Mechanisms of Branched Actin Network Formation through Coordinate Activation of Arp2/3 Complex
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Fundamental cellular processes such as motility and endocytosis rely on the actin cytoskeleton to translate biochemical protein interactions into mechanical forces. Cells utilize an extensive collection of actin binding proteins to comprehensively regulate actin networks during these dynamic cell operations. Branched actin networks, which are geometrically and functionally disparate from linear networks, are required for numerous cellular actions. Actin-related protein 2/3 complex (Arp2/3 complex) nucleates branched actin filaments upon activation by regulatory proteins known as nucleation promoting factors (NPFs). Often, several biochemically distinct NPFs are required for the same cellular structure, leading us to hypothesize that multiple NPFs can coordinately activate Arp2/3 complex to regulate the nucleation, architecture and assembly of branched networks. We identified and dissected the mechanisms of two sets of NPFs which coordinately activate Arp2/3 complex. Overall, these findings provide a better understanding of how Arp2/3 complex is activated and how cells control branched actin networks. In chapters II and III, we investigated the mechanism of synergistic activation of Arp2/3 complex by the NPFs cortactin and WASP family proteins. We found that cortactin accelerates the release of WASP family proteins from a branching intermediate, a previously unknown rate limiting step. Further dissection of the mechanism revealed that cortactin is specifically suited to displace WASP family proteins through a unique Arp2/3 complex binding region and target stalled branching intermediates with high affinity. Three different WASP family members were tested for their capacity to synergize with cortactin in Arp2/3 complex activation, establishing a list of cellular structures where cortactin-mediated synergistic activation is likely occurring. In chapter IV, we investigated the ability of Dip1 and Wsp1 to coordinately activate Arp2/3 complex during branched network formation. We established that Dip1 activation of Arp2/3 complex results in the formation of linear filaments which can template Wsp1 mediated branching. Subsequent kinetic data and modeling revealed that Dip1 and Wsp1 likely increase the rate of network formation by simultaneously binding to and co-activating Arp2/3 complex. These findings suggest that, together, Dip1 and Wsp1 regulate the initiation and rate of branched network assembly. This dissertation includes previously published and unpublished co-authored material and videos files.