Ena/VASP proteins capture actin filament barbed ends

Ena/VASP (vasodialator-stimulated protein) proteins regulate many actin-dependent events, including formation of protrusive structures, fibroblast migration, neurite extension, cell-cell adhesion, and Listeria pathogenesis. In vitro, Ena/VASP activities on actin are complex and varied. They promote actin assembly, protect filaments from cappers, bundle filaments, and inhibit filament branching. To determine the mechanisms by which Ena/VASP proteins regulate actin dynamics at barbed ends, we monitored individual actin filaments growing in the presence of VASP and profilin using total internal reflection fluorescence microscopy. Filament growth was unchanged by VASP, but filaments grew faster in profilin-actin and VASP than with profilin-actin alone. Actin filaments were captured directly by VASP-coated surfaces via interactions with growing barbed ends. End-attached filaments transiently paused but resumed growth after becoming bound to the surface via a filament side attachment. Thus, Ena/VASP proteins promote actin assembly by interacting directly with actin filament barbed ends, recruiting profilin-actin, and blocking capping.     

Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes

Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament.     

Ena/VASP proteins have an anti-capping independent function in filopodia formation

Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at filopodial tips. Mutation of the G-actin-binding motif (GAB) partially compromised stabilization of Ena/VASP at filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.     

Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility

Cell motility requires lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria. In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during lamellipodial protrusion. Ena/VASP-deficient lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

Understanding the role of the G-actin-binding domain of Ena/VASP in actin assembly

The Ena/VASP and WASP family of proteins play distinct roles in actin cytoskeleton remodeling. Ena/VASP is linked to actin filament elongation, whereas WASP plays a role in filament nucleation and branching mediated by Arp2/3 complex. The molecular mechanisms controlling both processes are only emerging. Both Ena/VASP and WASP are multidomain proteins. They both present poly-Pro regions, which mediate the binding of profilin-actin, followed by G-actin-binding (GAB) domains of the WASP-homology 2 (WH2) type. However, the WH2 of Ena/VASP is somewhat different from that of WASP, and has been poorly characterized. Here we demonstrate that this WH2 binds profilin-actin with higher affinity than actin alone. The results are consistent with a model whereby allosteric modulation of affinity drives the transition of profilin-actin from the poly-Pro region to the WH2 and then to the barbed end of the filament during elongation. Therefore, the function of the WH2 in Ena/VASP appears to be to "process" profilin-actin for its incorporation at the barbed end of the growing filament. Conformational changes in the newly incorporated actin subunit, resulting either from nucleotide hydrolysis or from the G- to F-actin transition, may serve as a "sensor" for the processive stepping of Ena/VASP. Conserved domain architecture suggests that WASP may work similarly.     

Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening

Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP’s main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP’s flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP’s effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP’s ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP’s network stiffening activity may be tuned by the local concentration of monomeric actin.     

Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end

Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell "chooses" among these alternative modes of "molecular hardware," we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of filopodia. We propose a model for selection of lamellipodial versus filopodial organization in which CP is a negative regulator of filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.     

The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation.

Vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena/VASP family of proteins that are implicated in regulation of the actin cytoskeleton. All family members share a tripartite structural organization, comprising an N-terminal Ena/VASP homology (EVH) 1 domain, a more divergent proline-rich central part, and a common C-terminal EVH2 region of about 160-190 amino acids. Using chemical cross-linking, sucrose gradient sedimentation, and gel filtration analyses of different truncated VASP constructs, we demonstrate that the VASP EVH2 region is both necessary and sufficient for tetramerization. Moreover, co-sedimentation and fluorescent phalloidin staining showed that the EVH2 region binds and bundles F-actin in vitro and localizes to stress fibers in transfected cells. Analysis of the functional contribution of highly conserved blocks within this region indicated that residues 259-276 of human VASP are essential for the interaction with F-actin, whereas residues 343-380 are required for tetramerization, probably via coiled-coil formation. Interactions with F-actin are enhanced by VASP tetramerization. The results demonstrate that the C-terminal EVH2 segment is not only conserved in sequence but also forms a distinct functional entity. The data suggest that the EVH2 segment represents a novel oligomerization and F-actin binding domain.     

