Title: Molecular mechanism of Ena/VASP-mediated actin-filament elongation
Abstract: Article7 January 2011Open Access Molecular mechanism of Ena/VASP-mediated actin-filament elongation Dennis Breitsprecher Dennis Breitsprecher Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Antje K Kiesewetter Antje K Kiesewetter Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Joern Linkner Joern Linkner Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Marlene Vinzenz Marlene Vinzenz Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Theresia E B Stradal Theresia E B Stradal Signaling and Motility Group, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany Institute for Molecular Cell Biology, University of Münster, Münster, Germany Search for more papers by this author John Victor Small John Victor Small Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Ute Curth Ute Curth Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Richard B Dickinson Richard B Dickinson Department of Chemical Engineering, University of Florida, Gainesville, FL, USA Search for more papers by this author Jan Faix Corresponding Author Jan Faix Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Dennis Breitsprecher Dennis Breitsprecher Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Antje K Kiesewetter Antje K Kiesewetter Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Joern Linkner Joern Linkner Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Marlene Vinzenz Marlene Vinzenz Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Theresia E B Stradal Theresia E B Stradal Signaling and Motility Group, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany Institute for Molecular Cell Biology, University of Münster, Münster, Germany Search for more papers by this author John Victor Small John Victor Small Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Ute Curth Ute Curth Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Richard B Dickinson Richard B Dickinson Department of Chemical Engineering, University of Florida, Gainesville, FL, USA Search for more papers by this author Jan Faix Corresponding Author Jan Faix Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Author Information Dennis Breitsprecher1, Antje K Kiesewetter1, Joern Linkner1, Marlene Vinzenz2, Theresia E B Stradal3,4, John Victor Small2, Ute Curth1, Richard B Dickinson5 and Jan Faix 1 1Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany 2Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria 3Signaling and Motility Group, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany 4Institute for Molecular Cell Biology, University of Münster, Münster, Germany 5Department of Chemical Engineering, University of Florida, Gainesville, FL, USA *Corresponding author. Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover 30625, Germany. Tel.: +49 511 532 2928; Fax: +49 511 532 2909; E-mail: [email protected] The EMBO Journal (2011)30:456-467https://doi.org/10.1038/emboj.2010.348 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 Ena/VASP proteins are implicated in a variety of fundamental cellular processes including axon guidance and cell migration. In vitro, they enhance elongation of actin filaments, but at rates differing in nearly an order of magnitude according to species, raising questions about the molecular determinants of rate control. Chimeras from fast and slow elongating VASP proteins were generated and their ability to promote actin polymerization and to bind G-actin was assessed. By in vitro TIRF microscopy as well as thermodynamic and kinetic analyses, we show that the velocity of VASP-mediated filament elongation depends on G-actin recruitment by the WASP homology 2 motif. Comparison of the experimentally observed elongation rates with a quantitative mathematical model moreover revealed that Ena/VASP-mediated filament elongation displays a saturation dependence on the actin monomer concentration, implying that Ena/VASP proteins, independent of species, are fully saturated with actin in vivo and generally act as potent filament elongators. Moreover, our data showed that spontaneous addition of monomers does not occur during processive VASP-mediated filament elongation on surfaces, suggesting that most filament formation in cells is actively controlled. Introduction The precise control of actin-filament elongation in eukaryotic cells is fundamental to establish coordinated cell movement driven by the formation of protrusive structures like filopodia and lamellipodia, to assemble the contractile ring at the cleavage furrow during cell division and to coordinate endocytosis and phagocytosis (Chhabra and Higgs, 2007; Chesarone and Goode, 2009; Faix et al, 2009; Insall and Machesky, 2009). The