Self-assembly of filopodia-like structures on supported lipid bilayers

Filopodia are finger-like protrusive structures, containing actin bundles. By incubating frog egg extracts with supported lipid bilayers containing phosphatidylinositol 4,5 bisphosphate, we have reconstituted the assembly of filopodia-like structures (FLSs). The actin assembles into parallel bundles, and known filopodial components localize to the tip and shaft. The filopodia tip complexes self-organize—they are not templated by preexisting membrane microdomains. The F-BAR domain protein toca-1 recruits N-WASP, followed by the Arp2/3 complex and actin. Elongation proteins, Diaphanous-related formin, VASP, and fascin are recruited subsequently. Although the Arp2/3 complex is required for FLS initiation, it is not essential for elongation, which involves formins. We propose that filopodia form via clustering of Arp2/3 complex activators, self-assembly of filopodial tip complexes on the membrane, and outgrowth of actin bundles. Actin assembly is largely responsible forcell shape and movement in eukaryoticcells (1). Actin filaments underlie a range of morphological structures; among these, filopo-dia, which contain closely apposed bundled par-allel actin filaments, are of particular interest (2, 3). They have numerous biological roles, but their mechanism of formation is poorly under-stood. The persistence of actin growth in a par-allel bundle in filopodia suggests roles for proteins with anti-capping and/or processive elongation activity [such as vasodilator-stimulated phospho-protein (VASP) and formins] (4–8). But formins are also important in lamellipodia (9). The role of the Neural Wiskott-Aldrich syndrome protein (N-WASP)/Arp2/3 complex pathway in filopo-dia formation is also contentious (8, 10–14). Al-though there are in vitro models of actin bundling (15–17), no current experimental system recapit-ulates two physiological aspects of filopodia: (i) spontaneous formation of actin bundles in the presence of capping activity and (ii) the assembly of a membrane-localized tip complex. To investigate actin polymerization at mem-branes and to exploit advanced microscopic tech-niques like total internal reflection fluorescence (TIRF) microscopy, we have replaced liposomes as the source of lipidswith supported lipid bilayer