Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin.

Filopodia are exploratory finger-like projections composed of multiple long, straight, parallel-bundled actin filaments that protrude from the leading edge of migrating cells. Drosophila melanogaster Enabled (Ena) is a member of the Ena/vasodilator-stimulated phosphoprotein protein family, which facilitates the assembly of filopodial actin filaments that are bundled by Fascin. However, the mechanism by which Ena and Fascin promote the assembly of uniformly thick F-actin bundles that are capable of producing coordinated protrusive forces without buckling is not well understood. We used multicolor evanescent wave fluorescence microscopy imaging to follow individual Ena molecules on both single and Fascin-bundled F-actin in vitro. Individual Ena tetramers increase the elongation rate approximately two- to threefold and inhibit capping protein by remaining processively associated with the barbed end for an average of ∼10 s in solution, for ∼60 s when immobilized on a surface, and for ∼110 s when multiple Ena tetramers are clustered on a surface. Ena also can gather and simultaneously elongate multiple barbed ends. Collectively, these properties could facilitate the recruitment of Fascin and initiate filopodia formation. Remarkably, we found that Ena's actin-assembly properties are tunable on Fascin-bundled filaments, facilitating the formation of filopodia-like F-actin networks without tapered barbed ends. Ena-associated trailing barbed ends in Fascin-bundled actin filaments have approximately twofold more frequent and approximately fivefold longer processive runs, allowing them to catch up with leading barbed ends efficiently. Therefore, Fascin and Ena cooperate to extend and maintain robust filopodia of uniform thickness with aligned barbed ends by a unique mechanistic cycle.