Analysis of Z-band assembly and maintenance in living skeletal muscle cells

描述(申请人提供)：我们的长期目标是了解肌瘤蛋白如何组装成肌原纤维，以及当突变、截断或缺失时，这些肌瘤蛋白如何导致肌病。实验的重点是Z-带蛋白，它在肌原纤维的形成和维持中起着至关重要的作用。我们的第一个假设是，随着Z-小体发展成Z-带，结构组织的增加伴随着蛋白质动力学和结合作用的变化，从而产生能够支持收缩的结构。在这项建议中，我们实验方法的主要策略是通过编码荧光嵌合GFP-肌肉蛋白的探针来分析活的骨骼肌细胞内肌原纤维的Z-带的形成。第一个具体目的是利用各种显微镜方法研究斑马鱼骨骼肌细胞中Z-小体和Z-带中几种蛋白质的组装、动力学和亲和力。共聚焦显微镜和去卷积显微镜将被用来跟踪在活的斑马鱼肌原纤维形成过程中GFP-肌瘤蛋白的组装。FRAP(荧光漂白后恢复)实验将测量在肌原纤维形成过程中Z小体和Z带中关键蛋白质的变化动态。将分析活的骨骼肌细胞中共表达的蛋白质及其结合伙伴的稳态FRET(荧光共振能量转移)效率，以产生Z-带形成过程中蛋白质相互作用的详细图像。在许多肌肉疾病中，Z-带蛋白发生了突变。我们的第二个假设是，肌动蛋白、肌红蛋白、ZASP/Cypher和端乙醇胺的突变或端乙醇胺的缺失将导致Z带和I带其他部分的蛋白质的动力学和结合特性改变，从而改变肌原纤维的稳定性。第二个具体目标是在单细胞水平上分析这四个已知与肌病有关的Z-带蛋白的几个突变的影响。FRAP、FRET和生化分析将被用来确定突变是否影响Z带和I带(原肌球蛋白)细丝中突变蛋白质及其结合伙伴(α-Actinin、Fatz、myotilin)的动态和相互作用，从而揭示活肌肉细胞中肌肉疾病的分子基础。我们的实验应该会对肌原纤维的组装、维护和肌病产生新的见解。

DESCRIPTION (provided by applicant): Our long-term goals are to understand how sarcomeric proteins are assembled into myofibrils, and, how when mutated, truncated or deleted, these sarcomeric proteins result in myopathies. The experiments focus on Z-band proteins that have an essential role in the formation and maintenance of myofibrils. Our first hypothesis is that as z-bodies develop into Z- bands, the increasing structural organization is accompanied by changes in the dynamics and binding interactions of the proteins resulting in a structure capable of supporting contractions. The major strategy of our experimental approaches in this proposal is to analyze the formation of the Z-bands of myofibrils inside living skeletal muscle cells via probes encoding fluorescent chimeric GFP-muscle proteins. The first specific aim is to investigate the assembly, the dynamics and proximities of several proteins in the z-bodies and Z-bands in skeletal myocytes in living zebrafish by using various microscopical approaches. Confocal and deconvolution microscopy will be used to follow the assembly of GFP-sarcomeric proteins during myofibrillogenesis in living zebrafish. FRAP (Fluorescence Recovery After Photobleaching) experiments will measure the changing dynamics of key proteins in the z-bodies and Z-bands during myofibrillogenesis. Steady-state FRET (Fluorescence Resonance Energy Transfer) efficiencies of the proteins and their binding partners co-expressed in living skeletal muscle cells will be analyzed to produce a detailed picture of protein interactions during Z-band formation. There are a number of myopathies in which Z- band proteins are mutated. Our second hypothesis is that mutations of actin, myotilin, ZASP/cypher and telethonin, or deletions of telethonin will lead to altered dynamics and binding properties of proteins in the Z-band, and other parts of the I-bands, changing the stability of the myofibrils. The second specific aim is to analyze, on the single cell level, the effects of several mutations of these four Z-band proteins known to be involved in myopathies. FRAP, FRET and biochemical analyses will be used to determine if the mutations affect the dynamics and interactions of the mutated proteins and their binding partners (alpha-actinin, FATZ, myotilin) in the Z-bands, and in the thin filaments in the I-bands (tropomyosin), and thus reveal the molecular bases for the muscle disease in living muscle cells. Our experiments should yield novel insights into myofibril assembly, maintenance, and myopathies.