Cell-cell junctions adhere epithelial cells to one another, transmit forces from cell to cell, and generate biological barriers that selectively regulate what can pass between cells in an epithelial tissue. Fundamental questions about how epithelial cell-cell junctions dynamically remodel in response to physiological forces that that challenge cell adhesion and barrier function remain unanswered. In addition to being absolutely essential for development and maintenance of organ homeostasis, disruption of adhesion and barrier function contributes to diseases including cancer cell metastasis and Inflammatory Bowel Disease. Therefore, it is critical to determine the mechanisms that control cell-cell junction remodeling as epithelial cells change shape. This proposal builds on recent discoveries from the lab showing that Rho flares locally reinforce tight junctions following leaks in barrier function, and that adherens junctions are reinforced by recruitment of Vinculin to the cleavage furrow of dividing epithelial cells. The overall objective of this application is to identify mechanisms that promote maintenance of adhesion and barrier function at sites of epithelial cell division and junction elongation. Our central hypothesis is that locally applied mechanical forces challenge adherens junctions and tight junctions and elicit actomyosin-mediated reinforcement required for maintenance of adhesion and barrier function at these sites. The central hypothesis will be tested by pursuing three specific aims: 1) Identify how mechanically-induced tight junction leaks trigger Rho flares; 2) Determine how Rho flare-mediated junction contractility repairs tight junctions; 3) Define mechanisms that mediate tension transmission and barrier maintenance at sites of locally increased tension. The proposed research is innovative because it applies powerful experimental tools including: a developing vertebrate model system (Xenopus laevis embryos), a live imaging barrier assay recently developed in the lab, proven approaches to locally or globally manipulate tension in the intact epithelium, probes for live imaging of active Rho dynamics as well as a host of cytoskeletal proteins, junction proteins, Rho regulators, and cytoplasmic calcium, and specialized analysis tools to quantitatively analyze live imaging data. The proposed research is significant because it will advance our knowledge about a fundamentally important problem in epithelial cell biology: how epithelial cells undergo dramatic cell shape changes like cytokinesis yet maintain tissue integrity and barrier function.
The proposed research is relevant to public health because it will to build our understanding of the molecular mechanisms that underlie maintenance of cell-cell adhesion and barrier function in epithelial tissues when junctions are challenged by physiological forces using the model system Xenopus laevis (African clawed frog). Knowing the key molecular players involved is critical for understanding development and maintenance of epithelial homeostasis. Furthermore, it will provide insight into how these mechanisms are disrupted in cancer cell metastasis and inflammatory bowel diseases.
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