CYTOCHALASIN AND PROTEINS THAT AFFECT ENDS OF F-ACTIN

这个项目的长期目标是了解 肌动蛋白微丝的组装和与其他微丝的连接 用于产生和传递力的蜂窝结构。在过去 几年来，我们的研究重点一直集中在结构和 张力蛋白的功能，这是一种多功能蛋白质，其特性表明 它在细丝-膜结合和信号传递中起关键作用 转导：(1)Tensin已免疫定位于肌肉Z线和 细胞-细胞和细胞-底物黏附连接，肌动蛋白 灯丝末端与其他结构相关联。(2)Tensin是 只有这些位置上的蛋白质具有离散的功能结构域 与肌动蛋白细丝、纽蛋白和整合素末端的特异性结合， 在粘连连接处也发现了蛋白质。后者在以下方面发挥了关键作用 细胞-底物附着和信号转导。(3)Tensin还拥有 Sh2结构域，与含磷酸酪氨酸的蛋白结合，并且是 苏氨酸、丝氨酸和酪氨酸残基的磷酸化。酪氨酸 随着病毒的转化，细胞-底物的磷酸化增加 粘附性，以及生长因子的刺激。拟议的研究采用了 一种综合运用细胞生物学、生物化学方法的方法- 分子生物学和生物物理学来研究细胞的结构和功能 不同的张力域。该项目由以下几部分组成 成分；(A)使用生化和生物物理分析方法 详细描述了张力蛋白与其他化合物的体外相互作用 存在于细胞-底物和细胞-细胞连接处的蛋白质，包括 肌动蛋白、纽蛋白和塔林。(B)重组蛋白的活性分析 确定亚分子结构域和关键氨基酸的蛋白质和突变体 与这些蛋白质相互结合有关的酸性残基。(C) 通过以下方式研究结构域修改的影响 磷酸化、蛋白分解等，以及可能的相互作用 不同的域。(D)确定结构要求和 通过研究张力蛋白结构域的定位来研究它们的功能作用 经显微注射、转染法和使用 中和单抗。(E)厘定3- 张力素结构域三维原子结构的核磁共振波谱研究 X射线结晶学，并研究结构与功能的关系 在分子建模程序的帮助下。/建议的结果 这项工作将增加我们对肌动蛋白分子基础的理解 正常和病变细胞的细胞骨架和收缩功能 纸巾。

The long term goal of this project is to understand the regulation of the assembly of actin filaments and the attachment of the filaments to other cellular structures for force generation and transmission. In the past several years, the focus of our research has been on the structure and function of tensin, a multifunctional protein with properties suggesting that it plays a key role in filament-membrane association and in signal transduction: (1) Tensin has been immunolocalized to muscle Z-lines and cell-cell and cell-substrate adherens junctions, locations where actin filament ends are associated with other structures. (2) Tensin is the only protein at these locations with discrete functional domains for specific binding to the ends of actin filaments, vinculin, and integrin, proteins also found at adherens junctions. The latter plays a key role in cell-substrate attachment and signal transduction. (3) Tensin also has an SH2 domain that binds phosphotyrosine-containing proteins, and is phosphorylated at threonine, serine, and tyrosine residues. Tyrosine phosphorylation increases with viral transformation, cell-substrate adhesion, and stimulation by growth factors. The proposed research employs an integrative approach using methods of cell biology, biochemistry- molecular biology, and biophysics to study the structure and function of the different tensin domains. The project is composed of the following components; (a) The use of biochemical and biophysical assays to characterize in detail the in vitro interaction of tensin with other proteins present at cell-substrate and cell-cell junctions, including actin, vinculin, and talin. (b) Analysis of the activity of recombinant proteins and mutants to define the submolecular domains and critical amino acid residues involved in the binding of these proteins to each other. (c) Investigation of the effects of modifications of the domains by phosphorylation, proteolysis, etc., and the possible interplay among the different domains. (d) Determination of the structural requirements and functional roles of the tensin domains by studying their localization in cultured cells following microinjection, transfection, and the use of neutralizing monoclonal antibodies. (e) Determination of the 3- dimensional atomic structures of the tensin domains by NMR spectroscopy and X-ray crystallography, and to study structure-function relationships with the help of molecular modelling programs. / The results of the proposed work will increase our understanding of the molecular basis of actin-based cytoskeletal and contractile functions in normal and diseased cells and tissues.