CELL SURFACE CONTROL OF CELLULAR PHYSIOLOGY

黏附和运动是启动细胞转移的基本过程。 黏附的适度过度表达(比内源性水平高10-30%) 菌斑蛋白，纽蛋白，抑制细胞运动和增加细胞 黏附，而纽蛋白的下调则增加细胞的运动性和 抑制粘连。这项研究的长期目标是了解 纽蛋白是如何调节细胞黏附和运动的。假设是为了 测试的是纽蛋白的头部(VH)和尾部结构域的相互作用 (Vt)对纽蛋白在调节中的作用具有重要的机械意义 活细胞中的细胞黏附和运动。一种由四环素调控的 利用表达载体过表达6xHisEGFP标记的纽蛋白 和中国仓鼠卵巢(CHO)细胞中纽蛋白的功能突变体。 纽蛋白在细胞内的浓度与细胞周期的关系 抑制纤维连接蛋白(FN)介导的细胞运动(最大迁移 最佳受体占有率的速度)、更高的粘附性(平均 脱离强度作为受体占有率的函数)，以及速率和 细胞扩散的程度将被确定。纽蛋白的作用 过表达对肌动蛋白细丝、分子组成的稳定作用 局灶性粘连斑块、粘连斑块的动力学、丝状伪足和 片状脂溢症、纽蛋白的定位以及纽蛋白与 细胞蛋白质将通过细胞学分析固定和 活细胞和生化方法。纽蛋白的贡献 将探讨过表达表型的配体结合活性。 通过定点突变来敲除个体活动。 Vt的肌动蛋白、酸性磷脂和VH结合功能的突变 将通过在一个窗口中系统地更改所有聚集电荷来实现 从5个氨基酸到丙氨酸(20个突变)。突变者将被描述为 鉴定保留“天然”结构但至少丢失的Vt突变体 Vt的三个测量功能之一。血管紧张素转换酶和他林的突变- 结合功能将基于敲除的已知点突变 这些在体外的活性。突变将在CHO细胞中表达 单个纽蛋白亚域的上下文和在完整的上下文中 纽卡林。这些细胞的性质在细胞黏附和 运动分析将作为过量的量的函数进行测试 表达突变蛋白，并对其表型进行分析 细胞学和生物化学上所描述的，将表型与 特殊的配体结合功能。控制血吸虫病的新治疗靶点 转移可以通过运动性和粘附性的基础研究来定义。

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.