Listeria protein ActA mimics WASp family proteins: it activates filament barbed end branching by Arp2/3 complex

Actin-based propulsion of the bacteria Listeria and Shigella mimics the forward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filament branching through Arp2/3 complex, the Listeria surface protein ActA directly activates Arp2/3 complex by an unknown mechanism. Here we show that the N-terminal domain of ActA binds one actin monomer, in a profilin-like fashion, and Arp2/3 complex and mimics the C-terminal domain of WASp family proteins in catalyzing filament barbed end branching by Arp2/3 complex. No evidence is found for side branching of filaments by ActA-activated Arp2/3 complex. Mutations in the conserved acidic (41)DEWEEE(46) and basic (146)KKRRK(150) regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living cells and in the medium reconstituted from pure proteins confirm the conclusions of biochemical experiments. Filament branching is followed by rapid debranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA than by the C-terminal domain of N-WASp. VASP is required for efficient propulsion of ActA-coated beads in the reconstituted motility medium, but it does not affect the rates of barbed end branching/debranching by ActA-activated Arp2/3 nor the capping of filaments. VASP therefore affects another still unidentified biochemical reaction that plays an important role in actin-based movement.     

Doing (F/L)PPPPs: EVH1 domains and their proline-rich partners in cell polarity and migration.

Actin filament assembly is a tightly regulated process that functions in many aspects of cell physiology. Members of the Ena/VASP (Drosophila Enabled/vasodilator-stimulated phosphoprotein) family are key players in regulating actin filament assembly, in many cases through their association with binding partners that display a particular proline-rich motif, FPPPP. Ena/VASP proteins interact with these partners via the highly conserved Ena/VASP homology 1 (EVH1) domain. The diverse array of binding partners for EVH1 domains, including cytoskeletal proteins such as zyxin, transmembrane guidance receptors such as Roundabout, and the T-cell signaling protein Fyb/SLAP, shows that these interactions are likely to be important in a number of cellular processes that require regulated actin filament assembly.     

VASP is a processive actin polymerase that requires monomeric actin for barbed end association

Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and promote assembly of both filopodial and lamellipodial actin networks. To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro. We observed multiple filament binding modes: (1) static side binding, (2) side binding with one-dimensional diffusion, and (3) processive barbed end tracking. Actin monomers antagonize side binding but promote high affinity (K(d) = 9 nM) barbed end attachment. In low ionic strength buffers, VASP tetramers are weakly processive (K(off) = 0.69 s(-1)) polymerases that deliver multiple actin monomers per barbed end-binding event and effectively antagonize filament capping. In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti-capping activity. Based on our observations, we propose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly.     

Contribution of Ena/VASP proteins to intracellular motility of listeria requires phosphorylation and proline-rich core but not F-actin binding or multimerization

The Listeria model system has been essential for the identification and characterization of key regulators of the actin cytoskeleton such as the Arp2/3 complex and Ena/vasodilator-stimulated phosphoprotein (VASP) proteins. Although the role of Ena/VASP proteins in Listeria motility has been extensively studied, little is known about the contributions of their domains and phosphorylation state to bacterial motility. To address these issues, we have generated a panel of Ena/VASP mutants and, upon expression in Ena/VASP-deficient cells, evaluated their contribution to Ena/VASP function in Listeria motility. The proline-rich region, the putative G-actin binding site, and the Ser/Thr phosphorylation of Ena/VASP proteins are all required for efficient Listeria motility. Surprisingly, the interaction of Ena/VASP proteins with F-actin and their potential ability to form multimers are both dispensable for their involvement in this process. Our data suggest that Ena/VASP proteins contribute to Listeria motility by regulating both the nucleation and elongation of actin filaments at the bacterial surface.     

