Ena/VASP Proteins Enhance Actin Polymerization in the Presence of Barbed End Capping Proteins
2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês
10.1074/jbc.m503957200
ISSN1083-351X
AutoresMelanie Barzik, Tatyana I. Kotova, Henry N. Higgs, Larnele Hazelwood, Dorit Hanein, Frank B. Gertler, Dorothy A. Schafer,
Tópico(s)RNA Research and Splicing
ResumoEna/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins. Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins. The vertebrate Ena/VASP proteins Mena, VASP, and Evl play important functions in regulating the cytoskeleton during cell motility, axon outgrowth, and guidance (1.Reinhard M. Jarchau T. Walter U. Trends Biochem. Sci. 2001; 26: 243-249Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 2.Krause M. Dent E.W. Bear J.E. Loureiro J.J. Gertler F.B. Annu. Rev. Cell Dev. Biol. 2003; 19: 541-564Crossref PubMed Scopus (525) Google Scholar) and for actin-based motility of the pathogenic bacterium Listeria monocytogenes (3.Laurent V. Loisel T.P. Harbeck B. Wehman A. Grobe L. Jockusch B.M. Wehland J. Gertler F.B. Carlier M.F. J. Cell Biol. 1999; 144: 1245-1258Crossref PubMed Scopus (297) Google Scholar, 4.Smith G.A. Theriot J.A. Portnoy D.A. J. Cell Biol. 1996; 135: 647-660Crossref PubMed Scopus (186) Google Scholar, 5.Niebuhr K. Ebel F. Frank R. Reinhard M. Domann E. Carl U.D. Walter U. Gertler F.B. Wehland J. Chakraborty T. EMBO J. 1997; 16: 5433-5444Crossref PubMed Scopus (333) Google Scholar, 6.Loisel T.P. Boujemaa R. Pantaloni D. Carlier M.F. Nature. 1999; 401: 613-616Crossref PubMed Scopus (805) Google Scholar). Ena/VASP proteins influence the dynamics of lamellipodia and formation of filopodial protrusions in fibroblasts and neuronal growth cones (7.Mejillano M.R. Kojima S. Applewhite D.A. Gertler F.B. Svitkina T.M. Borisy G.G. Cell. 2004; 118: 363-373Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 8.Lebrand C. Dent E.W. Strasser G.A. Lanier L.M. Krause M. Svitkina T.M. Borisy G.G. Gertler F.B. Neuron. 2004; 42: 37-49Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 9.Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). In Dictyostelium, VASP is required for efficient chemotaxis and formation of filopodia (10.Han Y.H. Chung C.Y. Wessels D. Stephens S. Titus M.A. Soll D.R. Firtel R.A. J. Biol. Chem. 2002; 277: 49877-49887Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Ena/VASP proteins regulate the architecture of actin filaments in the actin tails of motile Listeria (11.Skoble J. Auerbuch V. Goley E.D. Welch M.D. Portnoy D.A. J. Cell Biol. 2001; 155: 89-100Crossref PubMed Scopus (113) Google Scholar) or those associated with beads coated with the Listeria ActA protein (12.Plastino J. Olivier S. Sykes C. Curr. Biol. 2004; 14: 1766-1771Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 13.Samarin S. Romero S. Kocks C. Didry D. Pantaloni D. Carlier M.F. J. Cell Biol. 2003; 163: 131-142Crossref PubMed Scopus (125) Google Scholar). The mechanisms by which Ena/VASP proteins regulate these diverse actin-dependent events are unknown. Clues to a possible mechanism for Ena/VASP function during cell migration came from electron microscopic studies of actin filaments in lamellipodia of fibroblasts and neuronal growth cones. Depletion of Ena/VASP proteins from their normal locations in fibroblasts or neurons promoted formation of dense actin networks with short, highly branched filaments. In contrast, enrichment of Ena/VASP proteins at the plasma membrane resulted in sparse networks containing primarily long, unbranched filaments, which in growth cones coalesced into filopodia (8.Lebrand C. Dent E.W. Strasser G.A. Lanier L.M. Krause M. Svitkina T.M. Borisy G.G. Gertler F.B. Neuron. 2004; 42: 37-49Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 9.Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). These studies support the hypothesis that Ena/VASP proteins influence actin networks by promoting formation of long, unbranched actin filaments in the cell periphery. Ena/VASP proteins could influence the length of actin filaments and the extent of filament branching at the cell periphery via several mechanisms: by increasing the rate of actin filament elongation, reducing the frequency of forming branched actin filaments, increasing the dissociation rate of branched filament junctions, or decreasing barbed end capping activity. Biochemical experiments in which recombinant VASP enhanced actin polymerization in the presence of heterodimeric capping protein (CP) 1The abbreviations used are: CP, capping protein; DTT, dithiothreitol; SAS, spectrin F-actin seeds; PKA, protein kinase A. supported the hypothesis that Ena/VASP proteins promote the formation of long, unbranched actin filaments, in part, by protecting actin filament barbed ends from capping (9.Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). To determine the mechanism by which Ena/VASP proteins regulate actin polymerization and barbed end capping, we studied His6-tagged VASP and several mutant forms of VASP in biochemical assays of actin assembly in the presence of bacterially expressed capping proteins and profilin. Structural features of the VASP EVH2 domain essential for regulating actin assembly in the presence of barbed end capping proteins in vitro were similar to those required for Mena to restore normal motility to Ena/VASP-deficient fibroblasts (14.Loureiro J.J. Rubinson D.A. Bear J.E. Baltus G.A. Kwiatkowski A.V. Gertler F.B. Mol. Biol. Cell. 2002; 13: 2533-2546Crossref PubMed Scopus (110) Google Scholar). Recombinant Protein Expression and Purification—All VASP proteins were recombinant, His6-tagged and purified from Escherichia coli. Plasmids to express N-terminal His6-tagged murine VASP and mutant VASP proteins were constructed from PCR fragments cloned into pQE-80L (Qiagen). The wild-type and mutant VASP proteins used in this study are shown in schematic form in supplementary materials Fig. 1. VASP proteins were expressed in E. coli strain BL21 (DE3) CodonPlus and purified by chromatography on TALON resin (BD Biosciences) and Superdex-200 in MKEI-200 buffer (20 mm imidazole, pH 7.0, 200 mm KCl, 1 mm EGTA, 2 mm MgCl2, and 1 mm DTT). VASP proteins were stored on ice and used within 2 weeks of purification. Recombinant murine CP (α1β2) was purified as described (15.Palmgren S. Ojala P.J. Wear M.A. Cooper J.A. Lappalainen P. J. Cell Biol. 2001; 155: 251-260Crossref PubMed Scopus (133) Google Scholar). Plasmids for expression of human profilin I, profilin I-Y6D, and profilin I-R88E were constructed by D. Kaiser and J. Lu and were gifts from the laboratory of T. D. Pollard; profilins were purified as described (16.Lu J. Pollard T.D. Mol. Biol. Cell. 2001; 12: 1161-1175Crossref PubMed Scopus (115) Google Scholar). The actin binding and polyproline binding activities of wild-type and mutant profilins were verified. Recombinant gelsolin and CapG were gifts of M. Wear and F. Southwick, respectively. Gelsolin-actin complex was prepared as a 1:2 molar ratio of gelsolin and actin in 20 mm imidazole, pH 7.0, 100 mm KCl, 1 mm EGTA, 2 mm MgCl2, 1 mm DTT, and 0.2 mm CaCl2. Actin was prepared from rabbit muscle (17.Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) and gel filtered on Sephacryl S200 equilibrated in 2 mm Tris/HCl, pH 8.0, 0.2 mm ATP, 0.1 mm DTT, and 0.2 mm CaCl2. Pyrenyl-actin was prepared as described (18.Bryan J. Coluccio L.M. J. Cell Biol. 1985; 101: 1236-1244Crossref PubMed Scopus (51) Google Scholar). All proteins except actin were quantified from absorbance at 280 nm using extinction coefficients predicted from the amino acid sequence (Protean, DNAStar software): VASP, 32,650 m-1 cm-1, considered a tetramer (based on analytical ultracentrifugation experiments below); CP, 76,290 m-1 cm-1; and profilin, 18,100 m-1 cm-1. Actin was quantified from absorbance at 290 nm and the extinction coefficient of 26,600 m-1 cm-1. Other Proteins—Spectrin F-actin seeds (SAS) were prepared from human erythrocytes as described (19.DiNubile M.J. Cassimeris L. Joyce M. Zigmond S.H. Mol. Biol. Cell. 1995; 6: 1659-1671Crossref PubMed Scopus (63) Google Scholar). The concentration of SAS was determined from the initial rate of actin polymerization using kon for actin = 11.6 m-1 s-1 (20.Pollard T.D. J. Cell Biol. 1986; 103: 2747-2754Crossref PubMed Scopus (601) Google Scholar), assuming no pointed end growth. Actin Polymerization Assays—Seeded polymerization reactions contained 0.5-2 μm actin (5% pyrene-labeled), 0.2 nm SAS, 4 nm CP, VASP/mutant VASP protein, and profilin/mutant profilin, as indicated, in 20 mm imidazole, pH 7.0, 100 mm KCl, 2 mm MgCl2, 1mm EGTA, 0.2 mm ATP, and 0.1 mm DTT (MKEI-100 