The overarching goal of this project is to understand how molecular-scale interactions at cellular adhesion complexes dictate the organization of cells and tissues. In the first portion of this project, we focus on the proteins that make up adherens junctions (AJs) and tight junctions (TJs). AJs and TJs link the cytoskeletons of neighboring cells and allow epithelial tissues to fulfill their essential function as physical barriers, respectively. Importantly, these complex molecular assemblies are exquisitely responsive to the mechanical forces generated during embryonic development and tissue repair, and in the context of diseases such as cancer and heart disease. However, only a few of the protein-protein interactions that make up these adhesion complexes have been characterized biochemically, and even less is known about the underlying mechanisms by which these structures respond to mechanical load. This lack of quantitative data presents an unavoidable roadblock in the collective effort to understand how cells build and remodel multicellular tissues. In the past funding period, we used single-molecule biophysical assays to discover multiple unanticipated mechanisms by which the proteins present in these complexes sense and respond to mechanical force. Here, we will build on these results to discover the mechanisms by which AJ and TJ proteins may act to seed larger- scale organization at the cell and tissue levels. Based on strong preliminary data, we will examine how adhesion complexes templated by αE-catenin and afadin regulate the assembly of multicellular actomyosin cables that power collective cellular motions during embryonic development and wound healing. Preliminary data likewise demonstrate that PDZ domains, a widespread class of protein domains that mediate protein- protein interactions, can exhibit striking forms of mechanosensitivity. Building on this result, we will elucidate the function of mechanosensitive PDZ-mediated interactions in controlling the assembly and dynamics of TJs, and work to discover additional forms of force sensing employed by junctional proteins. The second portion of this proposal focuses on a class of specialized cellular adhesion complexes that mediate planar cell polarity (PCP). PCP refers to the long-range, front-back polarization of cells in the tissue plane. PCP is essential in multiple developmental contexts, and aberrations in PCP signaling are a prevalent source of birth defects. Previous work shows that the core PCP components assemble into clusters at cell-cell junctions, with specific proteins asymmetrically localized to opposite sides. The molecular mechanisms that mediate the induction of this key asymmetry have remained elusive. Here, we will combine the power of Drosophila genetics with quantitative imaging approaches to test the hypothesis that multivalent protein-protein interactions within individual clusters lead to a nonlinear increase in asymmetry with cluster size. In total, work acro...