne Guillaume Romet-Lemonne Cytoskeleton Dynamics and Motility Group, Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France Search for more papers by this author Hans-Michael Müller Hans-Michael Müller Heidelberg University Biochemistry Center, Heidelberg, Germany Search for more papers by this author Walter Nickel Walter Nickel Heidelberg University Biochemistry Center, Heidelberg, Germany Search for more papers by this author Joern Linkner Joern Linkner Hannover Medical School, Institute for Biophysical Chemistry, Hannover, Germany Search for more papers by this author Davy Waterschoot Davy Waterschoot Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium Search for more papers by this author Christophe Ampè Christophe Ampè Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium Search for more papers by this author Salvatore Cortellino Salvatore Cortellino IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Andrea Palamidessi Andrea Palamidessi IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Roberto Dominguez Roberto Dominguez Department of Physiology, Perelman School of Medicine Philadelphia, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Marie-France Carlier Marie-France Carlier Cytoskeleton Dynamics and Motility Group, Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France Search for more papers by this author Jan Faix Corresponding Author Jan Faix Hannover Medical School, Institute for Biophysical Chemistry, Hannover, Germany Search for more papers by this author Giorgio Scita Corresponding Author Giorgio Scita IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Dipartimento di Scienze della Salute, Universita’ degli Studi di Milano, Milan, Italy Search for more papers by this author Andrea Disanza Andrea Disanza IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Sara Bisi Sara Bisi IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Moritz Winterhoff Moritz Winterhoff Hannover Medical School, Institute for Biophysical Chemistry, Hannover, Germany Search for more papers by this author Francesca Milanesi Francesca Milanesi IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, ItalyPresent address: Center for Genomic Science of [email protected], Istituto Italiano di Tecnologia, at the IFOM-IEO Campus, Milan, Italy Search for more papers by this author Dmitry S Ushakov Dmitry S Ushakov Hannover Medical School, Institute for Biophysical Chemistry, Hannover, GermanyPresent address: Faculty of Biological Sciences, University of Leeds, Leeds, UK Search for more papers by this author David Kast David Kast Department of Physiology, Perelman School of Medicine Philadelphia, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Paola Marighetti Paola Marighetti IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Guillaume Romet-Lemonne Guillaume Romet-Lemonne Cytoskeleton Dynamics and Motility Group, Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France Search for more papers by this author Hans-Michael Müller Hans-Michael Müller Heidelberg University Biochemistry Center, Heidelberg, Germany Search for more papers by this author Walter Nickel Walter Nickel Heidelberg University Biochemistry Center, Heidelberg, Germany Search for more papers by this author Joern Linkner Joern Linkner Hannover Medical School, Institute for Biophysical Chemistry, Hannover, Germany Search for more papers by this author Davy Waterschoot Davy Waterschoot Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium Search for more papers by this author Christophe Ampè Christophe Ampè Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium Search for more papers by this author Salvatore Cortellino Salvatore Cortellino IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Andrea Palamidessi Andrea Palamidessi IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Search for more papers by this author Roberto Dominguez Roberto Dominguez Department of Physiology, Perelman School of Medicine Philadelphia, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Marie-France Carlier Marie-France Carlier Cytoskeleton Dynamics and Motility Group, Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France Search for more papers by this author Jan Faix Corresponding Author Jan Faix Hannover Medical School, Institute for Biophysical Chemistry, Hannover, Germany