Structural basis for the recruitment of profilin-actin complexes during filament elongation by Ena/VASP

Cells sustain high rates of actin filament elongation by maintaining a large pool of actin monomers above the critical concentration for polymerization. Profilin-actin complexes constitute the largest fraction of polymerization-competent actin monomers. Filament elongation factors such as Ena/VASP and formin catalyze the transition of profilin-actin from the cellular pool onto the barbed end of growing filaments. The molecular bases of this process are poorly understood. Here we present structural and energetic evidence for two consecutive steps of the elongation mechanism: the recruitment of profilin-actin by the last poly-Pro segment of vasodilator-stimulated phosphoprotein (VASP) and the binding of profilin-actin simultaneously to this poly-Pro and to the G-actin-binding (GAB) domain of VASP. The actin monomer bound at the GAB domain is proposed to be in position to join the barbed end of the growing filament concurrently with the release of profilin.     

Molecular mechanism of Ena/VASP-mediated actin-filament elongation

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.     

Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts

Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. In this report, we explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. We extended the observation that Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts to demonstrate that E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, we identified two distinct pools of Mena within individual homophilic adhesions that cells made when they adhered to cadherin-coated substrata. These Mena pools localized with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity was necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherin-based bundles perturbed the bundles without affecting the protrusive F-actin pool. We propose that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts.     

Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.     

WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP''s ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.     

Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein

The focal adhesion protein vinculin is an actin-binding protein involved in the mechanical coupling between the actin cytoskeleton and the extracellular matrix. An autoinhibitory interaction between the N-terminal head (Vh) and the C-terminal tail (Vt) of vinculin masks an actin filament side-binding domain in Vt. The binding of several proteins to Vh disrupts this intramolecular interaction and exposes the actin filament side-binding domain. Here, by combining kinetic assays and microscopy observations, we show that Vt inhibits actin polymerization by blocking the barbed ends of actin filaments. In low salt conditions, Vt nucleates actin filaments capped at their barbed ends. We determined that the interaction between vinculin and the barbed end is characterized by slow association and dissociation rate constants. This barbed end capping activity requires C-terminal amino acids of Vt that are dispensable for actin filament side binding. Like the side-binding domain, the capping domain of vinculin is masked by an autoinhibitory interaction between Vh and Vt. In contrast to the side-binding domain, the capping domain is not unmasked by the binding of a talin domain to Vh and requires the dissociation of an additional autoinhibitory interaction. Finally, we show that vinculin and the formin mDia1, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends.     

Critical roles of phosphorylation and actin binding motifs,but not the central proline-rich region,for Ena/vasodilator-stimulated phosphoprotein (VASP) function during cell migration

The Ena/vasodilator-stimulated phosphoprotein (VASP) protein family is implicated in the regulation of a number of actin-based cellular processes, including lamellipodial protrusion necessary for whole cell translocation. A growing body of evidence derived largely from in vitro biochemical experiments using purified proteins, cell-free extracts, and pathogen motility has begun to suggest various mechanistic roles for Ena/VASP proteins in the control of actin dynamics. Using complementation of phenotypes in Ena/VASP-deficient cells and overexpression in normal fibroblasts, we have assayed the function of a panel of mutants in one member of this family, Mena, by mutating highly conserved sequence elements found in this protein family. Surprisingly, deletion of sites required for binding of the actin monomer-binding protein profilin, a known ligand of Ena/VASP proteins, has no effect on the ability of Mena to regulate random cell motility. Our analysis revealed two features essential for Ena/VASP function in cell movement, cyclic nucleotide-dependent kinase phosphorylation sites and an F-actin binding motif. Interestingly, expression of the C-terminal EVH2 domain alone is sufficient to complement loss of Ena/VASP function in random cell motility.