ABSTRACT Cellular rearrangements during tissue formation, tissue regeneration and cancer metastasis occur via the coupled movement of groups of cells, a phenomenon known as collective cell migration. A key protein involved in the collective migration of epithelial cells is E-cadherin (Ecad), an essential cell-cell adhesion protein. Ecad adhesion is carefully regulated to orchestrate complex movement of cell monolayers and dysregulation of adhesion is a characteristic of certain cancers. However, little is known about how Ecad structure and adhesion are regulated and how this manifests in collective cell migration. The extracellular region of Ecad mediates adhesion, while its intracellular domain binds to regulatory proteins such as p120-catenin, α-catenin and vinculin, which link Ecad to the actin cytoskeleton. We hypothesize that α-catenin, vinculin and p120-catenin regulate adhesion by allosterically controlling the conformation of the Ecad extracellular region. We also propose that some Ecad cancer mutations impede the inside-out regulation of Ecad conformation and that understanding their mechanism of action will provide molecular insights into adhesion regulation in tumorigenesis. In aim 1 of this proposal, we will assign distinct roles to α-catenin, p120-catenin, and vinculin binding in modulating Ecad conformation and measure how cancer mutations impede inside-out regulation of adhesion. In aim 2, we will establish how cytoplasmic proteins alter the conformation of the Ecad extracellular region by building detailed structural models for intercellular junctions, with and without Ecad cancer mutations. Finally, in aim 3, we will resolve the role of different Ecad conformations and cancer mutants on the migratory patterns of epithelial cells. Our proposed research will provide a detailed mechanistic understanding of Ecad mediated adhesion and resolve adhesive states that are important in epithelial tissue formation, wound healing, and cancer. This knowledge will enable selective targeting and inhibition of specific Ecad structures, which can compromise cancer progression.