Search for more papers by this author Giorgio Scita Corresponding Author Giorgio Scita IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Dipartimento di Scienze della Salute, Universita’ degli Studi di Milano, Milan, Italy Search for more papers by this author Author Information Andrea Disanza1,‡, Sara Bisi1,‡, Moritz Winterhoff2, Francesca Milanesi1, Dmitry S Ushakov2, David Kast3, Paola Marighetti1, Guillaume Romet-Lemonne4, Hans-Michael Müller5, Walter Nickel5, Joern Linkner2, Davy Waterschoot6, Christophe Ampè6, Salvatore Cortellino1, Andrea Palamidessi1, Roberto Dominguez3, Marie-France Carlier4, Jan Faix 2 and Giorgio Scita 1,7 1IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy 2Hannover Medical School, Institute for Biophysical Chemistry, Hannover, Germany 3Department of Physiology, Perelman School of Medicine Philadelphia, University of Pennsylvania, Philadelphia, PA, USA 4Cytoskeleton Dynamics and Motility Group, Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France 5Heidelberg University Biochemistry Center, Heidelberg, Germany 6Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium 7Dipartimento di Scienze della Salute, Universita’ degli Studi di Milano, Milan, Italy ‡These authors contributed equally to this work *Corresponding authors. Hannover Medical School, Institute for Biophysical Chemistry, OE 4350, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Tel.:+49 511 532 2928; Fax:+49 511 532 5966; E-mail: [email protected] di Scienze della Salute, Universita’ degli Studi di Milano, IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, via Adamello, 16, Milan 20141, Italy. Tel.:+39 02574303277; Fax:+39 02574303231; E-mail: [email protected] The EMBO Journal (2013)32:2735-2750https://doi.org/10.1038/emboj.2013.208 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high-density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53-dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair. Introduction Cells move and interact with the environment by forming migratory structures composed of self-organized polymers of actin. These protrusions can be flat-sheet lamellipodia, or elongated, finger-like filopodia. Cells use lamellipodia as expanding membrane sheets driving cell locomotion (Wu et al, 2012), while filopodia act as compasses navigating cell migration (Faix et al, 2009; Yang and Svitkina, 2011a), as well as mechanical sensors (Chan and Odde, 2008). In lamellipodia, actin is organized in networks of branched filaments (Svitkina et al, 2003; Yang and Svitkina, 2011b), generated by the concerted action of the Arp2/3 complex, capping proteins, and filament elongation factors (Svitkina and Borisy, 1999; Wiesner et al, 2003; Akin and Mullins, 2008). Conversely, filopodia are composed of parallel actin bundles that emanate from the cell periphery (Adams, 2004). However, in several cell types, filopodia and lamellipodia rapidly interchange during protrusion. Furthermore, filopodia are frequently embedded into, or arise from, pre-existing lamellipodia (Rottner et al, 1999; Svitkina et al, 2003). This finding suggests that filopodia form through a reorganization of the underlying actin network by convergent elongation of pre-existing filaments, or by de novo actin nucleation and elongation (Faix et al, 2009; Yang and Svitkina, 2011a). Whatever the case, the occurrence of lamellipodia-to-filopodia transition suggests commonalities between the two structures. For example, while it appears that capping proteins, including CP and EPS8, determine which actin-based protrusive organelle dominates at the cell periphery (Mejillano et al, 2004; Akin and Mullins, 2008; Vaggi et al, 2011), they do not function in isolation. The ENA/VASP family of proteins (Gertler et al, 1995; Reinhard et al, 1995; Gertler et al, 1996), which in mammals includes MENA, VASP, and EVL, also influence the underlying actin architecture of migratory protrusions (Bear et al, 2002). Notably, by cellular localization alone, ENA/VASP proteins appear to be vital to this lamellipodial–filopodial transition since they localize to active sites of actin assembly, such as the tips of protruding lamellipodia and filopodia (Lanier et al, 1999; Rottner et al, 1999). At the leading edge, the intensity of GFP-VASP has been shown to increase locally in puncta that