MYOFIBRILLOGENESIS IN LIVING SKELETAL MUSCLE CELLS

描述（改编自研究者摘要）：表格的核心， 肌肉的功能是提供细胞骨架结构的肌原纤维 和骨骼肌特有的收缩力。长期 所提出的实验的目标是了解步骤和蛋白质 控制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显微技术， 分析单个活骨骼肌细胞内的肌原纤维形成 用表达与绿色连接的肌节蛋白的探针转染 荧光蛋白。我们的第二种方法是集中在分子分析， 与组装的Z体和Z带相互作用的蛋白质 肌原纤维第一个具体的目标将测试的假设，形成 肌管中的肌原纤维从前肌原纤维发展为成熟肌原纤维， 需要非肌肉肌球蛋白II和α-辅肌动蛋白分子。我们将使用 延时荧光显微镜分析肌节的组装 在活的骨骼肌细胞培养物中，三 独立的途径将被用来阻止活动或存在的 非肌肉肌球蛋白II，试图测试其在从 前肌原纤维到成熟肌原纤维。α-辅肌动蛋白的蛋白水解片段（27 和53 kD）将被显微注射到肌管中，试图干扰 前肌原纤维的形成和前肌原纤维向 成熟肌原纤维第二个具体目标是使用GFP靶向测定， 确定肌联蛋白的N-末端的哪些亚结构域可以定位于 骨骼肌细胞的Z带。我们将探讨不同的角色， 在靶向Z带和costameric蛋白中， 到Z波段蛋白质-蛋白质相互作用测定将用于鉴定 Z带和costameric蛋白，与肌联蛋白的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， 允许关于肌原纤维形成的假设直接在 单个活肌管。这些方法应该产生新的见解， 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.