The long-term goal of this project is to understand the spatial and temporal regulation of signal transduction pathways that underlie mammalian cell polarization. Polarity establishment is vital for every aspect of metazoan development. For example, the segregation of cell fate determinants to the opposite poles of a stem cell, coupled with oriented cell division, permits the specification of distinct daughter cell phenotypes. Cell polarization is also required for morphogenesis, directional motility, antigen presentation, and axon guidance; and the loss of cell polarity is a critical step in cancer progression. Remarkably, the novel signaling pathways involved in polarization have been highly conserved throughout metazoan evolution. Cdc42 binds to a protein called Par6, and Par6 binds to atypical protein kinases C (aPKC). These proteins form a complex that regulates many types of cell polarization. We propose that Par6 behaves as a targeting subunit for aPKC, recruiting substrates for phosphorylation. Three putative effectors for Par6 have been found: Par3, Lgl, and Pals1. Each of these 3 proteins is essential for epithelial cell polarization, and each interacts with several other proteins that have also been implicated in polarization. These three sets of proteins (Par, Pals, and Lgl) interact both genetically and physically to create the distinct membrane domains that define a polarized epithelial cell, and they also participate in other types of cell polarity. How do they accomplish these tasks? The central questions to be addressed are: how do these proteins associate with and regulate each other? How do they localize to their appropriate positions during polarization (what are their targeting cues)? Which proteins need to arrive first at the cell junctions? And how do they execute the polarization program? X-ray crystallography will be used to determine the structures of polarity protein complexes. For cell biological assays, MDCK cells will be used as an established, well-accepted model system. These cells form highly polarized confluent monolayers, but in response to scatter factor, or wounding, they lose their apical/basal polarity, and become motile, with an anterior/posterior polarity axis Stable, inducible cell lines will be developed that express fluorescent fusions of polarity proteins, and used to quantify the dynamics of cell junction assembly and disassembly.
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