subsequently give rise to filopodia, suggesting that higher order clustering of this protein may be critical for generating linear filaments to support filopodia protrusions (Lanier et al, 1999; Rottner et al, 1999; Svitkina et al, 2003; Applewhite et al, 2007). Biochemical studies have recently shown that soluble VASP displays weak, processive polymerase activity (Hansen and Mullins, 2010). High-density VASP clustering, however, is required to enhance processive filament elongation, even in the presence of high concentrations of capping proteins (Breitsprecher et al, 2008, 2011). Not surprisingly, genetic evidence in various organisms showed that ENA/VASP proteins are essential players in filopodia formation (Schirenbeck et al, 2006; Gates et al, 2007; Kwiatkowski et al, 2007). Whether and how the activity and higher order clustering of ENA/VASP family proteins is differentially tuned to control lamellipodia-to-filopodia transition remains, however, poorly defined. Molecules sitting at the actin:membrane interface are predicted to be important in this process, albeit their nature and mechanisms of action remain unclear. One candidate is IRSp53 (Insulin Receptor Substrate of 53 kDa) (also called BAIAP2, brain angiogenic inhibitor interacting protein 2) (Abbott et al, 1999; Oda et al, 1999; Okamura-Oho et al, 1999). IRSp53 possesses an inverted Bin-Amphiphysin-Rvs167 (I-BAR) domain that binds to PI(4,5)P2-rich lipid inducing negative curvatures, such as the ones required for filopodia protrusions (Zhao et al, 2011). Consistently, IRSp53 expression is sufficient to induce filopodia-like structures (Bockmann et al, 2002; Yamagishi et al, 2004; Disanza et al, 2006). Furthermore, IRSp53 binds activated CDC42 and, through its SH3 domain, a number of actin regulatory proteins that are involved in filopodia protrusions (Ahmed et al, 2010). Among them, IRSp53 was reported to bind to MENA in vitro (Krugmann et al, 2001) and to VASP in FRET-based and co-immunoprecipitation assays (Lim et al, 2008; Vaggi et al, 2011). Interestingly, the interaction between VASP and IRSp53 enhances the bundling activity of the former (Lim et al, 2008; Vaggi et al, 2011). However, it is unknown whether IRSp53 affects other key biochemical activities of MENA and VASP, and what the functional consequences of these interactions are. Here, we show that IRSp53 alone slows down barbed end growth. Binding to CDC42 relieves this inhibition and promotes IRSp53-dependent recruitment and clustering of VASP to the plasma membrane to initiate processive F-actin elongation. These events result in spatial restriction of VASP activity to initiate filopodia, drive cell migration, and promote tissue repair. Results Characterization of the IRSp53–VASP interaction We initially characterized the interaction between IRSp53 and VASP using purified proteins and various binding assays. IRSp53 associates with VASP, as well as EVL, through its SH3 domain (Supplementary Figure S1a–f) that contacts VASP on proline-rich sites partially overlapping, but distinct from the Profilin binding sites (Supplementary Figure S1c and g). Next, since VASP has recently been shown to acquire processive elongation activity upon clustering and IRSp53 can form dimer through its I-BAR domain (Millard et al, 2005), we tested whether in solution the two proteins can form high order oligomer by dynamic light scattering. Purified IRSp53 produced a single major species with a hydrodynamic radius of about 7.2 nm (Figure 1A) consistent with its dimeric structure (similar results were obtained by hydrodynamic measurements; Supplementary Figure S2a), while the VASP tetramer gave rise to a single species with a radius of about 14.3 nm (Figure 1A). Notably, the mixture of both proteins led to the formation of large clusters with an average diameter of about 200 nm (Figure 2A, left). Importantly, the VASP-ΔPRD mutant, which is unable to bind to IRSp53 (Supplementary Figure S1e), and the IRSp53 W413G mutant, which lacks VASP binding ability (Supplementary Figure S1c), did not form heterocomplexes (Figure 1A, right, and data not shown). In contrast, a VASP mutant lacking the central three GP5 motifs (VASP-ΔGP5), which retains IRSp53 binding ability, albeit with reduced affinity (Supplementary Figure S1d and f), was still able to hetero-oligomerize