buffer). SAS were omitted from reactions monitoring spontaneous actin polymerization. Fluorescence of pyrenyl-actin (excitation at 365 nm, emission at 386 nm) was monitored for 600 s at 25 °C. All components except actin and SAS were mixed in MKEI-100 buffer; reactions were initiated by the simultaneous addition of actin (primed with 1 mm EGTA and 0.1 mm MgCl2 for 90 s) and SAS. The delay between mixing reactants and recording fluorescence was <16 s. In experiments with profilin, VASP and profilin were incubated in MKEI-100 buffer for 5 min prior to addition of other components. Fluorescence was converted to the molar concentration of F-actin from the fluorescence of completely polymerized and unpolymerized actin, assuming a critical concentration of 0.1 μm. Initial rates of actin assembly were determined from linear fits of data collected during the initial 60 s. In control experiments, VASP did not alter the fluorescence of either pyrenyl-G-actin or pyrenyl-F-actin. Effect of VASP on Steady State Levels of G-actin—The effect of VASP on the amount of G-actin present in steady state solutions of 1 μm actin in MKEI-100 buffer was determined in reactions containing varying amounts of VASP with or without 10 nm CP in MKEI-100 buffer. Samples were incubated for 16 h at room temperature to reach equilibrium. The reactions were overlaid on a 10% sucrose cushion and subjected to centrifugation for 20 min at 80,000 × g in a TLA 120.1 rotor at 20 °C. Aliquots of the supernatant fraction were subjected to SDS-PAGE on 15% gels and stained with Coomassie Brilliant Blue. The amount of G-actin present in the supernatant fraction was quantified from the Coomassie-stained gels using ImageJ software and gels containing known amounts of actin. Quantitation of Actin Filament Barbed Ends—To determine if barbed ends were created de novo, reactions containing VASP or EVH2 and either 1 μm actin (for VASP) or 0.5 μm actin (for EVH2) were prepared without pyrenyl-actin as described for seeded polymerization assays. Profilin (10 μm) was included as indicated. After 90 s, reactions were diluted 25-fold into 0.5 μm actin (5% pyrene-labeled), and fluorescence was monitored for 600 s. Actin Depolymerization Assays—F-actin (1 μm, 50% pyrene-labeled) was incubated with 17 nm CP and varying amounts of VASP for 5 min. Care was taken to minimize filament breakage during mixing by using wide-bore pipette tips, triturating a defined number of strokes, and transferring F-actin solutions slowly. Aliquots were diluted 20-fold into stirring MKEI-100, and fluorescence of pyrene-actin was monitored for 600 s. Phosphorylation of VASP by Protein Kinase A (PKA)—VASP at a final concentration of 2 μm was incubated for 30 min at 30 °C with 200 units/ml of cAMP-dependent protein kinase, catalytic subunit (New England Biolabs) in 50 mm Tris/HCl, pH 7.5, 10 mm MgCl2, and 1 mm ATP. Mock-treated VASP was prepared similarly in a reaction without added PKA. Analytical Ultracentrifugation Analyses—For velocity analytical ultracentrifugation, 4 μm VASP in 150 mm NaCl, 0.5 mm EGTA, 10 mm NaH2PO4, pH 7.0, 0.5 mm DTT, was centrifuged at 40,000 × g at 20 °C in a Beckman Proteomelab XL-A rotor; 280 nm absorbance was monitored every minute by continuous scan at 0.003 cm. Protein partial specific volume, buffer density, and buffer viscosity were determined using Sednterp. 2D. Hayes, T. Laue, and J. Philo, personal communication. Scans 1-200 were analyzed using Sedfit87 (www.analyticalultracentrifugation.com). For sedimentation equilibrium ultracentrifugation, three concentrations of VASP (4, 2, and 1 μm) were centrifuged at 7,000, 10,000, and 14,000 × g for 15, 10, and 10 h, respectively, in an AN-60 rotor. Scans at 280 nm and 0.001-cm steps were recorded every hour. Winmatch, Winreedit, and Winnonln software were used to analyze and fit the data, assuming a single species. The resulting fit gave a σ of 7.99 and root mean square deviation of 0.00409 with no systematic deviation of residuals. Microscopy of Actin Filaments—Actin filaments were stained with rhodamine-phalloidin and prepared for light microscopy as described (21.Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Samples for electron microscopy contained 4 μm G-actin (10% pyrene-labeled) and 6 μm VASP in F-buffer (2 mm imidazole, pH 7.0, 2 mm MgCl2, 1 mm EGTA, 0.2 mm DTT, 0.1 mm ATP, 0.02% sodium azide) with varying concentrations of KCl. At steady state, determined from observations of pyrene fluorescence, aliquots were diluted in F-buffer, applied to glow-discharged 400-mesh EM carbon-coated grids, and stained with 2% uranyl acetate. Low dose images (∼10 e-/Å2) were recorded with a Tecnai 12 G2 electron microscope (FEI Electron Optics) at 120 keV with nominal magnification of 52,000 and 1.5-μm defocus. Kodak SO-163 plates were developed for 12 min in full D19 developer (Eastman Kodak). The bundles were processed and analyzed using the EMAN (22.Ludtke S.J. Baldwin P.R. Chiu W. J. Struct. Biol. 1999; 128: 82-97Crossref PubMed Scopus (2102) Google Scholar). Images were scanned at a raster of 6.4 μm pixel-1 using a SCAI scanner (Intergraph Corporation, Englewood, CO) with a final 2.45 Å pixel size on the image. The bundles were selected, boxed, and displayed with a modified version of Ximdisp (23.Smith J.M. J. Struct. Biol. 1999; 125: 223-228Crossref PubMed Scopus (81) Google Scholar). Fourier transforms were calculated using the Brandeis helical imaging package (24.Owen C.H. Morgan D.G. DeRosier D.J. J. Struct. Biol. 1996; 116: 167-175Crossref PubMed Scopus (86) Google Scholar). VASP Protects Actin Filament Barbed Ends from Capping—To understand how Ena/VASP proteins regulate actin filament assembly in the presence of barbed end-capping proteins, we studied actin polymerization from SAS in the presence of recombinant CP and His6-tagged VASP. We confirmed our previous findings that VASP increased the rate of actin polymerization from SAS in the presence of CP in a dose-dependent manner (9.Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar) (Fig. 1, A and B). The ability of VASP to enhance actin polymerization in the presence of CP, defined here as anti-capping activity, also depended on the concentration of capping protein (Fig. 1C). The apparent inhibition of capping by VASP could occur by several mechanisms: 1) VASP could compete directly with CP for binding barbed ends; 2) VASP could promote formation of new filament barbed ends via de novo filament nucleation or severing; or 3) VASP could increase the rate of filament elongation. A fourth possibility that VASP binds CP and prevents its interaction with barbed ends was ruled out in previous studies (9.Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). 3M. Barzik, unpublished data. To determine if VASP anti-capping activity involved enhanced filament elongation, we examined the effects of VASP on filament elongation in actin polymerization assays seeded by SAS. VASP increased slightly the initial rate of actin polymerization from SAS (Fig. 1D), suggesting that VASP enhances the rate of actin subunit association or decreases the rate of subunit dissociation. The increased rate of actin polymerization was apparent after a short lag period (∼30 s) indicated by the slight upward curvature in the reaction time courses. This lag period may reflect the fact that short actin filaments nucleated by SAS are required for VASP to associate with elongating filaments. The ability of VASP to increase the rate of actin polymerization did not result from de novo formation of actin filaments. Under the experimental conditions, spontaneous actin nucleation activity of VASP was negligible at concentrations below 150 nm (Fig. 1E). An increased rate of spontaneous actin polymerization was apparent at higher concentrations of VASP (≥200 nm) (Fig. 1E), however, this activity required a prolonged lag period and did not account for the increased initial rates of actin polymerization in reactions containing VASP. To confirm that VASP did not generate new actin filaments within the time required to determine initial rate measurements, aliquots of polymerization reactions containing SAS and varying amounts of VASP were removed at 90 s and diluted into 0.5 μm actin to observe filament elongation at barbed ends selectively. The rate of actin polymerization after dilution was independent of VASP (Fig. 1F), indicating that filament-barbed ends were not generated. These results confirm previous findings that VASP exhibits negligible actin nucleation activity at physiological salt conditions (3.Laurent V. Loisel T.P. Harbeck B. Wehman A. Grobe L. Jockusch B.M. Wehland J. Gertler F.B. Carlier M.F. J. Cell Biol. 1999; 144: 1245-1258Crossref PubMed Scopus (297) Google Scholar, 9.Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 25.Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (105) Google Scholar, 26.Bearer E.L. Prakash J.M. Manchester R.D. Allen P.G. Cell Motil. Cytoskeleton. 2000; 47: 351-364Crossref PubMed Scopus (51) Google Scholar, 27.Lambrechts A. Kwiatkowski A.V. Lanier L.M. Bear J.E. Vandekerckhove J. Ampe C. Gertler F.B. J. Biol. Chem. 2000; 275: 36143-36151Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and rules out that the apparent competition between capping proteins and VASP in seeded polymerization assays results from spontaneous actin nucleation as suggested by Samarin and colleagues (13.Samarin S. Romero S. Kocks C. Didry D. Pantaloni D. Carlier M.F. J. Cell Biol. 2003; 163: 131-142Crossref PubMed Scopus (125) Google Scholar). The mammalian Ena/VASP proteins, Mena and Evl, also exhibited anti-capping activity and enhanced the rate of actin filament elongation under conditions where actin nucleating activity was negligible (data not shown). VASP anti-capping activity depended on the concentrations of VASP and CP (Fig. 1, B and C), suggesting that CP and VASP compete for binding barbed ends. VASP also protected barbed ends from two other proteins with barbed end capping activity: gelsolin-actin complex and CapG (Fig. 2, A and B). The ability of VASP to inhibit capping by several different barbed end-binding proteins suggests that VASP prevents capping by directly associating with barbed ends. In support of this hypothesis, VASP decreased the amount of G-actin present in actin solutions containing CP under steady state conditions, indicating that VASP blocked CP from binding barbed ends without interfering with actin subunit addition (Fig. 2C). VASP alone increased slightly the amount of G-actin at steady state (Fig. 2C), consistent with a low-affinity interaction of VASP and G-actin (28.Walders-Harbeck B. Khaitlina S.Y. Hinssen H. Jockusch B.M. Illenberger S. FEBS Lett. 2002; 529: 275-280Crossref PubMed Scopus (80) Google Scholar). VASP also competed with CP for binding pre-formed actin filaments. Actin filaments capped by CP depolymerized slowly when diluted below the barbed end critical concentration, as expected. VASP competed with CP, resulting in increased rates of depolymerization upon dilution of F-actin incubated with both CP and VASP (Fig. 2D). VASP did not alter the rate of depolymerization of uncapped actin filaments, indicating that VASP does not sever filaments (data not shown). The EVH2 Domain of VASP Is Sufficient to Protect Barbed Ends from CP—The C-terminal EVH2 domain of VASP, which contains separate motifs that interact with G-actin (28.Walders-Harbeck B. Khaitlina S.Y. Hinssen H. Jockusch B.M. Illenberger S. FEBS Lett. 2002; 529: 275-280Crossref PubMed Scopus (80) Google Scholar), F-actin (25.Huttelmaier S. Harbeck B. Steffens O. Messerschmidt T. Illenberger S. Jockusch B.M. FEBS Lett. 1999; 451: 68-74Crossref PubMed Scopus (105) Google Scholar, 29.Bachmann C. Fischer L. Walter U. Reinhard M. J. Biol. Chem. 1999; 274: 23549-23557Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), and confer VASP tetramerization (29.Bachmann C. Fischer L. Walter U. Reinhard M. J. Biol. Chem. 1999; 274: 23549-23557Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), was more active than full-length VASP in enhancing actin polymerization in the presence of CP (Fig. 3, A and B). Actin nucleation activity by EVH2 was slightly higher than that of VASP (Fig. 3C), however, anti-capping activities of EVH2 and VASP could be compared in assays using 0.5 μm actin, where actin nucleation activity by both proteins was negligible. We confirmed that EVH2 did not promote formation of new filaments under these conditions using dilution assays that selectively monitor elongation at actin filament barbed ends (Fig. 3D). Thus, the VASP EVH2 domain contains the structural elements sufficient for anti-capping activity. The EVH2 domain of Mena similarly exhibited potent anti-capping activity (data not shown). Interactions via G-actin and F-actin Binding Motifs of VASP Are Required for Anti-capping Activity—The F-actin and G-actin binding motifs are required for Ena/VASP function during whole cell motility (14.Loureiro J.J. Rubinson D.A. Bear J.E. Baltus G.A. Kwiatkowski