MYOFIBRILLOGENESIS IN LIVING SKELETAL MUSCLE CELLS

活体骨骼肌细胞中的肌纤维发生

• 批准号: 6031559
• 负责人: Joseph William Sanger
• 金额: $ 28.23万
• 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/6031559
• 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（改编自研究者的摘要）：形式和内容的核心 肌肉的功能是提供细胞骨架结构的肌原纤维 以及骨骼肌特征的收缩力。长期来看 拟议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肌原纤维的形成 用表达与 Green 连接的肌节蛋白的探针转染 荧光蛋白。我们的第二种方法侧重于分子分析 与组装的 Z 体和 Z 带相互作用的蛋白质 肌原纤维。第一个具体目标将检验以下假设： 肌管中的肌原纤维从前肌原纤维发展为成熟肌原纤维，并且 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 在活体骨骼肌细胞培养物中将亚基转化为肌原纤维。三 将使用独立的途径来阻止该人的活动或存在 非肌肉肌球蛋白 II，以测试其在该途径中的作用 前肌原纤维到成熟肌原纤维。 α-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以试图干扰 前肌原纤维的形成和前肌原纤维转化为 成熟的肌原纤维。第二个具体目标是使用 GFP 靶向测定 确定 titin N 末端的哪些子结构域可以定位到 骨骼肌细胞的 Z 带。我们将探讨不同的作用 肌联蛋白此区域的结构域靶向 Z 带和肋蛋白 到Z带。蛋白质-蛋白质相互作用测定将用于鉴定 Z 带和肋蛋白，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 允许直接在实验中测试有关肌原纤维形成的假设 单活肌管。这些方法应该会对基本原理产生新的见解 和骨骼肌细胞中肌原纤维组装的病理过程。