PROTEINS & ARCHITECTURE OF AN ELASTIC SARCOMERE MATRIX

这项建议的长期目标是理解结构和 肌节相关细胞骨架在纹状体中的生理作用 肌肉细胞。肌动蛋白和肌动蛋白两种巨型结构蛋白的最新研究 星云蛋白表明，每种蛋白质都构成了一套分子 细丝，在肌节中形成弹性基质。肌节 基质可能起着重要的生理作用，包括Long的发生 射程弹性、肌节稳定性的维持和组装 在发育中的肌肉细胞中的新生肌节。 在拟议的研究中，我们解决了中央的几个问题 重要性，并系统地和批判性地检查它们 综合实验方法： (A)Titin弹性的构象基础是什么？我们将测试 假设特定的和可逆的构象转变 Titin多肽的折叠和未折叠状态是其作为 产生弹性的分子弹簧。分子结构及其应用 构象转变将在cDNA序列水平上确定， 氨基酸序列，蛋白质折叠，结构域组织，轮廓长度， 在各种实验条件下的灵活性和可扩展性。 (B)肌球蛋白和肌球蛋白是否作为模板或支架 肌动蛋白细丝分别是什么？我们将寻找特定的细丝间 蛋白质相互作用，并测试它们对肌动球蛋白相互作用的影响。 此外，他们在组装和长度方面的潜在参与 肌球蛋白和肌动蛋白细丝的调节将在 自组装、成核组装和选择性重组 提取的肌节？ (C)肌动蛋白和雾化蛋白在肌节中是如何组织的？我们将评估一项 高分辨电子显微镜下的四丝肌节模型 结合免疫标记。此外，系统地搜索 Z线和M线上的结合蛋白将被用来解释它们的 在肌节上的安克雷奇。 (D)肌节基质对压力有何反应？我们将评估 并完善了我们的工作假设，即Titin的节段性可扩展性 这是它作为双程分子弹簧的基础。一组 将使用针对非重复表位的单抗来追踪 不同类型肌节的动态易位和微丝应变 长度。实验条件将被设计为促进 分子构象、肌节构型与肌节构型的相关性 骨骼肌和心肌的肌肉力学。

The long range goal of this proposal is to understand the structural and physiological roles of sarcomere-associated cytoskeletons in the striated muscle cells. Recent studies of two giant structural proteins, titin, and nebulin, suggest that each protein constitutes a set of molecular filaments, forming an elastic matrix in the sarcomere. The sarcomere matrix may play major physiological roles, including the genesis of long range elasticity, the maintenance of sarcomere stability and the assembly of nascent sarcomeres in developing muscle cells. In the proposed research, we address several questions of central importance and examine them systematically and critically with an integrated experimental approach: (a) What is the conformational basis of titin elasticity? We will test the hypothesis that specific and reversible conformational transition between folded and unfolded states of titin polypeptides underlies its capacity as a molecular spring to generate elasticity. The molecular structure and its conformational transition will be determined at the level of cDNA sequence, amino acid sequence, protein folding, domain organization, contour length, flexibility and extensibility under a variety of experimental conditions. (b) Do titin and nebulin serve as templates or scaffolds for myosin and actin filaments, respectively? We will search for specific interfilament protein interactions and test their effects on actomyosin interaction. Additionally, their potential involvement in the assembly and length regulation of myosin and actin filaments will be examined at the level of self-assembly, nucleated assembly and reconstitution of selectively extracted sarcomeres? (c) How are titin and nebulin organized in sarcomere? We will evaluate a four-filament sarcomere model by high resolution electron microscopy combined with immuno-labeling. Additionally, a systematic search for binding proteins at the Z-and M-lines will be made t elucidate their anchorage in the sarcomere. (d) How does the sarcomere matrix respond to stress? We will evaluate and refine our working hypothesis that the segmental extensibility of titin underlies its capacity as a dual-range molecular spring. A battery of monoclonal antibodies to non-repetitive epitopes will be used to track dynamic translocations and filament strain in sarcomeres of various lengths. Experimental conditions will be designed to facilitate correlation between molecular conformation, sarcomere architecture and muscle mechanics of skeletal and cardiac muscles.