Project Summary E-cadherin is the primary mediator of strong cell-cell adhesion between epithelial cells and plays an essential role in the morphogenesis and maintenance of epithelial tissues. E-cadherin is also a known mechanosensor that actively responds to the levels of inter-cellular forces and resides in a microenvironment formed by adjoining epithelial cells. The long-term goal of the project is to understand how the mechanical regulation of E-cadherin adhesion leads to a cohesive yet dynamic multi-cellular architecture in epithelial tissues. The goal of the proposed project is to delineate the mechanism by which forces are transmitted via E-cadherin adhesions and to uncover how epithelial cells sense cell-like stiffness laterally via E-cadherin adhesions. The E-cadherin-β-catenin-α-catenin complex directly and indirectly couples to actin to transmit cell-generated forces. Firstly, while the α-catenin-vinculin link, which plays a role in force sensing is thought to be the primary force transmission pathway, we recently found that, surprisingly, α-catenin is not essential for force transmission. Therefore, we will test the hypothesis that the less studied β-catenin-vinculin link is an alternate significant mechanism of force transmission at E-cadherin adhesions. We will test this by using mutant versions of vinculin and α-catenin deficient in binding β-catenin and vinculin, respectively, and corresponding knockout cell lines. We will use traction force microscopy with E-cadherin-coated soft substrates to avoid the confounding factor of vinculin’s mechanical role in cell-matrix contacts. We will also use magnetic pulling cytometry with E-cadherin-coated beads and biaxial stretching of cell islands to assess the adhesion strength at multiple scales. Secondly, while E-cadherin has been shown to sense the stiffness of E-cadherin-coated soft substrates, it is still unclear if epithelial cells sense cell-like stiffness laterally via E-cadherin adhesions. To test this, we will devise a biomimetic model of E- cadherin in a physiologically relevant geometry, in which cells interface laterally with an E-cadherin-coated, stiffness tunable, soft surface. We will test the hypothesis that lateral sensing of cell-like stiffness modulates E-cadherin density, cell dynamics, Rho and YAP levels. We will also test whether this lateral stiffness sensing is dependent on the α-catenin-vinculin mechanotransduction axis. We will use biomimetic soft substrate fabrication, mutant version of α-catenin deficient in binding vinculin in α-catenin knockout cells, sensors/indicators of Rho and YAP, and perform live-cell imaging and immunofluorescence to accomplish this. Knowledge gained on cell-to-cell force transmission and epithelial cell sensing of neighboring cell mechanical properties will be crucial in understanding the context-dependent biophysical control of E- cadherin adhesion. This will be essential to better understand the functional basis of the role of E-cadhe...