CELL SURFACE CONTROL OF CELLULAR PHYSIOLOGY

微生物学相关的细胞：基质和细胞：细胞连接是微生物学的主要特征。 非肌肉细胞中的主要承重、力传递结构。 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、细胞迁移和细胞凋亡， 粘连 细胞粘附和形成细胞间粘附的能力丧失 细胞：基质连接与侵袭性和 转移性肿瘤细胞行为。 因为黏着斑蛋白是一种 它被认为是粘附连接的一个组成部分， 在其装配、结构或功能上。 这项提案的目的是 来定义黏着斑蛋白的特性在其中的重要性 粘附连接生理学。 了解黏着斑蛋白如何从细胞质募集到粘附连接 以及黏着斑蛋白与其配体的相互作用是如何调节的， 头部和尾部（H/T）结构域的分子内缔合和 粘着斑蛋白结合酸性磷脂的特性。 抗体 检测被H/T相互作用掩蔽的表位的酶探针将 开发并用于识别假设的开放构象， 黏着斑蛋白，其中头部从尾部移位，暴露出高亲和力 塔林蛋白和酸性磷脂的结合位点。 这些网站映射在 分离的头部和尾部结构域。 体外结合测定 将用于：评估是否分子内相互作用的 黏着斑蛋白头部和尾部是黏着斑蛋白与 α-辅肌动蛋白和肌动蛋白;评估46 kDa片段 调节190 kDa talin片段结合 黏着斑蛋白;确定蛋白激酶C介导的 talin磷酸化对黏着斑蛋白/talin相互作用Kd的影响 并表征保留以下特征的最小域： 在尾部发现的高亲和力酸性磷脂结合位点 黏着斑蛋白的结构域;表征黏着斑蛋白与 磷脂酰肌醇-4 '5 '-双P（PIP 2）;并确定P1和PIP 2是否 调节黏着斑蛋白与talin、肌动蛋白或α-辅肌动蛋白的结合。 黏着斑蛋白在生成或维持中起作用的假说 将通过下调浓度来测试跨膜力 黏着斑蛋白反义核酸对RDA 2人横纹肌肉瘤细胞增殖的影响 寡脱氧核苷酸 这种扰动对跨膜的影响 将通过测量单元的能力来评估部队的生成 来收缩胶原蛋白凝胶。 了解E钙粘蛋白触发粘附的必要性， 在L929细胞中的连接组装和功能，假定的连接- 依赖于细胞致密化、细胞表面发育 极性、运动限制和侵袭性将在 E钙粘蛋白转染的克隆。 缺乏钙粘蛋白相关的γ- 这些细胞中的连环蛋白将被探讨其需求 E钙粘蛋白触发的连接组装。

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.