upon addition of IRSp53 (Figure 1A, middle). Thus, IRSp53 promotes clustering of VASP in solution, and might therefore be a decisive factor in the regulation of VASP function in vitro and in vivo. Figure 1.IRSp53 slows down barbed end growth: an effect relieved by CDC42. (A) IRSp53 and VASP form large clusters in solution. Intensity weighted diameter distributions for IRSp53 and VASP showed single species for either IRSp53 (red lines) or VASP WT and mutant proteins alone at concentrations of up to 30 μM (green lines). Aggregates formed in mixtures of 10 μM IRSp53 and 10 μM VASP WT or VASPΔ(GP5)3, whereas no clusters were observed with VASP-ΔPRD (blue lines). (B) VASP increases the association rate constant of profilin-actin to barbed ends. Barbed end growth was measured in bulk pyrenyl-actin polymerization assays using 1.25 μM MgATP-G-actin (5% pyrenyl-labelled), 5 μM profilin, and VASP (V) at 0 (blue lines), 64 nM (red lines), and 169 nM (green lines), in the absence (dotted lines) or in the presence (continuous lines) of 0.16 nM spectrin-actin seeds. Inset shows cumulated data from additional assays performed with different VASP concentrations. (C) IRSp53 slows down actin polymerization by weakly capping barbed ends. Kinetics of actin polymerization induced by spectrin-actin seeds, measured as described in (A), in the presence of the indicated concentrations of IRSp53. (D) IRSp53 and VASP inhibit barbed end depolymerization. Depolymerization of actin filaments (2.5 μM F-actin, 50% pyrenyl-labelled) was induced by 50-fold dilution into polymerization buffer in the presence of 0.29 μM IRSp53 (red curve) or 0.34 μM VASP (green curve). (E) Summary of barbed end growth inhibition (Cap) by the indicated IRSp53 constructs and mutants (see also Supplementary Figure S2). (F) Active CDC42 relieves IRSp53 capping activity. Kinetics of actin polymerization induced by spectrin-actin seeds was measured as described in (A) in the presence of 0.58 μM IRSp53, alone or with 10 μM CDC42-GTP or CDC42-GDP. Control (CTR) sample contained no IRSp53. (G) Kinetics of actin polymerization induced by spectrin-actin seeds was measured as described in (A) in the presence of the indicated concentrations of IRSp53 or its truncated mutant 1–375. Right graph represents a magnification of the initial phase of elongation by seeds (inset in the left graph). Download figure Download PowerPoint Figure 2.IRSp53 recruits and clusters Ena/VASP proteins to drive processive actin filament elongation in the presence of capping protein. (A) IRSp53 inhibits barbed end elongation. (Left) Polymerization of 1.0 μM G-actin (20% Atto-488 labelled) in the absence or presence of 1 μM IRSp53 in 1 × TIRF buffer monitored by TIRF microscopy. Time is indicated in seconds in the top right corner of each frame. Scale bar, 10 μm. (Right) Quantification of actin filament elongation rates in the absence or presence of IRSp53 as monitored by TIRF microscopy. Elongation rates are presented as mean±s.e.m. from three independent experiments. At least 24 filaments per condition/experiment were measured. *, P<0.05 and **, P<0.01 (T-test, compared to 1 μM actin). (B) TIRF micrographs of the assembly of 1.0 μM G-actin (20% Atto-488 labelled) on uncoated (red arrowhead) or His-IRSp53-saturated Ni-NTA beads in the presence of 50 nM capping protein and 200 nM VASP, EVL, or hVASP DdGAB. Blue arrowheads indicate buckling filaments (also see Supplementary Movie 1). Only filaments that were attached with their barbed ends to the beads were observed, whereas capping protein largely abolished filament growth in solution. Time is indicated in seconds in the upper right corner of each frame. Scale bar, 10 μm. (C) Quantification of actin filament elongation rates from experiment shown in (B). Data are presented as mean±s.d. of three independent experiments, in which at least 16 filaments/experiment were measured. ***, P<0.005 (T-test, compared to His-VASP control). Download figure Download PowerPoint IRSp53-mediated inhibition of barbed end growth is relieved by CDC42 Given the established role of VASP as an actin elongation factor with a relatively weak filament nucleation activity (Huttelmaier et al, 1999; Samarin et al, 2003; Barzik et al, 2005; Breitsprecher et al, 2008; Pasic et al, 2008), we next assessed whether