Project Summary The process of Epithelial to Mesenchymal Transition (EMT) is a transdifferentiation event in which epithelial cells switch their phenotype to a mesenchymal one, which facilitates individual cell migration, cell invasion, and tissue assembly. This dynamic process is critical during embryonic development, and, when coupled with the reverse MET process, facilitates correct spatial and temporal development of organs. While EMT is critical for development, its misregulation is implicated in many diseases, including cardiac fibrosis, cirrhosis, and cancer. The events that initiate EMT are not well understood, but appear to be linked to changes in the intracellular biochemical signaling and gene expression, soluble factor section, autocrine signaling, mechanical and paracrine signaling between neighboring cells, and mechanical signaling between the epithelial sheet and the underlying extracellular matrix (ECM). The Conway lab has demonstrated that a drop in force on adherens junctions is necessary for progression of EMT. The Lemmon lab has demonstrated that progression of EMT requires assembly of the ECM protein fibronectin (FN) into new fibrils, which contain binding sites for several pro-EMT growth factors. Our recent study suggests that FN fibrils drive EMT by clustering these growth factors at the cell surface. These findings have led us to a hypothesis in which EMT is initiated by disruption of forces on adherens junctions, which in turn redistributes forces to the underlying matrix, initiates FN fibril assembly, clustering soluble signals that promote EMT at the cell surface, and driving intracellular biochemical signaling. In this work, we will: 1) develop a computational model that predicts junctional forces, matrix forces and matrix assembly, autocrine and paracrine signaling, and epithelial and mesenchymal cell markers, and uses these to predict spatial localization of EMT in an assembling and confluent cell sheet. Our model will start from a recently developed cell-based model of cell-matrix interactions, using the previously developed Lemmon-Romer model of traction force prediction, and extend this to predict force redistribution of cell-matrix forces in neighboring epithelial cells and the forces acting on adherens junctions in each cell. We will couple these mechanical forces with a model of intracellular signaling pathways. 2) We will use a novel suite of tools to establish an experimental system that can simultaneously measure junctional forces, matrix forces, FN assembly, and EMT markers. Colonies of epithelial cells will be generated using microcontact printing, which allows for repeatable colony size and shape. We will measure spatial regulation of EMT markers within the colonies while also quantifying both junctional forces (using a FRET-based bi...