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.