CELL SURFACE CONTROL OF CELLULAR PHYSIOLOGY

细胞生理学的细胞表面控制

• 批准号: 2180936
• 负责人: SUSAN W CRAIG
• 金额: $ 19.83万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-08-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180936
• 关键词: actins; binding proteins; cell adhesion; cell adhesion molecules; cell cell interaction; cell motility; chickens; circular dichroism; electron microscopy; gap junctions; human tissue; immunofluorescence technique; immunoprecipitation; intermolecular interaction; laboratory mouse; laboratory rabbit; neoplastic cell culture for noncancer research; phosphatidylinositols; phospholipids; phosphorylation; protein kinase C; vinculin

Microfilament-associated, cell:substrate and cell:cell junctions are the major load-bearing, force-transducing structures in non-muscle cells. The assembly and regulation of these adherens junctions is essential to many cell processes including wound healing, cell migration, and cellular adhesion. Loss of cell adhesion and the ability to form intercellular and cell:substrate junctions is tightly correlated with invasive and metastatic tumor cell behavior. Because vinculin is a diagnostic component of adherens junctions it is thought to play an important role in their assembly, structure, or function. The goal of this proposal is to define specific ways in which the properties of vinculin are important to adherens junction physiology. To learn how vinculin is recruited from cytoplasm to adherens junctions and how the interactions of vinculin with its ligands are regulated, the intramolecular association of head and tail (H/T) domains and the ability of vinculin to bind acidic phospholipids will be characterized. Antibody and enzyme probes that detect epitopes masked by the H/T interaction will be developed and used to identify the hypothesized open conformation of vinculin in which head is displaced from tail exposing the high affinity binding sites for talin and acidic phospholipid. These sites map in isolated head and tail domains, respectively. In vitro binding assays will be used to: assess whether the intramolecular interaction of vinculin head and tail is regulatory for the interaction of vinculin with alpha-actinin and actin; evaluate the hypothesis that the 46 kDa fragment of talin regulates the ability of the 190 kDa talin fragment to bind to vinculin; determine the effect of protein kinase C-mediated phosphorylation of talin on the Kd of vinculin/talin interaction define and characterize the smallest domain that retains the characteristics of the high-affinity, acidic phospholipid binding site found in the tail domain of vinculin; characterize the interaction between vinculin and phosphatidylinosito-4'5'-bisP (PIP2); and determine whether P1 and PIP2 modulate the binding of vinculin to talin, actin, or alpha-actinin. The hypothesis that vinculin plays a role in generation or maintenance of transmembrane force will be tested by downregulating the concentration of vinculin in RDA2 human rhabdomyosarcoma cells with antisense oligodeoxynucleotides. The effect of this perturbation on transmembrane force generation will be evaluated by measuring the ability of the cells to contract collagen gels. To gain insight on the requirement for E cadherin to trigger adherens junction assembly and function in L929 cells, the putative junction- dependent functions of cell compaction, development of cell surface polarity, restriction of motility, and invasiveness will be evaluated in E cadherin-transfected clones. The absence of cadherin-associated gamma- catenin in these cells will be explored with respect to its requirement for E cadherin-triggered junction assembly.

微丝相关的细胞：基质和细胞：细胞连接是 非肌肉细胞中主要的承重、力传导结构。 这些粘附连接的组装和调节对于 许多细胞过程，包括伤口愈合、细胞迁移和细胞 附着力。 细胞粘附和形成细胞间质的能力丧失 细胞：基质连接与侵袭性和侵袭性密切相关 转移性肿瘤细胞行为。 因为纽蛋白是一种诊断剂 粘附连接的组成部分被认为发挥着重要作用 它们的组装、结构或功能。 该提案的目标是 定义纽蛋白特性重要的具体方式 粘附连接生理学。 了解纽蛋白如何从细胞质募集至粘附连接 以及如何调节纽蛋白与其配体的相互作用， 头尾 (H/T) 结构域的分子内关联及其能力 将表征纽蛋白结合酸性磷脂的特性。 抗体 检测 H/T 相互作用掩盖的表位的酶探针将 被开发并用于识别假设的开放构象 纽蛋白的头部从尾部移位，暴露出高亲和力 talin 和酸性磷脂的结合位点。 这些站点映射在 分别分离头域和尾域。 体外结合测定 将用于： 评估是否存在分子内相互作用 纽蛋白头和尾部调节纽蛋白与 α-肌动蛋白和肌动蛋白；评估 46 kDa 片段的假设 talin 调节 190 kDa talin 片段结合的能力 纽蛋白;确定蛋白激酶C介导的作用 talin 磷酸化对纽蛋白/talin 相互作用的 Kd 的影响定义 并表征保留特征的最小域 尾部发现的高亲和力酸性磷脂结合位点 纽蛋白结构域；描述纽蛋白之间的相互作用 磷脂酰肌醇-4'5'-bisP (PIP2)；并判断P1和PIP2是否 调节纽蛋白与踝蛋白、肌动蛋白或α-辅肌动蛋白的结合。 纽蛋白在生成或维持中发挥作用的假设 通过下调浓度来测试跨膜力 纽蛋白在 RDA2 人横纹肌肉瘤细胞中的反义作用 寡脱氧核苷酸。 这种扰动对跨膜的影响 将通过测量细胞的能力来评估力的产生 收缩胶原蛋白凝胶。 深入了解 E 钙粘蛋白触发粘附的要求 L929 细胞中的连接组装和功能，假定的连接 细胞压实、细胞表面发育的依赖功能 极性、运动限制和侵袭性将在 E钙粘蛋白转染的克隆。 钙粘蛋白相关γ-的缺失 将探索这些细胞中的连环蛋白的需求 用于 E 钙粘蛋白触发的连接组装。