MYOFIBRILLOGENESIS IN LIVING SKELETAL MUSCLE CELLS

描述(改编自调查员摘要)：表格的核心和 肌肉的功能是提供细胞骨架结构的肌原纤维 以及骨骼肌特有的收缩力量。长期的 拟议实验的目标是了解步骤和蛋白质 管理骨骼肌细胞中肌原纤维的组装。第一 我们实验方法的策略是使用微观技术来 分析单个活的骨骼肌细胞内肌原纤维的形成 用表达与Green连锁的肌瘤蛋白的探针 荧光蛋白。我们的第二种方法集中在分子分析上 与Z-小体和Z-带相互作用的蛋白质 肌原纤维。第一个具体目标将检验这样一个假设，即 肌管中的肌原纤维从前肌原纤维到成熟的肌原纤维，以及 需要非肌肉肌球蛋白II和α-肌动蛋白分子。我们将使用 用时移荧光显微镜分析肌瘤的组装 在活的骨骼肌细胞培养中形成肌原纤维的亚基。三 将使用独立的途径来阻止 非肌肉肌球蛋白II，试图测试它在从 从前肌原纤维到成熟的肌原纤维。α-肌动蛋白水解酶片段(27 和53kD)将被显微注射到肌管内，试图干扰 前肌原纤维的形成和前肌原纤维向 成熟的肌原纤维。第二个具体目标是使用GFP靶向分析来 确定肌动蛋白N末端的哪些亚域可以定位到 骨骼肌细胞的Z带。我们将探索不同的角色 肌动蛋白这一区域靶向Z-带和胞核蛋白的结构域 到Z波段。将使用蛋白质-蛋白质相互作用分析来确定 Z-带和胞核蛋白，与Titin的Z-带区域相互作用。 先进的光学方法与分子生物学技术相结合将 允许关于肌原纤维形成的假说直接在 单个活的肌管。这些方法应该产生对基本知识的新见解 以及骨骼肌细胞中肌原纤维组装的病理过程。

DESCRIPTION (adapted from investigator's abstract): Central to the form and function of muscles are the myofibrils that provide the cytoskeletal structure and the contractile force that characterize skeletal muscles. The long-term goal of the proposed experiments is to understand the steps and proteins governing the assembly of myofibrils in skeletal muscle cells. The first strategy of our experimental approaches is to use microscopic techniques to analyze myofibril formation inside single living skeletal muscle cells transfected with probes expressing sarcomeric proteins linked to Green Fluorescent Proteins. Our second approach is focused on a molecular analysis of proteins interacting with the Z-bodies and Z-bands of the assembling myofibrils. The first specific aim will test the hypothesis that the formation of myofibrils in myotubes proceeds from premyofibrils to mature myofibrils, and requires nonmuscle myosin II and alpha-actinin molecules. We will use time-lapse fluorescence microscopy to analyze the assembly of sarcomeric subunits into myofibrils in cultures of living skeletal muscle cells. Three independent avenues will be used to block the activity or presence of the non-muscle myosin II in an effort to test the its role in the pathway from premyofibrils to mature myofibrils. Proteolytic fragments of alpha-actinin (27 and 53 kD) will be microinjected into myotubes in an attempt to interfere with the formation of premyofibrils and the transformation of premyofibrils into mature myofibrils. The second specific aim is to use a GFP targeting assay to determine which subdomains of the N-terminus of titin can localize to the Z-bands of skeletal muscle cells. We will probe the role of the different domains of this region of titin in targeting the Z-band and costameric proteins to the Z-bands. Protein-protein interaction assays will be used to identify the Z-band and costameric proteins, that interact with the Z-band regions of titin. The advanced optical methods coupled with molecular biological techniques will allow hypotheses about the formation of myofibrils to be tested directly in the single living myotube. These approaches should yield new insights into basic and pathologic processes in myofibril assembly in skeletal muscle cells.