The precise spatiotemporal control of the actin cytoskeleton at the leading edge is critical for cell migration, and many of the `players' (molecules) involved have been identified, including those involved in regulating actin assembly to drive leading edge protrusion. However, many of the interacting components of this actin assembly machinery (e.g., IQGAP, APC, and Formins) are large molecules with complex domain structures and interactions, making them difficult to study. For this reason, there persists a fundamental gap in our understanding of the molecular basis of actin assembly regulation at the leading edge. Adenomatous polyposis coli (APC) localizes to the leading edge (Nathke et al., 1996, JCB), and recent work from the Goode Lab has shown that a C-terminal domain in APC potently stimulates actin assembly in vitro, in direct collaboration with formins, via a `Rocket Launcher' mechanism (Okada et al., 2010, JCB; Breitsprecher et al., 2012, Science). Further, the Goode Lab used a powerful separation-of-function point mutation in this domain to demonstrate that APC-mediated actin assembly is critical for directed cell migration (Juanes et al., 2017, JCB). A major challenge now is to determine how the potent actin assembly-promoting activities of APC and Dia1 are harnessed and controlled, spatially and temporally, to orchestrate leading edge advancement. This proposal investigates the role of IQGAP in this process. IQGAP is a multi-domain scaffolding protein that directly binds to and bundles F-actin, binds to APC and Dia1, and colocalizes with APC at the leading edge (Watanabe et al, 2004, Dev Cell, & Brandt et al, 2007, JCB). My preliminary in vitro observations using purified full-length IQGAP show that it directly attenuates actin assembly induced by APC and Dia1. Additionally, using labeled IQGAP, I observed that it transiently associates with the barbed end of the actin filament, suppressing growth. Consistent with these inhibitory biochemical effects, I found that RNAi silencing of IQGAP in HeLa cells results in excessive F-actin accumulation at the leading edge. Thus, my in vitro and in vivo observations are in good agreement, and indicate that IQGAP attenuates, or `restrains' actin assembly by APC and Dia1. My working model is that IQGAP scaffolds APC and Dia1 to spatiotemporally control actin assembly at the leading edge until the correct signals are received (possibly Rac1, Cdc42, and/or microtubule plus end-associated proteins, all of which bind to IQGAP). Using in vitro multi-wavelength single-molecule imaging, I will next define the stoichiometry and kinetics of IQGAP's interactions with actin filaments, APC, and Dia1, and their combined regulatory effects on actin assembly in vitro. In parallel, I will use genetic perturbations and live-cell imaging to determine the precise role of IQGAP, and its interactions with actin, APC, and Dia1, in tuning actin dynamics at the leading edge to drive cell migration. I will quantify the cellular effects caused by silencing each protein - on the localization dynamics of the other components, on the assembly and turnover kinetics of leading edge actin networks, and on directed cell migration. This work will clarify the mechanisms controlling actin network assembly in cell migration. Further, it will offer novel insights into the mechanisms of human disease, since mutations in APC cause >80% of all colorectal cancers, and there is mounting evidence that its role in cancer progression involves not only the loss of its Wnt signaling function, but also its cytoskeletal functions.
Relevance to Public Health: The proposed grant investigates the mechanistic basis of human IQGAP and APC in regulating actin dynamics at the leading edge of migrating cells. This work has direct relevance to human health because mutations in APC cause over 80% of colorectal cancers. Further, cell migration plays a fundamental role in human development, immunity, and cancer metastasis. Additionally, IQGAP has crucial roles in normal kidney, cardiac, lung and nervous system functions. For these reasons, this work will define the molecular underpinnings of human disease, and potentially facilitate the development of new therapies.