only proteins known so far that directly enhance filament elongation by interaction with the growing barbed end and recruitment of monomeric actin for polymerization are formins and Ena/VASP proteins. Ena/VASP family members were previously shown to localize in a protrusion-dependent manner to lamellipodia tips (Rottner et al, 1999) and to influence the length of actin filaments as well as their apparent branching density within lamellipodia (Bear et al, 2002), and Listeria monocytogenes comet tails (Plastino et al, 2004). Ena/VASP proteins are required for the formation of filopodia in mammals and Dictyostelium (Schirenbeck et al, 2006; Applewhite et al, 2007; Dent et al, 2007) and were also shown to enhance the actin-driven propulsion of L. monocytogenes (Laurent et al, 1999; Loisel et al, 1999; Geese et al, 2002) as well as of beads coated with ActA (Samarin et al, 2003). Additionally, they are implicated in neuritogenesis and cortex development (Kwiatkowski et al, 2007, 2009) as well as in tumour development and progression (Hu et al, 2008; Philippar et al, 2008). Ena/VASP proteins display a conserved tripartite architecture encompassing an N-terminal Ena/VASP homology 1 (EVH1) domain required for subcellular targeting followed by a central proline-rich domain implicated in recruitment of profilin–actin complexes (Jonckheere et al, 1999; Ferron et al, 2007), and a C-terminal EVH2 domain mediating tetramerization and interaction with monomeric and filamentous actin (Bachmann et al, 1999; Hüttelmeier et al, 1999; Breitsprecher et al, 2008). The G-actin-binding site (GAB) within the EVH2 domain displays close sequence homologies to WASP homology 2 (WH2) motifs, which are present in many actin regulators (Supplementary Figure S1; Paunola et al, 2002; Dominguez, 2007, 2009). In addition, the adjacent F-actin-binding site (FAB) was also proposed to posses WH2-like properties (Dominguez, 2007, 2009). While both Ena/VASP proteins and formins accelerate actin filament barbed end elongation in vitro, the underlying molecular mechanisms are different (Faix and Grosse, 2006; Breitsprecher et al, 2008; Chesarone and Goode, 2009; Dominguez, 2010). Formin dimers remain processively associated with the growing filament barbed end by virtue of their dimeric FH2 domain while inserting thousands of actin monomers, thereby efficiently protecting the filament from heterodimeric capping proteins (CPs) (Zigmond et al, 2003; Harris et al, 2004; Schirenbeck et al, 2005; Kovar et al, 2006). Moreover, formin-mediated enhancement of filament elongation strictly depends on the recruitment of profilin–actin complexes by the adjacent proline-rich FH1 domain (Chang et al, 1997; Sagot et al, 2002; Kovar et al, 2006). Surface-immobilized VASP captures growing barbed ends (Pasic et al, 2008) and, when clustered on the surface at sufficiently high density, can translocate processively with the growing filament end (Breitsprecher et al, 2008). Unlike formins, profilin appears dispensable for VASP-mediated filament elongation in vitro (Samarin et al, 2003; Schirenbeck et al, 2006; Breitsprecher et al, 2008). A notable property of VASP is the different influence of CPs when VASP is free in solution or clustered on the surface of a bead. Upon clustering, VASP triggers processive filament elongation at high concentrations of CP that block VASP-mediated filament elongation in solution (Breitsprecher et al, 2008). Collectively, this suggests that clustered VASP tetramers cooperate in tethering and elongating actin filaments at surfaces, such as on the cell membrane and the surface of pathogens such as L. monocytogenes (Laurent et al, 1999; Breitsprecher et al, 2008; Footer et al, 2008). Although the filament elongation activity of VASP could be attributed to its GAB and FAB motifs, the underlying general mechanisms of VASP-mediated actin assembly remained obscure, as human VASP (hVASP) showed a drastically reduced elongating activity in vitro when compared with the orthologue from the highly motile soil amoeba Dictyostelium discoideum (DdVASP) (Breitsprecher et al, 2008). To dissect the molecular mechanism of Ena/VASP-mediated filament elongation, we employed a domain shuffling approach, replacing the GAB, FAB and their connecting linker region of hVASP by those of the fast-elongating DdVASP and by WH2 motifs from other actin-binding proteins. Our results allowed us to develop a quantitative mathematical model for an affinity based, WH2-domain-mediated actin assembly used by Ena/VASP proteins, whereby the filament elongation rate is correlated to the saturation of the GAB with actin monomers. Results VASP, Mena and EVL enhance filament elongation to similar extends Previously, it was shown that