IRSp53 regulates VASP filament elongation in bulk pyrenyl-actin polymerization assays. In this assay, VASP increased the rate (up to 2.5-fold) of spectrin-actin seeded barbed end growth from both ATP-G-actin (data not shown) and profilin-ATP-G-actin in an identical and dose-dependent fashion, under conditions in which its nucleating effect was negligible (Figure 1B). In contrast, IRSp53 slowed down barbed end growth by ∼10-fold, in a substoichiometric concentration range with respect to G-actin, indicating that the interaction of IRSp53 with barbed ends, rather than with G-actin, mediates this inhibitory effect (Figure 1C). The interaction of VASP and IRSp53 with barbed ends also inhibited dilution-induced depolymerization of filaments (Figure 1D). A structure/function analysis of IRSp53 domains mediating barbed end growth inhibition revealed that the SH3 domain binding activity is dispensable. Indeed, an IRSp53-W413G mutant inhibited barbed end growth to the same extent as wild-type (WT) IRSp53, but bound to barbed ends instantaneously with a Kd of 0.29 μM (Figure 1E, scheme; Supplementary Figure S2b and c). In contrast, the isolated I-BAR domain of IRSp53 had no effect on barbed end growth (Figure 1E-scheme; Supplementary Figure S2b). We determined that the minimal region that mediates barbed end growth inhibition includes the I-BAR domain and a stretch of 30 amino acids just before the SH3 domain (Figure 1E, scheme; Supplementary Figure S2d). These results suggest that IRSp53 uses multiple interaction surfaces for inhibiting barbed end growth (e.g., the I-BAR domain to bind to filaments and additional surfaces to dock onto the barbed end protomers) or that it adopts an appropriate three-dimensional conformation centred on the dimeric I-BAR domain. Indeed, IRSp53 has recently been shown by the Dominguez group (in a work submitted elsewhere) to fold into a closed conformation held together by an intramolecular interaction between the SH3 domain and a proline-rich region that is part of an atypical CDC42 binding interface. Mutation of these prolines (P278DA, P281D) leads to an ‘open’ IRSp53 conformation, which was capable of inhibiting barbed end growth, albeit slightly less efficiently than WT IRSp53 (Supplementary Figure S2d). CDC42-GTPγS abolished the slowing down of barbed end growth by IRSp53-WT and the proline mutant, but not by a CDC42 binding-deficient mutant (IRSp53-I267A-S268A; Figure 1E; Supplementary Figure S2e). Importantly, only active GTP-loaded CDC42, but not the GDP-bound form, relieved IRSp53-mediated inhibition of barbed end elongation (Figure 1F). Thus, IRSp53 can inhibit barbed end growth in both the ‘closed’ and ‘open’ conformation, and CDC42 relieves this inhibition presumably by sterically hindering the IRSp53:barbed end interaction. Notably, inhibition of barbed end elongation by IRSp53 developed slowly during filament growth (Figure 1C). This finding is further supported by data showing that steady-state amounts of assembled actin were unaffected by the presence of IRSp53 and/or VASP at the concentrations tested (Supplementary Figure S2f). Furthermore, an IRSp53 mutant retaining the minimal surfaces for barbed end inhibition, but lacking the entire SH3 domain (and thus presumably adopting an open conformation) instantaneously inhibited barbed end growth (IRSp53-1–374; Figure 1G), similarly to IRSp53-W413G (Supplementary Figure S2b). Finally, we monitored filament elongation in real time by in vitro TIRF microscopy in the presence of IRSp53. Similarly to our bulk spectrin-actin seed assays, addition of increasing concentrations of IRSp53 significantly slowed down barbed growth by ∼30% (Figure 2A). This effect was reverted by the addition of activated CDC42 (Supplementary Figure S2g). It must be noted, however, that TIRF data were collected in the early phase of filament elongation, between 0 and 400 s. In this time frame, we also detected partial inhibition of growth in bulk polymerization spectrin-actin seed assays. These data provide further evidence for the slow kinetics of IRSp53 association to barbed ends. To detect more extensive barbed end growth inhibition by TIRF, we would need to use concentrations of IRSp53 much higher than those we can achieve given the relatively low Kd (∼0.3 μM) of IRSp53 for barbed ends (Supplementary Figure S2c). The low affinity of IRSp53 for barbed ends could also result in short t1/2 of the IRSp53:barbed end complex, which may allow growth of barbed ends that are only transiently occupied by IRSp53. Alternatively, IRSp53 may bind slowly to barbed ends because it needs to undergo a slow conformational change for efficient binding, or a slow conformational change might follow IRSp53 binding to the sides of filaments, close to the barbed ends, resulting in a reduction in the rate of filament growth. Although VASP and IRSp53 bind to each other, they have independent and opposing effects on barbed end growth. The addition of VASP to IRSp53-blocked barbed ends resulted in restoration of fast filament growth to rates obtained with VASP alone (Supplementary Figure S3a). Hence, in this assay, the possible association of VASP and IRSp53 to barbed ends does not change the kinetic behaviour of VASP-bound barbed ends. Thus, the formation of a VASP::IRSp53 complex has negligible effects on the filament elongation rate of VASP in solution. IRSp53 promotes VASP recruitment and clustering on supported surfaces to drive processive filament elongation High-density clustering of VASP onto functionalized beads allows processive, WH2 domain-mediated actin filament elongation, even in the presence of high concentrations of capping protein (Breitsprecher et al, 2008, 2011). Thus, we assessed whether beads saturated with IRSp53 could recruit and cluster VASP to drive processive actin assembly in the presence of capping protein using TIRF microscopy. To facilitate the visualization of actin filament growth, we initially used a chimaeric VASP (VASP-DdGAB), bearing the GAB motif of the Dictyostelium discoideum VASP homologue. This DdGAB motif, due to its high affinity for G-actin, markedly enhances filament elongation compared to WT mammalian VASP at the low G-actin concentration (1 μM) used in the TIRF assay (Figure 2B and C; Supplementary Figure S3d; Breitsprecher et al, 2011). We observed that high-density crowding or clustering of VASP-DdGAB on IRSp53-coated beads relieved inhibition of actin growth by capping protein and promoted marked processive filament elongation (Figure 2B and C; Supplementary Figure S3d). Buckling actin filaments grew away from the bead surfaces with elongation rates ranging from 37.1±7 to 45.4±5.5 actin subunits/s, which is comparable to the rate obtained with VASP-DdGAB-coated control beads (Figure 2B and C; Supplementary Figure S3d; Supplementary Movie 1). This elongation rate is about four times faster than the elongation rate (∼10 actin subunits/s) of spontaneously growing actin control filaments (Breitsprecher et al, 2008, 2011). The recruitment of VASP-DdGAB to the beads was specifically mediated by IRSp53, since no filament growth was observed when uncoated beads were incubated with soluble VASP (Figure 2B and C). Likewise, no filament growth was observed when beads were coated with IRSp53-W413G, which possesses a defective SH3 domain (Supplementary Figure S3d). We extended these observations to human VASP (hVASP) and EVL (hEVL). In both cases, the recruitment of the proteins to IRSp53-coated beads in the presence of capping proteins promoted processive filament elongation in the presence of capping proteins (Figure 2B and C). As expected, the rate of filament elongation in the presence of hVASP or hEVL was 3- to 4-fold slower compared to that of hVASP-DdGAB, due to the lower affinities of their GAB domains for G-actin (Breitsprecher et al, 2011). Collectively, these results demonstrate that bead-immobilized IRSp53 is necessary and sufficient to recruit and cluster VASP from solution, which in turn drives processive actin filament assembly in the presence of CP. CDC42 favours the formation of an IRSp53–VASP complex in vivo IRSp53 can bind both activated RAC (Miki et al, 2000) and CDC42 (Govind et al, 2001; Krugmann et al, 2001). MENA, another member of the ENA/VASP family protein, has been shown to bind to IRSp53 in a CDC42-dependent manner in vitro (Krugmann et al, 2001). Using recombinant purified proteins, we first examined whether IRSp53 serves as a link between VASP and active CDC42. Binding of IRSp53 to GTP-CDC42 was readily detected, while VASP interacted with GTP-CDC42 in an IRSp53-dependent manner,
Referência(s)