The Cytoskeletal Drivers of Cell Polarity

Project Summary/Abstract The polarity of cells is a fundamental biological necessity, involving the creation of compositionally and functionally distinct domains that form cellular asymmetry. This is required for mechanisms such as cell division, motility, and the establishment of cell morphology during development. Disruption of proper polarity regulation results in tissue or cell-type-specific human disease. Examples include cancer metastasis through the epithelial to mesenchymal transition and neurological disorders through failure to establish proper dendritic or axonal processes. Creation and maintenance of polarized cell morphology involves the cooperative actions of multiple interlaced cellular pathways. These pathways include the assembly of cytoskeletal filaments into polarized networks, force production by molecular motors, and the regulation by signaling molecules. Although a foundational understanding of their operation is available, there exists a void in our mechanistic understanding of how these components are coordinated temporally and spatially. We focus on the regulation and organization of the cell cortex by Rho family and kinase signaling proteins, specifically in the region on epithelial cells that contains three distinct actin bases structures: bundles supporting the microvilli, the terminal web, and the junctional belt associated with the adheres junction. This system provides a unique opportunity to uncover detailed mechanisms underlying local signaling regulation. In this proposal, we focus on two critical regulators of apical cell polarity both of which are localized to, and regulate, microvilli: the Class IX myosins (Myo-9) and Lymphocyte Oriented Kinase (LOK). Myo-9 is unique among human proteins for the presence of both an actin binding molecular motor domain and a rho family GTPase activating protein (GAP) signaling domain. Using biochemistry, biophysics, and cell biology, we will examine the roles of the GAP and actin-binding domains on the structure of the apical domain, how the myosin is targeted, and define associated proteins and their function. The second project probes the function of LOK and its homologue SLK, the only known kinases to localize to the apical surface of epithelial cells where they act as leading regulators of cell shape and cytoskeletal organization. Despite their central role in apical polarity, the ezrin radixin, moesin (ERM) family of proteins are the only known kinase substrates and almost nothing is known about how they are targeted to the apical cortex. We will elucidate the mechanisms employed to localize these kinases, identify their kinase targets, and characterize their role in the spatial and temporal regulation of apical actin-based structures. By characterizing these polarity driving organizers in the model system of microvilli formation, this project will advance our understanding of actin cytoskeletal regulation's precise spatial and temporal nature in the formation of polarized cells.