hVASP only weakly accelerates actin elongation in vitro, whereas the Dictyostelium orthologue DdVASP enhanced the growth of single filaments about seven-fold (Breitsprecher et al, 2008). Mammalian cells express two additional Ena/VASP proteins, referred to as Ena (enabled) and EVL (Ena/VASP like), the latter of which is abundantly expressed in migrating neutrophils, suggesting that this particular paralogue might mediate faster filament elongation than hVASP. In search for the underlying cause of different filament elongation rates, we compared DdVASP and the three mammalian Ena/VASP proteins on the domain level and found that the lengths of the linkers separating the GAB and FAB motifs in hVASP, EVL, Mena and DdVASP differ considerably, encompassing 18, 25, 33 and 40 residues, respectively (Figure 1A and B). Recently, it was shown that the length of the linkers separating the three WH2 motifs in the protein Cobl was critical for its actin nucleation activity (Ahuja et al, 2007). Since models of VASP-mediated actin assembly propose that a GAB-bound actin monomer is handed over directly to the barbed end of the FAB-bound filament (Ferron et al, 2007; Breitsprecher et al, 2008; Dickinson, 2009), we assumed that the short 18 residues linker of hVASP might impair this transfer and hence cause the lower elongation activity compared with DdVASP. Whereas the GAB and FAB of DdVASP differ substantially from the mammalian Ena/VASP proteins, the sequences of GAB and FAB motifs of the latter are almost identical (Figure 1A and B; Supplementary Figure S1). This suggests that mammalian Ena/VASP proteins display comparable actin-binding properties, in turn making them well-suited candidates to investigate the effects of the linker lengths on the filament elongation rate. Figure 1.Effects of hVASP, Mena and hEVL on actin-filament elongation. (A) Domain organization of Ena/VASP proteins and sequence alignment of the corresponding GAB-linker-FAB region within the EVH2 domains of DdVASP and hVASP. The GAB and FAB are highlighted in yellow. Identical amino acids within these motifs are marked with an asterisk. G, GAB; F, FAB; T, tetramerization domain. (B) Sequence alignment of the GAB-linker-FAB region of hVASP, hEVL and Mena. The linker length differs in all three proteins. (C) Elongation rates of 1.3 μM actin (30% Oregon-Green (OG) labelled) in the presence of different concentrations of hVASP, Mena EVH2 and hEVL determined by single-filament TIRFM in TIRF buffer (see Materials and methods). (D) TIRFM micrographs of the assembly of 1.3 μM actin (30% OG labelled) on beads saturated with hVASP, Mena EVH2 and hEVL in the presence of 80 nM CP. Arrows indicate growing filaments. Time is indicated in seconds, scale=10 μm. (E) Comparison of the maximal elongation rates of 1.3 μM actin (30% OG labelled) for the three mammalian Ena/VASP paralogues and DdVASP determined by TIRFM in solution or immobilized on beads. Elongation rates are presented as mean values±s.e.m. Download figure Download PowerPoint We employed TIRF microscopy to quantify the effects of purified Ena/VASP isoforms on single actin-filament elongation in vitro. Control actin filaments grew spontaneously from 1.3 μM G-actin (30% OG labelled), with an average rate of 10.5 subunits s−1 (sub s−1) in our assays. Similar to hVASP, the EVH2 domain from Mena and full-length EVL-bundled actin filaments and enhanced their elongation rate, both in solution and when clustered on beads in the presence of CP (Supplementary Movie 1). However, Mena and EVL again only slightly increased the elongation rate of actin filaments by ∼1.5-fold like hVASP (Figure 1C and D). Thus, despite different linkers, all three mammalian Ena/VASP paralogues possess low actin-filament elongation activities and mediate considerably slower elongation rates when compared with DdVASP in vitro (Figure 1E). Domain shuffling of GAB and FAB motifs reveals the molecular requirement for fast filament elongation Replacement of the GAB and FAB from hVASP (hGAB and hFAB) by the corresponding motifs from DdVASP (DdGAB and DdFAB) (Figure 2A) allowed hVASP to assemble actin filaments with significantly higher elongation rates when compared to the wild-type protein. Chimera hVASP DdGABFAB mediated virtually the same elongation rates as DdVASP, both in solution and when clustered on beads, enhancing filament elongation rates up to ∼70 sub s−1 (Figure 2B–D; Supplementary Movies 2 and 3). Almost identical results were obtained with hVASP DdGAB-L-FAB, additionally containing the entire linker region (L) of DdVASP, corroborating our previous finding that the linker region does not affect filament elongation (Figure 2C). Most notably, chimera hVASP DdGAB already enhanced the filament