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 anticapping 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, 2) and for actinbased motility of the pathogenic bacterium Listeria monocytogenes (3-6). Ena/VASP proteins influence the dynamics of lamellipodia and formation of filopodial protrusions in fibroblasts and neuronal growth cones (7-9). In Dictyostelium, VASP is required for efficient chemotaxis and formation of filopodia (10). Ena/VASP proteins regulate the architecture of actin filaments in the actin tails of motile Listeria (11) or those associated with beads coated with the Listeria ActA protein (12, 13). 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, 9). 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 whic...

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.

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. INTRODUCTIONActin-based protrusions known as filopodia, first described by Porter, Claude, and Fullam as early as 1945 (Porter et al., 1945;Albrecht-Buehler, 1976), are composed of tightly bundled parallel filaments 5-50 m long and 0.1-0.5 m thick (Small, 1988;Mitchison and Cramer, 1996). Filopodia have been implicated in a number of cellular processes including neuronal growth cone pathfinding, embryonic development, wound healing, and metastasis (Jacinto et al., 2000(Jacinto et al., , 2001Vasioukhin and Fuchs, 2001;Dent and Gertler, 2003;Hashimoto et al., 2005). Despite the importance of filopodia to so many diverse cellular functions, the exact mechanism governing their initiation and formation has yet to be fully explained. A number of actin-binding proteins have been implicated in the formation of filopodia, most recently myosin X and the formin family of proteins, which catalyze the formation of long unbranched actin filaments Zigmond, 2004;Bohil et al., 2006;Kovar, 2006). However, in several cell types, filopodia and the sheet-like Arp2/3-based lamellipodia, rapidly interchange during protrusion, suggesting commonalities between the two structures . The type of protrusion that dominates in the subcellular compartment critically depends on whether filaments are allowed to elongate persistently or are capped shortly after nucleation. Accordingly, when the heterodimeric Capping Protein (CP), the major barbed end terminator in lamellipodia, is silenced by small interfering RNA (siRNA), the balance between the two structures, is shifted to favor filopodial formation (Mejillano et al., 2004). A similar phenomenon has been observed in vitro where a decrease in CP levels led to a transition from a dendritic-like network...

Nonmusclemyosin 2 (NM-2) powers cell motility and tissue morphogenesis by assembling into bipolar filaments that interact with actin. Although the enzymatic properties of purified NM-2 motor fragments have been determined, the emergent properties of filament ensembles are unknown. Using single myosin filament in vitro motility assays, we report fundamental differences in filaments formed of different NM-2 motors. Filaments consisting of NM2-B moved processively along actin, while under identical conditions, NM2-A filaments did not. By more closely mimicking the physiological milieu, either by increasing solution viscosity or by co-polymerization with NM2-B, NM2-A containing filaments moved processively. Our data demonstrate that both the kinetic and mechanical properties of these two myosins, in addition to the stochiometry of NM-2 subunits, can tune filament mechanical output. We propose altering NM-2 filament composition is a general cellular strategy for tailoring force production of filaments to specific functions, such as maintaining tension or remodeling actin.

Filopodia are actin-based cell extensions that contribute to cell adhesion and spreading. The cytoskeleton regulators mDia2 and VASP have distinct roles in filopodia assembly and function, and VASP controls the length, dynamics, stability, and integrin-dependent adhesion of filopodia formed by mDia2.

The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips.

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Highlights d Mechanotransduction regulates actin at the tips of mammalian cochlear stereocilia d With transduction onset, ADF/cofilin localize to mechanotransducing stereocilia tips d Actin-severing proteins increase available F-actin barbed ends at stereocilia tips d Normal stereocilia length and width depend on ADF and cofilin-1