Mutational Analysis of Intermediate Filaments in Muscle

中间纤维是横纹肌的主要结构成分。为了研究肌肉IF的组织和功能，我们构建了IF蛋白波形蛋白的两种突变形式。Vimentin的C-末端截短与IF共同组装并破坏IF。这种“破坏性”突变体在大鼠成肌细胞系L6中的表达将被用来研究在体外肌肉分化过程中破坏IF组织的作用。结合光学显微镜和电子显微镜将被用来确定是否需要完整的IF系统来进行成肌细胞融合、肌节组织的建立、胞腔的形成和稳定性(即Z线和质膜之间的相互作用区域)，或者肌原纤维进入肌纤维的横向排列。为了补充这些体外研究，我们将检测破坏性波形蛋白突变对发育中的非洲爪哇蝌蚪骨骼肌组织的影响。在游泳蝌蚪的背景下，IF与肌肉和肌肉成分的机械整合相关的功能可能比在培养细胞中更明显。为了确定肌肉IF组织中涉及的蛋白质，我们制造了不与内源性IF共同组装的波形蛋白的突变形式。这些可溶的“非组装”突变体保持了波形蛋白分子的C-和/或N-末端结构域不变。如果蛋白质被认为与其他分子保持完整，就是通过这些结构域。因此，非配对突变体有望与IF与Z-Disc、质膜、微管和线粒体相互作用中涉及的特定IF相关蛋白相互作用，甚至可能隔离。我们将确定非组装波形蛋白突变体的表达是否影响肌肉和肌源性细胞的IF组织或细胞分化。然后我们将确定特定的细胞蛋白是否与外源的、非组装的、突变的蛋白共分离。对这些蛋白质的研究将扩展我们对肌发生过程中IF变化的组织是如何控制的理解。我们的研究结果应该能够清楚地定义IF在横纹肌中的功能，并通过IF相关蛋白的鉴定，更好地理解肌肉的分子结构。

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.