elongation rate up to 41 sub s−1 in solution and up to 49 sub s−1 on saturated beads, respectively, pointing towards a key role of actin monomer recruitment during VASP-mediated filament elongation (Supplementary Movies 2 and 3). In contrast, chimera hVASP DdFAB mediated only a moderate acceleration of the elongation rate up to 23.2 sub s−1 in solution and 19.2 sub s−1 on beads when compared with hVASP, suggesting that the contribution of the FAB motif to filament elongation is smaller than that of the GAB motif (Figure 2B–D; Supplementary Movie 4). The differential enhancement of the filament elongation rate by the VASP chimeras was confirmed by seeded pyrene actin polymerization assays (Supplementary Figure S2). Figure 2.Replacement of the GAB and FAB in hVASP with the corresponding DdVASP motifs markedly accelerates VASP-mediated filament elongation. (A) Scheme of hVASP chimeras bearing different domains of DdVASP. DdVASP is shown in colour and hVASP is shown in greyscale. G, GAB; F, FAB; T, tetramerization domain. (B) TIRFM micrographs of the assembly of 1.3 μM actin (30% OG labelled) in TIRF buffer containing 500 nM of the chimera are indicated. Time is indicated in seconds, scale=10 μm. (C) Elongation rates of the chimeras in solution in a concentration rage from 25 nM to 1 μM. (D) (Left) TIRFM micrographs of the assembly of 1.3 μM actin (30% OG labelled) in TIRF buffer in the presence of 200 nM CP on beads saturated with the hVASP chimeras are indicated. Time is shown in seconds, scale=10 μm. (Right) Plots of the lengths of individual filaments versus time yield filament elongation rates. Elongation rates are presented as mean values±s.e.m. Download figure Download PowerPoint The GAB has a pivotal role in specifying Ena/VASP elongation properties Since the transplantation of the DdGAB motif into the hVASP backbone was already sufficient to enhance actin-filament elongation 4–5-fold, we hypothesized that GAB-mediated actin monomer recruitment determines the filament elongation rates driven by Ena/VASP proteins. Therefore, we sought to analyse the affinities of DdGAB and hGAB peptides for G-actin under buffer conditions that were used in the TIRF experiments. Binding of GAB peptides to OG-actin monomers caused a 10–20% increase in OG-fluorescence, which was instrumental to detect the formation of GAB–actin complexes by fluorescence titrations. Determination of the equilibrium dissociation constants (KD) for DdGAB and hGAB peptides to latrunculin-A (LatA) sequestered Mg2+–ATP–actin in the presence of 50 mM KCl showed that their affinities towards G-actin differed by as much as ∼20-fold, yielding KD values of 0.63 and 10.3 μM, respectively (Figure 3A–C). Concomitant binding of LatA to actin had virtually no effect on the GAB–actin interaction (Figure 3C). Competition experiments with OG labelled and unlabelled actin in G-buffer revealed that both the DdGAB and hGAB peptides bound ∼1.7–2-fold stronger to OG-actin than to unlabelled actin (data not shown). The KD value determined for the DdEVH2–actin interaction was virtually identical to the KD value obtained for the DdGAB peptide, showing that the monomer affinity of the GAB motif is not changed within the VASP tetramer (Supplementary Figure S3). In addition to the equilibrium constants, we analysed the kinetics of actin monomer binding to the EVH2 domains of hVASP and DdVASP by stopped-flow analysis, yielding kon values of 53 μM−1 s−1 for the DdEVH2 domain and significantly lower rates of ∼12–18 μM−1 s−1 for the hEVH2 domain, respectively (Figure 3D). Figure 3.Actin-binding properties of the GAB motifs and EVH2 domains of hVASP and DdVASP. (A) Determination of the KD value of the DdGAB–actin interaction by fluorescence titration of 100 nM OG-actin (left) and 500 nM OG-actin (right) with the DdGAB peptide at the buffer conditions is indicated (for details see Materials and methods section). The solid lines represent calculated binding isotherms. (B) Determination of the KD value of the hGAB–actin interaction by fluorescence titration of 300 nM OG-actin (left) and 3 μM OG-actin (right) with the hGAB peptide at conditions as in (A). The solid lines represent calculated binding isotherms. (C) Summary of KD values determined from experiments similar as shown in (A, B) at the conditions indicated. KD values for the GAB–Ca2+–ATP–actin interaction are given in brackets. KD values obtained under conditions used in TIRF assays (50 mM KCl, Mg2+–ATP–actin) are shown in bold. (D) (Left) Kinetics of the DdEVH2–G-actin interaction. Time course of the binding of DdEVH2 to latA-sequestered, Mg2+–ATP–OG-actin was monitored by a stopped-flow apparatus at a final actin concentration of 2.5 μM actin and the DdEVH2 concentrations