STRUCTURE AND FUNCTION OF SKELETAL MUSCLE NEBULIN

我们提出的研究的长期目标是了解分子 星云的结构，并定义星云如何对 骨骼肌细胞的力学特性和发育程序。 星云蛋白是一种巨大的肌动蛋白结合蛋白，由近2%的 肌原纤维蛋白。这种模块化的蛋白质被认为是一种 调节细丝长度的蛋白质尺子，作为一种钙- 钙调素介导的肌动球蛋白相互作用调节蛋白和AS 一种用于组装细丝格子的模板 发展。我们建议使用一个 生化、生物物理、结构和细胞的结合 接近了。具体目标是： (L)将星云蛋白亚型的大小与肌动蛋白的长度相关联 广泛存在于骨骼肌中的细丝。扫描传输 电子显微镜和质谱仪将被用来帮助确定 成熟和发育中星云蛋白亚型的大小和轮廓长度 兔、鼠和鸡的骨骼肌。的免疫本地化 肌动蛋白和原肌球蛋白将用来帮助定义肌动蛋白的长度 细丝。 (2)含云母细丝的制备及排布研究 用高分辨低温电子研究细丝上的星云 显微镜。 (3)探讨星云蛋白-肌动蛋白相互作用的机制 雾化蛋白对肌动球蛋白相互作用的抑制作用及钙拮抗作用 和钙调蛋白及其通过磷酸化和磷酸化的潜在调节 磷脂。共沉淀，固相结合，ATPase分析，在 体外运动分析和荧光光谱分析将用于 揭示分子间的相互作用。 (4)确定星云蛋白与肌动蛋白、肌球蛋白和肌球蛋白的分子界面。 钙调素。化学交联、蛋白质测序和突变型星云 片段将被用来识别界面上的重要氨基酸。 (5)完成人骨骼肌球蛋白的编码序列， 定义产生星云蛋白亚型的机制，并 对功能域进行分类。 (6)阐明星云蛋白在肌原纤维形成和发育中的作用 培养中的小鼠骨骼肌。雾化蛋白的合成与组装 及其在正常细胞和过度表达Neulin的细胞中的转录 将监测碎片，并确定细丝长度 通过免疫电子显微镜观察。 雾化蛋白在骨骼肌收缩机制中的存在 提出了新的挑战，并重新引起了人们对 细丝。这项拟议的研究将定义生理学 星云蛋白的功能及其对地球运动的贡献的分子基础 骨骼肌中细丝的结构、调节和组装 在健康和患病的状态下。

The long term goal of our proposed research is to understand the molecular structure of nebulin and to define how nebulin contributes to the mechanical properties and developmental programs of skeletal muscle cells. Nebulin is a giant actin binding protein that comprises nearly 2% of myofibrillar proteins. This modular protein has been proposed to act as a protein ruler to regulate the length of thin filaments, as a calcium- calmodulin-mediated regulatory protein of actomyosin interaction, and as a template for the assembly of the thin filament lattice during development. We propose to evaluate these hypotheses critically using a combination of biochemical, biophysical, structural and cellular approaches. The specific aims are: (l) to correlate the size of nebulin isoforms and the length of actin filaments in a wide range of skeletal muscles. Scanning transmission electron microscopy and mass spectrometry will be used to help determine the size and contour length of nebulin isoforms of mature and developing skeletal muscles of rabbit, mouse, and chicken. lmmunolocalization of actin and tropomyosin will be used to help define the length of actin filaments. (2) to prepare nebulin-containing thin filaments and study the arrangement of nebulin on the thin filaments by high resolution cryoelectron microscopy. (3) to investigate the mechanisms of nebulin-actin interaction and nebulin's inhibition of actomyosin interaction, its reversal by calcium and calmodulin and its potential regulation by phosphorylation and phospholipids. Cosedimentation, solid phase binding, ATPase assays, in vitro motility assays, and fluorescence spectroscopy will be used to reveal molecular interactions. (4) to define molecular interfaces of nebulin with actin, myosin, and calmodulin. Chemical crosslinking, protein sequencing and mutant nebulin fragments will he used to identify important amino acids at the interface. (5) to complete the coding sequence of human skeletal muscle nebulin, to define the mechanisms with which nebulin isoforms are generated and to catalog functional domains. (6) to elucidate the role of nebulin in myofibrillogenesis in developing mouse skeletal muscle in culture. The synthesis and assembly of nebulin and its transcripts in normal cells and in cells that overexpress nebulin fragments will be monitored, and thin filament length will be determined by immunoelectron microscopy. The presence of nebulin in the contractile machinery of skeletal muscles poses new challenges and elicits renewed interest in the understanding of thin filaments. The proposed research would define the physiological function of nebulin and the molecular basis for its contribution to the structure, regulation, and assembly of thin filaments in skeletal muscles in healthy and diseased states.