CONTROL OF EPITHELIAL CELL MOTILITY BY E CADHERIN

钙依赖黏附介导的细胞-细胞接触 E-钙粘附素分子强烈影响极性和 上皮细胞的分化。我们发现E-钙粘附素 也会抑制上皮细胞的运动。令人惊讶的是，这 抑制不需要E-钙粘附素介导的黏附，但 相反，似乎涉及到细胞内运动性的产生- 抑制信号。我们已经确定了E的一部分- 钙粘蛋白胞质片段，即膜旁区域，是 对于产生这个信号来说是必不可少的。我们的总体目标是 确定该信号通路的组件，以确定 细胞运动的哪些方面受到信号的抑制，以及 明确该信号在表皮发育过程中的重要性 和伤口愈合。为了定义JM域的边界， 我们将分析JM缺失对细胞运动的影响 JM区的突变体和显性负性形式。 与JM域交互的组件将由 与免疫共沉淀的蛋白质模式的比较 通过亲和力，含有或不含有该区域的E-钙粘附素的形式 用JM结构域融合蛋白进行纯化，并利用 酵母双杂交系统。我们将测试这些JM是否- 相关的组件通过以下方式参与信号通路 表达显性负性多肽，将阻断其 绑定到JM域。我们将测试是否有同性恋E- 钙粘附素结合激活MAP激酶。要定义蜂窝 可能受此信号调控的反应，我们将确定 E-钙粘附素是否影响膜皱缩和丝状尿路 分机。最后，要确定这一规定的作用 在皮肤发育和伤口愈合过程中，我们将 建立和分析显性阴性的转基因小鼠 E-钙粘蛋白的不同形式在基底细胞中特异表达 有核细胞。总之，这些研究将产生更好的 对细胞-细胞接触机制的理解 调节上皮细胞的运动。这样的知识将证明 有益于促进伤口愈合和控制癌细胞 入侵。

Cell-cell contacts mediated by the calcium-dependent adhesion molecule, E-cadherin, strongly affect the polarity and differentiation of epithelial cells. We have found that E-cadherin also suppresses the movement of epithelial cells. Strikingly, this suppression does not require adhesion mediated by E-cadherin, but instead appears to involve generation of an intracellular motility- suppressing signal. We have determined that part of the E- cadherin cytoplasmic segment, the juxtamembrane 9JM) region, is essential for generating this signal. Our overall goals are to identify the components of this signaling pathway, to determine which aspects of cell motility are suppressed by the signal, and to define the importance of this signal during epidermal development and wound-healing. To define the boundaries of the JM domains, we will analyze the effects on cell movement of JM deletion mutants and dominant-negative forms of the JM region. Components that interact with the JM domain will be identified by comparing the pattern of proteins co-immunoprecipitated with forms of E-cadherin that contain or lack this region, by affinity purification with JM domain fusion proteins, and by use of the yeast two-hybrid system. We will test whether these JM- associated components are involved in the signaling pathway by expressing dominant-negative polypeptides that will block their binding to the JM domain. We will test whether homophilic E- cadherin binding activates MAP kinase. To define cellular responses that may be regulated by this signal, we will determine whether E-cadherin affects membrane ruffling and filopodial extension. Finally, to determine the role of this regulation of motility during skin development and wound-healing, we will create and analyze transgenic mice in which dominant-negative forms of E-cadherin are expressed specifically in basal karatinocytes. Together, these studies will result in a better understanding of the mechanisms by which cell-cell contact regulates epithelial cell motility. Such knowledge will prove useful for enhancing wound healing and controlling carcinoma cell invasion.