are indicated. Noisy curves represent experimental data, and solid lines are fits for a reversible, bimolecular reaction with KD=0.6 μM, yielding kon=53 μM−1 s−1. (Right) kon values of the interaction of the hEVH2 and DdEVH2 domain with OG-actin. Due to the low affinity of the hGAB, high actin concentrations had to be used (10 μM final), which resulted in more noisy signals (not shown) that could not be fitted as accurately as those for the higher-affinity DdEVH2. Download figure Download PowerPoint As expected, the DdGAB peptide inhibited actin-filament nucleation in pyrene assays according to its relatively high affinity to monomers, but did not noticeably sequester actin monomers even at a high molar excess (Supplementary Figure S4), indicating that binding of the GAB to monomers does not interfere with filament elongation. This finding is in line with previous results showing that WH2 motifs display profilin-like properties during barbed end assembly, and that they rapidly dissociate from the monomer after its incorporation into the filament (Hertzog et al, 2002, 2004). Collectively, these results let us hypothesize that the faster filament elongation mediated by DdVASP compared with hVASP is mainly caused by a higher affinity for monomers, resulting in a significantly higher saturation of the VASP tetramer at low actin concentrations typically used in in vitro polymerization assays. Replacement of the GAB of hVASP by other WH2 motifs reveals a saturation-based mechanism of VASP-mediated actin assembly To further investigate the dependence of the filament elongation rates on the affinity of the G-actin-binding motif, we replaced the GAB in hVASP with foreign WH2 core motifs from WASP-interacting protein (WIP), which binds monomers with a high affinity in G-buffer (KD=0.16 μM), and the WH2 motif from thymosin β4 (Tβ4), which displays a much weaker G-actin-affinity (KD=3.1 μM) (Chereau et al, 2005). We found that the affinity of the WIP-WH2-peptide to Mg2+–ATP–actin in the presence of 50 mM KCl was rather high, yielding a KD value of 0.84 μM (Figure 4B). Unfortunately, Tβ4 binding to OG-actin did not result in a detectable change in fluorescence (data not shown) as previously reported for pyrene- or NBD-labelled actin (Hertzog et al, 2002). Notwithstanding, we noted that the KD value of the interaction of Mg2+–ATP–actin with full-length Tβ4 in the presence of 50 mM KCl was previously determined to be 1.4 μM (De La Cruz et al, 2000). Assuming that the affinity of the Tβ4–WH2 motif for actin under our polymerization conditions is about a factor 4 lower (as reported for G-buffer; Chereau et al, 2005), we estimated the KD for the Tβ4–WH2–actin interaction to be in the range of ∼5.6 μM. Figure 4.Analysis of hVASP WH2 chimeras reveals VASP-mediated filament elongation is enhanced by saturable monomer binding to GAB sites. (A) Sequence alignment of the WH2 motifs is indicated. In the chimeric proteins, the GAB of hVASP was replaced by the WH2 motifs from Tβ4 and WIP. KD values of the actin–WH2 interaction in G-buffer where either determined in this study (*) or by Chereau et al (2005) (#). Conserved hydrophobic residues are highlighted in grey and the conserved LxxV/T motifs (x=basic amino acid) are boxed. (B) KD values determined as for Figure 3A and B under the conditions are indicated. (*) The KD values for the Tβ4–WH2 interaction were estimated on the basis of previous studies (De La Cruz et al, 2000; Hertzog et al, 2002; Chereau et al, 2005). (C) TIRFM micrographs of the assembly of 1.3 μM actin (30% OG labelled) on beads saturated with hVASP Tβ4 and hVASP WIP in TIRF buffer in the presence of 80 nM CP. Both chimeras processively elongate actin filaments. Time is indicated in seconds, scale=20 μm. (D) Elongation rates mediated by hVASP and the chimeras hVASP Tβ4, hVASP WIP and hVASP DdGAB at 1.3 μM G-actin either with 500 nM of the VASP constructs in solution or in the presence of 80 nM CP on saturated beads. Number of analysed filaments >20 for bead assays and >40 for assays in solution. Elongation rates are presented as mean values±s.e.m. (E) Elongation rates obtained from TIRF assays with beads coated with different hVASP constructs in the presence of 80 nM CP at the actin concentrations indicated. Solid lines represent best fits of the experimental data using the mathematical model for processive filament elongation by immobilized VASP as described in Figure 5 and the Materials and methods section. Elongation rates are presented as mean values±s.e.m. (F) Parameters derived from fitting of the data are shown in (E). Download figure Download PowerPoint Based on these different affinities, we tested the filament elongation properties of chimeras hVASP