MYOFIBRILLOGENESIS IN LIVING SKELETAL MUSCLE CELLS

活体骨骼肌细胞中的肌纤维发生

• 批准号: 6512026
• 负责人: Joseph William Sanger
• 金额: $ 29.94万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-17 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6512026
• 关键词: fluorescence microscopy; green fluorescent proteins; molecular assembly /self assembly; muscle cells; muscle proteins; myofibrils; myogenesis; protein protein interaction; sarcomeres; tissue /cell culture

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.

描述（根据调查员的摘要改编）：表格和形式的中心 肌肉的功能是提供细胞骨架结构的肌原纤维 以及特征骨骼肌的收缩力。长期 提出的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组装。第一个 我们实验方法的策略是使用微观技术 分析单个活骨骼肌细胞内的肌纤维形成 用表达与绿色相关的肉瘤蛋白的探针转染 荧光蛋白。我们的第二种方法集中于分子分析 蛋白质与组装的Z-Bodies和Z波段相互作用 肌原纤维。第一个特定目的将检验形成的假设 肌原纤维中的肌原纤维从乳房前纤维到成熟的肌原纤维进行，然后 需要非肌肉肌球蛋白II和α-肌动蛋白分子。我们将使用 延时荧光显微镜分析肌膜的组装 活着的骨骼肌细胞培养物中的肌原纤维亚基。三 独立途径将用于阻止活动或存在 非肌肉肌球蛋白II，以测试其在途径中的作用 前纤维到成熟的肌原纤维。 α-肌动蛋白的蛋白水解片段（27 53 kd）将被微注射到肌管中，以尝试干扰 乳房纤维的形成和乳房纤维的转化 成熟的肌纤维。第二个具体目的是使用GFP靶向测定 确定Titin N末端的哪些子域可以定位于 骨骼肌细胞的Z波段。我们将探究不同的角色 titin区域靶向Z波段和costameric蛋白的域 到Z波段。蛋白质 - 蛋白质相互作用测定将用于识别 Z频段和costameric蛋白，与Titin的Z波段相互作用。 与分子生物技术结合的高级光学方法将 允许对直接测试肌原纤维形成的假设 单身肌管。这些方法应该给基本的新见解 和骨骼肌细胞中肌膜组装中的病理过程。