Maintenance of Adhesion and Barrier Function during Epithelial Cell Shape Changes

项目摘要/摘要 细胞-细胞连接将上皮细胞相互粘连，在细胞之间传递力，并产生 生物屏障，选择性地调节上皮组织中细胞之间的传递。基本原理 关于上皮细胞-细胞连接如何动态重塑以响应生理力量的问题 对细胞黏附和屏障功能的挑战仍然没有得到回答。除了是绝对必要的 发展和维持器官动态平衡，破坏黏附和屏障功能 导致包括癌细胞转移和炎症性肠病在内的疾病。因此，它是 对于确定当上皮细胞改变形状时控制细胞-细胞连接重塑的机制至关重要。 这一建议是建立在实验室最近的发现基础上的，这些发现表明Rho耀斑局部加强了紧密连接。 在屏障功能发生泄漏后，粘连连接通过将纽蛋白重新聚集到 分裂上皮细胞的裂沟。此应用程序的总体目标是识别机制 促进上皮细胞分裂和连接部位的黏附和屏障功能的维持 伸长率。我们的中心假设是，局部施加的机械力挑战粘连连接和 紧密连接和肌动球蛋白介导的加强是维持黏附和屏障所必需的 在这些网站上发挥作用。核心假设将通过追求三个具体目标来检验：1)确定如何 机械诱导的紧密连接泄漏触发Rho耀斑；2)确定Rho耀斑如何介导连接 可伸缩性修复紧密连接；3)确定调节张力传递和屏障的机制 在局部紧张局势加剧的地点进行维护。这项拟议的研究具有创新性，因为它适用于 强大的实验工具包括：发展中的脊椎动物模型系统(非洲爪哇胚胎)，活的 最近在实验室开发的成像屏障分析，经过验证的局部或全球操作方法 完整上皮的张力，活动性Rho动态成像的探针以及一系列细胞骨架 蛋白质、连接蛋白、Rho调节剂和细胞质钙，以及专门的分析工具 定量分析实时成像数据。这项拟议的研究具有重要意义，因为它将推动我们的 对上皮细胞生物学中一个根本性的重要问题的认识：上皮细胞如何经历 细胞形态发生戏剧性的变化，如胞质分裂，但仍保持组织完整性和屏障功能。

PROJECT SUMMARY / ABSTRACT 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.