CYTOCHALASIN & PROTEINS THAT AFFECT ENDS OF F-ACTIN

这个项目的长期目标是理解规则 通过研究细胞骨架和收缩功能的控制 肌动蛋白细丝的组装和拆卸(F-肌动蛋白)。这个 我们一直采用的方法是使用细胞松弛素(真菌 具有抗运动活性的代谢物)作为识别 影响肌动蛋白聚合的细胞成分。我们 先前发现这些化合物与高亲和力结合 F-肌动蛋白的快速生长末端，从而强烈影响 长丝的装卸速度以及长丝 体外实验中的长度分布。此外，我们能够分离出 来自骨骼肌的一种蛋白质(指定为Capactin)，它 影响肌动蛋白的组装和拆卸 与细胞松弛素相似。在拟议的研究中，我们 计划研究肌肉中电容蛋白的结构和功能 和非肌肉细胞，通过以下方式。(A)我们会决定 通过对蛋白质一级结构的分析确定蛋白质的生化结构 亚基结构、活性中心、等电异构体等。 编码Capactin的基因将被分离和测序。 针对蛋白质不同表位的单抗 都会做好准备。我们将比较电容蛋白的特性 来自肌肉和非肌肉细胞，在这类细胞之间 蛋白质和其他肌动蛋白调节蛋白，如明胶蛋白。(B) 我们将研究Capactin在成人和发育中的定位 肌肉和非肌肉组织中的免疫荧光和 标记Capactin掺入细胞内和无细胞内 系统。蛋白质及其信使的出现将 也可以在各种生理条件下进行研究 使用西方和北方的墨迹。(C) Capactin与肌动蛋白和其他细胞成分将 用生物物理和药理学技术进行研究。

The long term goal of this project is to understand the regulation of cytoskeletal and contractile functions by studying the control of the assembly and disassembly of actin filaments (F-actin). The approach we have been taking is to use cytochalasins (fungal metabolites with anti-motility activity) as tools to identify cellular components that affect actin polymerization. We previously found that these compounds bind with high affinity to the fast growing end of F-actin, thereby strongly affecting the rates of filament assembly and disassembly as well as filament length distribution in vitro. Moreover, we were able to isolate from skeletal muscle a protein (designated as capactin) that affects actin assembly and disassembly in a manner that closely resembles that of the cytochalasins. In the proposed research, we plan to study the structure and function of capactins in muscle and nonmuscle cells in the following ways. (A) We will determine the biochemical structure of the protein by analyzing its primary and subunit structures, active site, isoelectric isoforms, etc. cDNA encoding capactin will be isolated and sequenced. Monoclonal antibodies against different epitopes on the protein will be prepared. We will compare the properties of capactins from muscle and nonmuscle cells, and between this class of proteins and other actin modulating proteins such as gelsolin. (B) We will study the localization of capactins in adult and developing muscle and in nonmuscle tissues by immunofluorescence and by incorporation of labelled capactin into cellular and cell-free systems. The appearance of the protein and its messengers will also be studied under a variety of physiological conditions with the use of Western and Northern blots. (C) The interaction of capactin with actin and with other cellular components will be studied with biophysical and pharmacological techniques.