Control of actin filament networks by Arp2/3 complex and its regulators

SUMMARY Branched actin filaments nucleated by Arp2/3 complex provide pushing forces that drive normal cellular processes like endocytosis and lamellipodial protrusion, as well as pathogenic processes like bacterial infection and tumor metastasis. Regulatory proteins control the activity of Arp2/3 complex and the architecture and stability of the actin networks it assembles. Elucidating how these regulators function is critical to understanding how cells orchestrate actin-based processes dependent on Arp2/3 complex. Here we propose to investigate key unresolved questions about Arp2/3 activators from the WASP and WISH/DIP/SPIN90 families, as well as the debranching protein, coronin 7. We will address these questions using a combination of biochemical reconstitution, single molecule fluorescence, live cell imaging, electron microscopy, molecular dynamic simulations, and filament-level modeling. WASP proteins trigger a major activating conformational change in Arp2/3 complex but cannot complete the activation process until the complex binds to a pre-existing actin filament. We propose to determine how the WASP bound-complex is held inactive without actin filaments, as this ensures that linear filament nucleation is repressed during WASP-mediated activation, allowing WASP-activated Arp2/3 complex to create exclusively branches. We will also investigate the mechanism by which WASP is released to complete the activation process. We anticipate these experiments will provide critical insights into the mechanism by which cells create actin networks with specific architectures. The WISH/DIP/SPIN90 (WDS) proteins activate Arp2/3 complex without a pre-existing actin filament, enabling them to initiate branched actin network assembly. However, they promote creation of linear actin filaments that could disrupt the dense branching thought to be important for effective pushing by Arp2/3- assembled actin networks. We will investigate how simultaneous filament nucleation by WDS and WASP proteins influences force-producing actin networks assembled by Arp2/3 complex. We anticipate these experiments will yield important insights into how WDS and WASP proteins coordinately regulate Arp2/3 complex, and how the architecture of actin networks assembled by Arp2/3 complex influences their function. Actin filament debranchers influence branched actin networks in multiple ways, including by facilitating their disassembly or remodeling them into bundles. Coronin 7, the most recently discovered debrancher, plays important roles in regulating actin during cellular motility, asymmetric cell division, and protein trafficking. While biochemical experiments showed that it can accelerate disassembly of individual branches, how it influences force-producing branched actin networks is unclear, and the debranching mechanism is not known. Here we propose to investigate the influence of coronin 7 on force-production, remodeling, and recycling of actin networks, and to reveal the molecular mechanism of this important debrancher.