WIP and hVASP Tβ4. Remarkably, both WH2 chimeras promoted actin assembly in an Ena/VASP-mediated manner, enhancing filament elongation in solution and processively elongating actin filaments in the presence of CP when clustered on polystyrene beads (Figure 4C; Supplementary Movie 5). As hypothesized, chimera hVASP WIP-mediated fast elongation rates both in solution and on beads (32.1 and 36.3 sub s−1, respectively), whereas chimera hVASP Tβ4 enhanced filament elongation only moderately to 20.1 and 19.5 sub s−1, respectively (Figure 4D). As shown in Figure 4E, the elongation rates mediated by VASP clustered on beads increased in order of their affinities for actin monomers. Notably, the rates appeared to approach saturation at higher actin concentrations, as can be most clearly seen for chimeras hVASP DdGAB and hVASP WIP. Thus, these results let us propose that the elongation rate of processive filament growth on VASP-saturated beads reaches a maximum rate when the available GAB regions are saturated (actin concentration ≫KD), suggesting that all Ena/VASP proteins are potent filament elongators at high actin monomer concentrations. Model of VASP-mediated actin-filament elongation The dependence of processive VASP-mediated filament elongation on its saturation with actin monomers can be explained by a mathematical model based on the ‘actoclampin’ model of actin-filament end-tracking proteins (Dickinson and Purich, 2002; Dickinson et al, 2004), in which processive elongation is achieved by multivalent affinity-modulated interactions between the VASP tetramer and the filament tip (Figure 5). At steady state, one VASP subunit of a tetramer binds to the filament terminal subunit by its GAB, leaving a number (N) of free GABs to capture monomers from solution (concentration C) with rate constant kon. Captured monomers either dissociate (at rate constant koff=kon × KD) or are transferred to the tip and irreversibly incorporated with rate constant kt. As shown in the Materials and methods section and Figure 5A, this kinetic model derives the VASP-mediated elongation rate rV to be Figure 5.Mathematical model of VASP-mediated actin-filament elongation. (A) General mechanism of VASP-mediated filament elongation. A VASP tetramer is attached to the filament barbed end by at least one EVH2 domain during filament elongation. The free EVH2 domains recruit actin monomers from solution with an on-rate constant kon, and subsequently either transfer the subunit onto the barbed end with a transfer rate kt or release the actin monomer back into solution with an off-rate koff. This model also assumes that upon binding of the GAB-associated monomer to the barbed end, the already bound GAB is quickly (or simultaneously) released from the now penultimate subunit so that it becomes immediately available to capture another monomer. By this cycle, the VASP tetramer is able to processively track the elongating filament tip while cyclically maintaining one GAB bound to the terminal subunit and three GABs free to capture monomers from solution. (B) VASP-mediated actin-filament elongation in solution. When VASP is free in solution, both, processive association of VASP with the barbed end and spontaneous addition of free actin monomers via the direct pathway with an independent transfer rate constant kf can occur. Free barbed ends produced by the direct pathway are accessible either for other VASP tetramers, actin monomers or, if present, capping proteins. The average elongation rate is therefore the sum of the VASP-mediated filament elongation rate and the rate of the direct pathway. (C) VASP-mediated filament elongation on surfaces. Upon clustering, the high density of VASP at the surface leads to processive association of VASP with the filament end, efficiently blocking binding of capping proteins and also preventing spontaneous addition of monomers via the direct pathway. Hence, filament elongation is exclusively fueled by actin monomers recruited and transferred by VASP. Download figure Download PowerPoint In Figure 4E, model predictions show excellent agreement with elongation rates observed on beads saturated with chimeras hVASP WIP, hVASP Tβ4, hVASP DdGAB and hVASP WT over the widest possible range of actin concentrations that is applicable to TIRF assays (0.5–4 μM actin). Importantly, the model anticipates a saturation of the rate with respect to monomer concentration, which will be achieved when all GAB sites are occupied with actin. All four curves were fitted simultaneously by weighted least-squares regression with only four fitted parameters: the number of GABs N, the transfer rate constant kt, which was shown to have nearly the same value for all hVASP constructs (Supplementary Figu