Mutational Analysis of Intermediate Filaments in Muscle

肌肉中间丝的突变分析

• 批准号: 9105523
• 负责人: Michael Klymkowsky
• 金额: $ 24万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9105523&HistoricalAwards=false
• 关键词: Mutational; Analysis; Intermediate; Filaments; Muscle

Intermediate filaments (IFs) are a major structural component of striated muscle. To study the organization and functions of muscle IFs, we have constructed two types of mutant forms of the IF protein vimentin. C-terminal truncations of vimentin co- assemble with and disrupt IFs. The expression of such "disruptive" mutants in the rat myogenic cell line L6 will be used to study the effects of disrupting IF organization during the course of muscle differentiation in vitro. A combination of light and election microscopy will be used to determine whether an intact IF system is required for myoblast fusion, the establishment of sarcomeric organization, the formation and stability of costameres (i.e. regions of interaction between Z- lines and the plasma membrane), or the lateral alignment of myofibrils into myofibers. To complement these in vitro studies, we will examine the effects of disruptive vimentin mutants on skeletal muscle organization in the developing Xenopus tadpole. In the context of the swimming tadpole, functions of IFs related to mechanical integration of muscle and muscle components may become more obvious than in cultured cells. To identify proteins involved in muscle IF organization, we have made mutants forms of vimentin that do not co-assemble with endogenous IFs. These soluble, "non-assemblying" mutants retain either the C- and/or N-terminal domains of the vimentin molecule intact. It is through these domains that IF proteins are thought to intact with other molecules. Non-asssemblying mutants are therefore expected to interact with, and perhaps sequester, specific IF-associated proteins involved in the interaction between IFs and the Z-disc, the plasma membrane, microtubules and mitochondria. We will determine whether the expression of non- assemblying vimentin mutants affects IF organization or cellular differentiation in muscle and myogenic cells. We will then determine whether specific cellular proteins co-isolate with the exogenous, non-assemblying, mutant proteins. The study of these proteins should extend our understanding of how the changing organization of IFs during myogenesis is controlled. The result of our studies should provide a clear definition of the functions of IFs in striated muscle and, through the identification of IF-associated proteins, a better understanding of the molecular architecture of muscle.

中间纤维（IF）是一个主要的结构组成部分， 横纹肌 研究的组织和职能 肌肉IF，我们已经构建了两种类型的突变形式的 IF蛋白波形蛋白。 波形蛋白的C-末端截短共 聚集并破坏IFs 这种表达方式 在大鼠肌原细胞系L6中的"破坏性"突变体将是 用于研究在干扰IF组织的过程中， 体外肌肉分化的过程。 的组合 光学和电子显微镜将用于确定是否 成肌细胞融合需要完整的IF系统， 肌节组织的建立、肌节的形成和 costameres的稳定性（即Z-之间的相互作用区域） 线和质膜），或横向排列的 肌原纤维转化为肌纤维。 为了补充这些体外研究， 我们将研究破坏性波形蛋白突变体对 非洲爪蟾蝌蚪发育中的骨骼肌组织。 在游泳蝌蚪的背景下，IF的功能相关 肌肉和肌肉成分的机械整合可以 比在培养的细胞中更明显。 为了鉴定参与肌肉IF组织的蛋白质，我们 制造了波形蛋白的突变体， 内源性干扰因子 这些可溶性的"非组装"突变体保留了 波形蛋白分子的C-和/或N-末端结构域 完整 正是通过这些结构域， 与其他分子结合 非融合突变体是 因此，预计将与之相互作用，也许会隔离， 参与相互作用的特异性IF相关蛋白 在IF和Z盘之间，质膜，微管和 线粒体 我们将决定是否表达非- 装配波形蛋白突变体影响IF组织或细胞 在肌肉和肌原细胞中的分化。 然后我们将 确定特定的细胞蛋白质是否与 外源的、非组装的突变蛋白质。 研究这些 蛋白质应该扩展我们的理解， 肌生成过程中IF的组织化受到控制。 我们的研究结果应该提供一个明确的定义， 横纹肌中IFs的功能，并通过 鉴定IF相关蛋白，更好地了解 肌肉的分子结构。