CYTOCHALASIN & PROTEINS THAT AFFECT ENDS OF F-ACTIN

本项目的长期目标是了解法规 的细胞骨架和收缩功能，通过研究控制 肌动蛋白丝（F-actin）的组装和分解。 的 我们一直采取的方法是使用细胞松弛素（真菌 具有抗运动活性的代谢物）作为鉴定 影响肌动蛋白聚合的细胞成分。 我们 先前发现这些化合物以高亲和力结合至 F-actin的快速生长端，从而强烈影响 灯丝组装和拆卸速率以及灯丝 体外长度分布。 此外，我们还分离出了 来自骨骼肌的一种蛋白质（称为capactin）， 影响肌动蛋白的组装和分解， 类似于细胞松弛素。 在这项研究中，我们 计划研究肌肉中capactin结构和功能 和非肌肉细胞之间的联系 (A)我们将确定 蛋白质的生化结构，通过分析其主要 以及亚基结构、活性位点、等电异构体等。 将分离和测序编码capactin的cDNA。 针对蛋白质上不同表位的单克隆抗体 会做好准备。 我们将比较电容特性 从肌肉细胞和非肌肉细胞中分离， 蛋白质和其它肌动蛋白调节蛋白如凝溶胶蛋白。 （B） 我们将研究在成人和发育中 肌肉和非肌肉组织的免疫荧光和 将标记的capactin掺入细胞和无细胞 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， Western和北方印迹法的使用。 (C)的相互作用 capactin与肌动蛋白和其他细胞成分将被 用生物物理学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.