Direct Actin Monomer Delivery is a WASP-specific Requirement for Arp2/3 Complex Activation.

WASP family proteins activate Arp2/3 complex to nucleate branched actin filaments in diverse cellular processes. To activate, WASP proteins recruit actin monomers to Arp2/3 complex through their conserved WASP homology 2 domains, but how these recruited monomers contribute to activation of Arp2/3 complex has been unclear. Previous studies suggested potential species-specific differences: work on human Arp2/3 complex indicated that direct actin monomer delivery by WASP is required to assemble the filament nucleus, while studies on budding yeast Arp2/3 complex showed that direct monomer recruitment is required specifically for WASP-mediated activation of the complex and is not a fundamental requirement for activation. Here we show that mutations that shift the actin-related subunits (Arp2 and Arp3) in human Arp2/3 complex toward a filament-like conformation enable nucleation without WASP. This demonstrates that direct monomer delivery by WASP is not a fundamental requirement for formation of the nucleus, but instead a specific requirement for WASP-mediated activation-consistent with findings in yeast Arp2/3 complex. Using kinetic models of actin polymerization, we provide evidence that while the Arp2-Arp3 filament-like dimer is insufficient for nucleation by human Arp2/3 complex, actin monomers diffuse and bind to the complex to create the filament nucleus in the absence of recruitment. We show that this diffusion-based mechanism can stimulate nucleation by mammalian Arp2/3 complex at rates near tethered actin monomer delivery. These results provide important insights into Arp2/3 complex activation with or without monomer delivery and demonstrate that fundamental aspects of activation are conserved across fungal and mammalian species.