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

描述（由申请人提供）：我们的长期目标是了解肌节蛋白如何组装成肌原纤维，以及当突变、截短或缺失时，这些肌节蛋白如何导致肌病。这些实验集中在Z带蛋白上，它们在肌原纤维的形成和维持中起着重要作用。我们的第一个假设是，随着Z体发展成Z带，结构组织的增加伴随着蛋白质的动力学和结合相互作用的变化，从而产生能够支持收缩的结构。我们的实验方法的主要策略是在这个建议是分析的Z-带的活骨骼肌细胞内的肌原纤维通过探针编码荧光嵌合GFP-肌肉蛋白的形成。第一个具体的目的是调查的装配，动力学和邻近的几种蛋白质的Z-体和Z-带在骨骼肌细胞在活斑马鱼通过使用各种显微镜的方法。共聚焦和去卷积显微镜将被用来跟踪在活斑马鱼肌原纤维形成过程中GFP-肌节蛋白的组装。FRAP（光漂白后的荧光恢复）实验将测量肌原纤维形成过程中Z体和Z带中关键蛋白的变化动态。将分析在活骨骼肌细胞中共表达的蛋白质及其结合伴侣的稳态FRET（荧光共振能量转移）效率，以产生Z带形成期间蛋白质相互作用的详细图像。有许多肌病中Z带蛋白发生突变.我们的第二个假设是，肌动蛋白，myotilin，ZASP/cypher和telethonin的突变，或telethonin的缺失将导致改变的动力学和结合特性的蛋白质的Z带，和其他部分的I带，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已知参与肌病的这四种Z带蛋白的几种突变的影响。FRAP、FRET和生物化学分析将用于确定突变是否影响Z带中突变蛋白及其结合伴侣（α-辅肌动蛋白、FATZ、肌球蛋白）和I带中细丝（原肌球蛋白）的动力学和相互作用，从而揭示活肌细胞中肌肉疾病的分子基础。我们的实验应该产生新的见解肌原纤维组装，维护和肌病。

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.