A.V. Gertler F.B. Mol. Biol. Cell. 2002; 13: 2533-2546Crossref PubMed Scopus (110) Google Scholar). To determine if VASP anti-capping activity depended on interactions via its G-actin and F-actin binding motifs, we examined the anti-capping activity of mutant proteins that either lack or alter these motifs. Mutant VASP with a deletion at the F-actin binding motif (VASP-ΔFAB, Δ256-273) did not protect barbed ends from CP, even when tested at concentrations exceeding 400 nm (supplementary materials Fig. 2A). Similarly, VASP with mutations that disrupt the G-actin binding site (VASP-GAB; R232E,K233E) (28.Walders-Harbeck B. Khaitlina S.Y. Hinssen H. Jockusch B.M. Illenberger S. FEBS Lett. 2002; 529: 275-280Crossref PubMed Scopus (80) Google Scholar) did not protect barbed ends from CP when used at concentrations up to 400 nm (supplementary materials Fig. 2B). To determine if VASP binding to free G-actin was required for anti-capping activity, we examined the dependence of anti-capping activity on the concentration of G-actin. VASP anti-capping activity was independent of the concentration of G-actin (supplementary materials Fig. 2C), indicating that anti-capping activity likely does not involve interactions of VASP and free G-actin. Instead, we suggest that VASP anti-capping activity requires interactions of the G-actin binding motif with actin subunits incorporated in filaments, possibly the terminal actin subunits situated at barbed ends. VASP Tetramers Are Required for Anti-capping Activity—VASP is predicted to assemble as a tetramer (29.Bachmann C. Fischer L. Walter U. Reinhard M. J. Biol. Chem. 1999; 274: 23549-23557Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 30.Zimmermann J. Labudde D. Jarchau T. Walter U. Oschkinat H. Ball L.J. Biochemistry. 2002; 41: 11143-11151Crossref PubMed Scopus (23) Google Scholar, 31.Kuhnel K. Jarchau T. Wolf E. Schlichting I. Walter U. Wittinghofer A. Strelkov S.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17027-17032Crossref PubMed Scopus (89) Google Scholar). We confirmed that full-length VASP is a tetramer using equilibrium sedimentation ultracentrifugation. A molecular mass of 173.2 kDa was calculated from the experimentally determined effective reduced molecular weight and partial specific volume of 0.7219 ml/g. The predicted molecular mass for His6-tagged monomeric VASP is 41.2 kDa, corresponding to a 164.8-kDa tetramer. No systematic deviation between experimental data and curve fit was present, suggesting that VASP is a stable tetramer. Sedimentation velocity ultracentrifugation experiments for VASP showed a single peak at 4.7 S with a frictional coefficient (f/f0) of 2.02, consistent with VASP being an asymmetric, elongated molecule (supplementary materials Fig. 3A). To determine if VASP tetramers are required for anti-capping activity, we tested a mutant form of VASP lacking the C-terminal coiled-coil region involved in tetramer formation (VASP-ΔCoCo, Δ331-375). VASP-ΔCoCo neither exhibited anti-capping activity (supplementary materials Fig. 3B) nor enhanced actin assembly in the absence of CP (data not shown). Although deletion of the C-terminal coiled-coil motif may disrupt other features within the EVH2 domain required for anti-capping activity (29.Bachmann C. Fischer L. Walter U. Reinhard M. J. Biol. Chem. 1999; 274: 23549-23557Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), other experiments showed VASP-ΔCoCo stimulated Listeria motility in fibroblasts lacking Ena/VASP proteins (32.Geese M. Loureiro J.J. Bear J.E. Wehland J. Gertler F.B. Sechi A.S. Mol. Biol. Cell. 2002; 13: 2383-2396Crossref PubMed Scopus (88) Google Scholar). Thus, VASP-ΔCoCo retains some structural features required for biological function. Profilin Enhances Actin Polymerization in the Presence of VASP—The central proline-rich domain of VASP binds profilin and this interaction enhances actin-based Listeria motility (32.Geese M. Loureiro J.J. Bear J.E. Wehland J. Gertler F.B. Sechi A.S. Mol. Biol. Cell. 2002; 13: 2383-2396Crossref PubMed Scopus (88) Google Scholar, 33.Reinhard M. Giehl K. Abel K. Haffner C. Jarchau T. Hoppe V. Jochusch B.M. Walter U. EMBO J. 1995; 14: 1583-1589Crossref PubMed Scopus (420) Google Scholar, 34.Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. 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