CELL SURFACE CONTROL OF CELLULAR PHYSIOLOGY

细胞生理学的细胞表面控制

• 批准号: 6180148
• 负责人: SUSAN W CRAIG
• 金额: $ 28.72万
• 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 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180148
• 关键词: CHO cells; SDS polyacrylamide gel electrophoresis; actins; cell adhesion; cell membrane; cell motility; chemical binding; fluorescence microscopy; green fluorescent proteins; immunoprecipitation; intermolecular interaction; laboratory rabbit; phospholipids; phosphorylation; point mutation; polymerase chain reaction; protein structure function; receptor expression; site directed mutagenesis; video microscopy; vinculin; western blottings

Adhesion and motility are fundamental processes that gate cell metastasis. Moderate over-expression (10-30% above endogenous levels) of the adhesion plaque protein, vinculin, inhibits cell motility and increased cell adhesion, whereas down-regulation of vinculin increase cell motility and inhibits adhesion. The long-term goal of this research is to understand how vinculin regulates cell adhesion and motility. The hypothesis to tested is that the interactions of vinculin's head (Vh) and tail domain (Vt) are mechanistically important to the role of vinculin in regulating cell adhesion and motility in living cells. A tetracycline-regulated expression vector will be used to over-express 6xHisEGFP-tagged vinculin and functional mutants of vinculin in Chinese hamster ovary (CHO) cells. The relationship between the intracellular concentration of vinculin and inhibition of fibronectin (FN)- mediated cell motility (maximum migration speed at the optimal receptor occupancy), increased adhesion (mean detachment strength as a function of receptor occupancy), and rate and extent of cell spreading will be established. The effects of vinculin over-expression on stabilization of actin filaments, molecular composition of focal adhesion plaques, dynamics of adhesion plaques, filopodia, and lamellipodia, localization of vinculin, and vinculin's association with cellular proteins will be assayed by cytological analysis of fixed and living cells and by biochemical methods. The contribution of vinculin's ligand-binding activities to the over-expression phenotype will be probed through site-directed mutagenesis to knock-out individual activities. Mutants in the actin, acidic phospholipid, and Vh binding functions of Vt will be made by systematically changing all clustered charges in a window of 5 amino acids to alanine (20 mutations). Mutants will be characterize to identify Vt mutants that retain "native" structure but lose at least one of the 3 measured functions of Vt. Mutations in the VASP- and talin- binding functions will be based on known point mutations that knock out these activities in vitro. Mutations will be expressed in CHO cells in the context of individual vinculin subdomains and in the context of intact vinculin. The properties of these cells in the cell adhesion and locomotion assays will be assayed as a function of the amount of over- expressed mutant protein, and the phenotypes will be analyzed cytologically and biochemically as described, to connect phenotype with a particular ligand-binding function. New therapeutic targets for control of metastasis may be defined by this basic research on motility and adhesion.

粘附和运动是门控细胞转移的基本过程。 中度过度表达（高于内源性水平10-30%） 斑蛋白，黏着斑蛋白，抑制细胞运动和增加细胞 粘附，而黏着斑蛋白的下调增加细胞运动性， 抑制粘附。这项研究的长期目标是了解 纽蛋白如何调节细胞粘附和运动。假设， 测试的是黏着斑蛋白的头部（Vh）和尾部结构域的相互作用， (Vt)对黏着斑蛋白在调节 活细胞中的细胞粘附和运动性。四环素调节的 表达载体将用于过表达6xHisEGFP标记的黏着斑蛋白 和中国仓鼠卵巢（CHO）细胞中黏着斑蛋白的功能突变体。 细胞内黏着斑蛋白浓度与 抑制纤连蛋白（FN）介导的细胞运动（最大迁移 最佳受体占有率的速度），增加的粘附（平均 作为受体占有率的函数的分离强度），以及速率和 将建立小区扩展的程度。纽蛋白的作用 过表达对肌动蛋白丝稳定性的影响，分子组成 局灶性粘连斑块，粘连斑块，丝状伪足， 板状伪足，黏着斑蛋白的定位，以及黏着斑蛋白与 细胞蛋白质将通过固定的细胞学分析进行测定， 活细胞和生物化学方法。黏着斑蛋白的贡献 将探测过表达表型的配体结合活性 通过定点诱变来敲除个体活性。 Vt的肌动蛋白、酸性磷脂和Vh结合功能的突变 将通过系统地改变窗口中的所有聚集电荷来进行 5个氨基酸突变为丙氨酸（20个突变）。将对突变体进行表征 为了鉴定保留“天然”结构但至少失去 Vt的3个测量函数之一。VASP和talin基因的突变 结合功能将基于已知的点突变， 这些活动在体外。突变将在CHO细胞中表达， 在单个黏着斑蛋白亚结构域的背景下和在完整黏着斑蛋白亚结构域的背景下， 黏着斑蛋白这些细胞的特性在细胞粘附和 运动测定将作为过量- 表达突变蛋白，并分析表型 细胞学和生物化学，如所述，以连接表型与 特殊的配体结合功能。新的治疗靶点，用于控制 转移可以通过对运动性和粘附性的基础研究来定义。