Filopodial protrusion driven by density-dependent Ena-TOCA-1 interactions.

Filopodia are narrow actin-rich protrusions with important roles in neuronal development where membrane-binding adaptor proteins have emerged as upstream regulators that link membrane interactions to actin regulators, for example I-BAR and F-BAR domain-containing proteins interacting with Ena/VASP and formins. To explore the significance of the F-BAR neuronal membrane adaptor TOCA-1 in filopodia we used quantitative analysis of TOCA-1 and filopodial dynamics in Xenopus retinal ganglion cells, where Ena/VASP proteins have a native role in filopodial extension. Both the adaptors and their binding partners are part of diverse and redundant protein networks that can functionally compensate for each other. Increasing density of TOCA-1 enhances Ena/VASP binding in vitro and an accumulation of TOCA-1, and its coincidence with Ena, correlates with filopodial protrusion in vivo. Two-colour single molecule localisation microscopy of TOCA-1 and Ena supports their nanoscale association. TOCA-1 clusters promote filopodial protrusion depending on a functional SH3 domain and activation of Cdc42, which we perturbed using small molecule inhibitor CASIN. We propose that TOCA-1 clusters act independently of membrane curvature to recruit and promote Ena activity for filopodial protrusion.