Epithelial cells, which line both the inside cavities and outside of the body, exist in tissues as monolayers, multilayers of cells, and three dimensional tube/duct structures. Proper formation and homeostasis of the epithelium is critical for tissue and organ function; dysregulation of the epithelium is associated with epithelial barrier loss (including sepsis), defective wound healing, and development and progression of cancer. Strong cell-cell junctions are critical to the integrity of the epithelium, including cell cohesion, barrier function, and ability to resist mechanical stress. Loss of junctions is associated with epithelial dysfunction including inflammatory-induced increases in permeability and epithelial to mesenchymal transition (EMT). Although formation cell-cell adhesions have been shown to be critical regulators of cell proliferation, migration, and tissue organization, very little is known how cell-cell junction forces contribute to these processes. In addition, the nucleus, which is physically connected to the cytoskeleton by the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex also experiences mechanical force resulting from both actomyosin contractility and externally applied forces across cell-cell contacts and cell-matrix adhesions. Nuclear forces have been shown to regulate the cell cycle, nuclear pore complex, and chromatin. Recent work by my group has also shown that the LINC complex is a critical structure for epithelial homeostasis. This proposal examines the role of force across proteins in both cell-cell junctions and the nucleus as mediators of epithelial homeostasis. The major research goals of this R35 MIRA renewal are to 1) examine how mechanical forces regulate epithelial homeostasis and morphogenesis, 2) identify the role of the LINC complex in epithelial homeostasis and mesenchymal stem cell differentiation, and 3) investigate how forces across nuclear lamins and nuclear pore complexes regulate epithelial physiology. Proposed experiments include in vitro models of ductal/glandular epithelium using FRET-based tension biosensors to directly measure forces across tight junctions, adherens junctions, and desmosomes, as well as the LINC complex, nuclear pores, and the nuclear lamina. New and existing technical approaches will be used to modulate these structures, with the objective of identifying both the upstream regulators of force and the downstream processes regulated by force. Additionally, in vivo mouse models will be used to assess the role of the LINC complex and desmosomes in 3D epithelial tissue homeostasis. This comprehensive study of cell adhesion and nuclear forces will greatly advance the understanding of how epithelial homeostasis is regulated, which is relevant to the processes of wound repair, inflammation, and epithelial tissue development and organization, as well as epithelial diseases, including cancer, fibrosis, and chronic inflammation. Furthermore, results from this study concerning t...