Formins have important roles in the nucleation of actin and the formation of linear actin filaments, but their role in filopodium formation has remained elusive. Dictyostelium discoideum Diaphanous-related formin dDia2 is enriched at the tips of filopodia and interacts with profilin II and Rac1. An FH1FH2 fragment of dDia2 nucleated actin polymerization and removed capping protein from capped filament ends. Genetic studies showed that dDia2 is important for cell migration as well as the formation, elongation and maintenance of filopodia. Here we provide evidence that dDia2 specifically controls filopodial dynamics by regulating actin turnover at the barbed ends of actin filaments.

Filopodia are highly dynamic finger-like cell protrusions filled with parallel bundles of actin filaments. Previously we have shown that Diaphanous-related formin dDia2 is involved in the formation of filopodia. Another key player for the formation of filopodia across many species is vasodilator-stimulated phosphoprotein (VASP). It has been proposed that the essential role of VASP for formation of filopodia is its competition with capping proteins for filament barbed-end interaction. To better understand the function of VASP in filopodium formation, we analyzed the in vitro and in vivo properties of Dictyostelium VASP (DdVASP) and extended our findings to human VASP. Recombinant VASP from both species nucleated and bundled actin filaments, but did not compete with capping proteins or block depolymerization from barbed ends. Together with the finding that DdVASP binds to the FH2 domain of dDia2, these data indicate that the crucial role of VASP in filopodium formation is different from uncapping of actin filaments. To identify the activity of DdVASP required in this process, rescue experiments of DdVASP-null cells with mutant DdVASP constructs were performed. Only WT DdVASP, but not a mutant lacking the F-actin bundling activity, could rescue the ability of these cells to form WT-like filopodia. Our data suggest that DdVASP is complexed with dDia2 in filopodial tips and support formin-mediated filament elongation by bundling nascent actin filaments.actin cytoskeleton ͉ Dictyostelium ͉ formin C ytoskeletal dynamics in moving fronts, lamellipodia, or filopodia require fast and highly coordinated actin polymerization, which leads to broad filamentous networks or to spiky membrane protrusions. The Arp2͞3 complex is known to nucleate branching of actin filaments, thus favoring the formation of dense F-actin meshworks, whereas formins efficiently elongate linear actin filaments (1). Although formin function could be clearly correlated with the establishment of actin cables and stress fibers (2, 3), we only recently began to understand its role in filopodium formation (4-6). The Dictyostelium Diaphanous-related formin dDia2 is associated with the distal tips of growing filopodia and is required for the elongation of filopodial actin filaments (6); however, growth and stabilization of filopodia coincide with recruitment of a number of actin-associated proteins, including the actin-bundling protein fascin (7), talin (8, 9), myosin VII and X (10, 11), Scar͞Wave (12), and Irsp53 (13, 14), as well as Ena͞vasodilator-stimulated phosphoprotein (VASP) proteins (15-18). It still remains to be shown how these proteins stabilize the emerging bundle (19). VASP is a member of the Ena͞VASP family of proteins, which are implicated in regulation of the actin cytoskeleton and control of cell motility (20)(21)(22). Ena͞VASP proteins were proposed to associate with the barbed ends of microfilaments, to protect them from capping protein (CP), and to support growth of filopodial actin filaments (17,18,23). Consistent with a crucial function...

Filopodia are finger-like cell protrusions composed of parallel arrays of actin filaments, which elongate through actin polymerization at their tips. These highly dynamic structures seem to be used by many cell types as sensing organs to explore environmental cues and have been implicated in cell motility as well as in cell-substrate adhesion. Formins are highly conserved multidomain proteins that play important roles in the nucleation of actin and the formation of linear actin filaments, yet their role in filopodia formation has remained poorly defined. The Dictyostelium diaphanous-related formin dDia2 is strongly enriched in filopodia tips. Genetic and biochemical analysis revealed that this protein is important for cell migration and cell adhesion, but most importantly for the formation of filopodia. Recently, we have identified the Dictyostelium VASP (vasodilator-stimulated phosphoprotein) orthologue as a binding partner of dDia2 and provide evidence for a co-operative role of both proteins in filopodia formation.

Filopodia are finger-like cell protrusions composed of parallel arrays of actin filaments, which elongate through actin polymerization at their tips. These highly dynamic structures seem to be used by many cell types as sensing organs to explore environmental cues and have been implicated in cell motility as well as in cell-substrate adhesion. Formins are highly conserved multidomain proteins that play important roles in the nucleation of actin and the formation of linear actin filaments, yet their role in filopodia formation has remained poorly defined. The Dictyostelium diaphanous-related formin dDia2 is strongly enriched in filopodia tips. Genetic and biochemical analysis revealed that this protein is important for cell migration and cell adhesion, but most importantly for the formation of filopodia. Recently, we have identified the Dictyostelium VASP (vasodilator-stimulated phosphoprotein) orthologue as a binding partner of dDia2 and provide evidence for a co-operative role of both proteins in filopodia formation.