Computational simulations of polymerizing actin filaments indicate that competition for a limiting pool of building blocks is not sufficient to control their length.

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.

Formins stimulate actin filament assembly for fundamental cellular processes including division, adhesion, establishing polarity, and motility. A formin inhibitor would be useful because most cells express multiple formins whose functions are not known and because metastatic tumor formation depends on the deregulation of formin-dependent processes. We identified a general small molecule inhibitor of formin homology 2 domains (SMIFH2) by screening compounds for the ability to prevent formin-mediated actin assembly in vitro. SMIFH2 targets formins from evolutionarily diverse organisms including yeast, nematode worm, and mice, with a half-maximal inhibitor concentration of ∼5 to 15 μM. SMIFH2 prevents both formin nucleation and processive barbed end elongation and decreases formin's affinity for the barbed end. Furthermore, low micromolar concentrations of SMIFH2 disrupt formin-dependent, but not Arp2/3 complex-dependent, actin cytoskeletal structures in fission yeast and mammalian NIH 3T3 fibroblasts.

How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly challenging in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review we explore recent studies in fission yeast that help unravel how different actin structures operate in cells.