PROJECT SUMMARY Polarity establishment is essential for development, from asymmetric cell division to the formation of tissues. Radial polarization of PAR polarity proteins occurs in early mammalian embryos and is required for separation of embryonic and extra-embryonic lineages. Early mammalian embryos and epithelial cells use cell contacts as cues for establishing polarity, but the molecular links between cell contact cues and the cortical PAR polarity proteins that polarize cells remain poorly understood. The C. elegans early embryo polarizes when the RhoGAP, PAC-1/ARHGAP21, is recruited to cell contacts (acting as the symmetry breaking cue) and inactivates CDC-42, a Rho-GTPase, resulting in the loss of PAR proteins from contact sites. E-cadherin, a homophilic adhesion protein, is required for contact-mediated polarity in many different cell types. In C. elegans, E-cadherin plays an instructive role in establishing polarity by helping to recruit PAC-1 to contact sites. E-cadherin acts with components of the cadherin-catenin complex (CCC) to drive polarization, and functions redundantly with an unidentified cadherin-independent pathway to localize PAC-1. The proteins involved in polarity establishment in C. elegans are highly conserved, so understanding their role in this genetically tractable system will provide insights into contact-mediated polarization in mammalian embryos and epithelia. The overarching goal of this proposal is to identify mechanisms that link cell contact with apicobasal polarity within the cell. To achieve this, I will identify the biochemical connections between E-cadherin and PAC-1 that recruit it to cell contacts (Aim 1). I hypothesize that the conserved polarity protein, afadin (AFD-1), aids in PAC-1 localization by functioning as a linker between a-catenin/HMP-1 (downstream of E-cadherin) and PAC-1. I will test this hypothesis using genetic manipulation of the CCC and immunoprecipitation to identify protein interactions. I will also use mass spec to identify proteins interacting with AFD-1 to drive PAC-1 localization and potentially function. I will identify the E-cadherin-independent pathway that also localizes PAC-1 (Aim 2). I hypothesize that another polarity protein recruits PAC-1, either directly (protein-protein interaction) or indirectly (through phosphorylation or changes in membrane curvature). I will examine candidate proteins that were identified through yeast two hybrid to interact with the PH domain of PAC-1 (the region known to be important for localization without E- cadherin) for their ability to localize PAC-1 in the absence of E-cadherin. I will also use mass spec to identify the proteins interacting with PAC-1 with and without E-cadherin. This, along with phosphorylation analysis, will allow us to understand how PAC-1 localizes without the aid of E-cadherin. An unbiased EMS mutagenesis screen in an E-cadherin null background will aid in determining how PAC-1 localizes in the absence of E-cadherin. ...