Structural-Functional Basis of Actin Cytoskeleton Dynamics

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Summary The actin cytoskeleton is a highly dynamic system consisting of hundreds of proteins. Cells use the actin cytoskeleton to move, divide, transport organelles and exchange materials with the environment. Many human diseases result from malfunctioning of actin cytoskeletal components. There is therefore intense interest in understanding molecular mechanisms that control actin cytoskeletal processes, which has both fundamental importance and the potential to accelerate the development of targeted therapies to treat human diseases. Among actin cytoskeletal components, none is more important than Arp2/3 complex, a 7-subunit actin filament nucleation and branching system conserved in eukaryotes from yeast to human. This grant addresses important gaps of knowledge of the mechanisms of Arp2/3 complex activation, inhibition, branch stabilization, and branch destabilization. Published work, extensive preliminary studies presented in the application, and new advances in the laboratory such as the implementation of cryo-electron microscopy (cryo-EM) to the Arp2/3 complex system, and innovative protein expression methods and biochemical assays provide the scientific and technical premises supporting the research plans. The specific aims focus on three major areas: 1) understand the molecular determinants of human Arp2/3 complex branch stability and mechanosensation using a microfluidics-TIRF microscopy assay, 2) determine the structural-functional mechanism of branch stabilization by cortactin using biochemical methods and cryo-EM, and 3) uncover whether one of the members of the coronin family acts as an Arp2/3 complex inhibitor, a branch stabilizer, or a branch destabilizer using biochemical approaches and cryo-EM