PROJECT SUMMARY The actin-related-protein (Arp) 2/3 complex is a 225-kDa seven-subunit actin filament nucleator that nucleates branched actin filaments. Polymerizing branched actin networks provide protrusive forces necessary to drive a myriad of cellular processes, including motility, vesicle trafficking, and endocytosis. To orchestrate these functions, cells utilize proteins that bind to and activate Arp2/3 complex known as nucleation promotion factors (NPFs). The most ubiquitous and well-studied class of NPFs, Wiskott-Aldrich syndrome proteins (WASP), are characterized by a conserved C-terminal “VCA” (verprolin homology, central, acidic) motif that binds to actin monomers (V) and Arp2/3 complex (CA). In the absence of WASP, Arp2/3 complex is held in an inactive conformation in which the two actin-related proteins Arp2 and Arp3 are arranged in an end-to-end orientation referred to here as the splayed state. Activation depends on a large conformational change that moves Arp2 and Arp3 into filament-like arrangement known as the short-pitch conformation. Previously, we demonstrated that WASP binding stimulates formation of the short-pitch conformation and that this is the main activating function of WASP. However, exactly how WASP binding shifts the splayed to short-pitch conformational equilibrium is unclear, and is the focus of this proposal. We will address this from a structure-function perspective using high resolution structures of the inactive state and hypothetical models of the active state to determine how the complex is held inactive in the absence of WASP (Aim 1). In addition, we will address two fundamental aspects of WASP-mediated regulation of the complex that are critical open questions in the field. First, while recent data indicated that WASP binds to two distinct binding sites on the complex, how engagement at each site contributes to activation of the complex and assembly of actin networks in vitro or in cells remains unknown. We will address this question in Aim 2, taking advantage of a recently determined map of the WASP binding sites on Arp2/3 complex determined by crosslinking/mass-spectrometry. Second, recent data show that to activate Arp2/3 complex, WASP must first bind to stimulate the short pitch conformation, but then must be released to allow nucleation to proceed. Because WASP binds membranes in cells, release of WASP is thought to play a critical role in the assembly of force-producing actin networks; i.e., it provides a transient connection between the network to the membrane that facilitates pushing, yet by releasing after nucleation ensures that polymerizing networks are not so tightly bound they cause network compression. No studies have addressed how the interactions of WASP with the complex are tuned to optimally balance its nucleation potency versus its ability to serve as a tether between actin networks and membranes, despite the fact that both of these activities are critical in assembling productive actin...