Loss of Ena/VASP Interferes with Lamellipodium Architecture, Motility and Integrin-Dependent Adhesion

Cell migration entails protrusion of the front comprising networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively. Ena/VASP proteins accumulate at their tips and in focal adhesions (FA), where they presumably act as potent filament polymerases. However, the precise contributions of this protein family to protrusion, migration and adhesion are unclear. Here, using combined CRISPR/Cas9-mediated disruption of genes encoding VASP, Mena and Evl in B16-F1 melanoma, we show that their removal abolishes the formation of microspikes normally embedded in lamellipodia, and perturbs lamellipodial architecture accompanied by reduced actin assembly and cell migration. Notably, microspikes could be rescued by any Ena/VASP family member, but neither by active variants of the GTPase Rif, the formins mDia2, FMNL2 and FMNL3, nor unconventional Myosin X. Since all of the latter can potently induce filopodia, these data suggest microspikes and filopodia to constitute distinct, molecularly separable, actin-based projections. Finally, Ena/VASP-deficient cells displayed impaired spreading and focal adhesion (FA) patterns accompanied by reduced traction forces.