Roles for Ena/VASP proteins in FMNL3-mediated filopodial assembly

Filopodia are actin-dependent finger-like structures that protrude from the plasma membrane. Actin filament barbed-end-binding proteins localized to filopodial tips are key to filopodial assembly. Two classes of barbed-end-binding proteins are formins and Ena/VASP proteins, and both classes have been localized to filopodial tips in specific cellular contexts. Here, we examine the filopodial roles of the FMNL formins and Ena/VASP proteins in U2OS cells. FMNL3 suppression reduces filopodial assembly by 90%, and FMNL3 is enriched at >95% of filopodial tips. Suppression of VASP or Mena (also known as ENAH) reduces filopodial assembly by >75%. However, VASP and Mena do not display consistent filopodial tip localization, but are enriched in focal adhesions (FAs). Interestingly, >85% of FMNL3-containing filopodia are associated with FAs. Two situations increase Ena/VASP filopodial localization: (1) expression of myosin-X, and (2) actively spreading cells. In spreading cells, filopodia often mark sites of nascent adhesions. Interestingly, VASP suppression in spreading cells causes a significant increase in adhesion assembly at filopodial tips. This work demonstrates that, in U2OS cells, Ena/VASP proteins play roles in filopodia beyond those at filopodial tips. This article has an associated First Person interview with the first author of the paper.