The long-term goal of this proposal is to understand the structural and functional mechanisms that control actin cytoskeleton dynamics in health and disease. Specifically, Aim-1 will tackle the long-standing problem of how WASP-family Nucleation Promoting Factors interact with and activate actin branch formation by the Arp2/3 complex. Because nucleation is the rate-limiting step in actin polymerization, cells use actin nucleators to precisely control the de novo formation of actin filaments in time and space. Among these, the Arp2/3 complex is the sole nucleator of branched actin networks. Branched actin assembly is important in myofibril development and is misregulated in Wiskott-Aldrich Syndrome causing thrombocytopenia and platelet dysfunction. My plans to understand the mechanism of Arp2/3 complex activation build upon the ability to perform biochemical and structural studies on the baculovirus-expressed Arp2/3 complex and subcomplexes. Extensive preliminary work provides the scientific premise and supports feasibility. In brief, the aims of this proposal tackle the following questions pertaining to Arp2/3 complex's mechanism of activation: (1) In what order do the Nucleation Promoting Factors that activate Arp2/3 complex bind and what is their molecular interaction with the complex? (2) How does Arp2/3 complex interact with the mother filament? (3) What are the functional consequences of these interactions? Aim 2 of this proposal focuses on Arp2/3 complex's newly appreciated role in focal adhesion maturation. Focal adhesions are macromolecular assemblies that link the extracellular matrix to the actin cytoskeleton. An unresolved and fundamental question in cell motility and adhesion is; how are actin networks recruited to focal adhesions? Recently, Arp2/3 was shown to interact with Kindlin, an important activator of focal adhesions. Based on this observation and my own preliminary results, here I dissect Arp2/3 complex's role in focal adhesion maturation, with the underlying hypothesis that Kindlin recruits Arp2/3 to initiate the formation of dendritic actin networks at adhesion sites. Towards this aim, my proposal will answer the following questions: (1) Does Kindlin bind free Arp2/3, branch junctions, or both? (2) Does Kindlin affect Arp2/3 nucleation activity? (3) At an atomic level, how does Kindlin bind Arp2/3? Improper focal adhesion formation is linked to various diseases including cardiomyopathy, hematopoic dysfunction and muscular dystrophy. Understanding Arp2/3 complex's role at focal adhesions is an important step towards understand platelet activation, muscle development and healthy physiology as a whole. These two aims are thematic of the laboratory's workflow, tackling mechanistic questions in vitro with biochemical and structural assays and subsequently understanding their functional consequences in vitro and in vivo. Taken together, these aims will have a far reaching impact and will train me in the skills of biochemistry, structural biology and molecular biophysics.
This research plan addresses major gaps of knowledge in the mechanisms that cells use to control branched actin assembly and focal adhesion maturation. The branched actin architecture drives essential biological processes, such as (but not limited to) cell division and motility, platelet function, and muscle development. Understanding the molecular basis of these processes is fundamental in developing strategies for the diagnosis and treatment of a myriad of human diseases associated with their dysfunction, including muscular dystrophies, cardiomyopathies, blood diseases, and many others.
