Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Lambda-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Lambda-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Lambda-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.
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