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原纤维蛋白是如何组装成肌原纤维的，以及当这些肌原蛋白发生突变、截断或删除时，这些肌原蛋白是如何导致肌病的。实验的重点是在肌原纤维形成和维持中起重要作用的z带蛋白。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。我们的主要实验方法是通过编码荧光嵌合gfp肌肉蛋白的探针来分析活骨骼肌细胞内肌原纤维z带的形成。第一个具体目的是利用各种显微方法研究活斑马鱼骨骼肌细胞中z-体和z-带中几种蛋白质的组装、动力学和接近性。共聚焦和反褶积显微镜将用于跟踪活斑马鱼肌原纤维形成过程中gfp -肉瘤蛋白的组装。FRAP（荧光恢复后光漂白）实验将测量z-体和z-带中关键蛋白质在肌纤维形成过程中的变化动态。蛋白质及其结合伙伴在活骨骼肌细胞中共表达的稳态FRET（荧光共振能量转移）效率将被分析，以产生z波段形成过程中蛋白质相互作用的详细图像。有许多肌病是由Z带蛋白突变引起的。我们的第二个假设是，肌动蛋白、肌动素、ZASP/cypher和端递蛋白的突变或端递蛋白的缺失将导致z带和i带其他部分蛋白质的动力学和结合特性的改变，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已知与肌病有关的这四种z带蛋白的几种突变的影响。FRAP、FRET和生化分析将用于确定突变是否影响突变蛋白及其z带结合伙伴（α -肌动蛋白、FATZ、肌动蛋白）和i带细丝（原肌球蛋白）的动力学和相互作用，从而揭示活肌肉细胞中肌肉疾病的分子基础。我们的实验应该对肌原纤维的组装、维持和肌病产生新的见解。公共卫生相关性：肌原纤维组装是精确的细胞工程的例证，它产生了一系列致力于调节骨骼肌力量产生的蛋白质。虽然组成蛋白排列在一个精确的结构框架中，但它们是动态的，能够在肌原纤维和细胞蛋白质池之间交换。我们认为，与骨骼肌疾病相关的突变蛋白改变了动态和结合特性，导致肌原纤维不稳定。含有这些突变蛋白的肌原纤维不能支持力的产生，因此肌原纤维变得不稳定并分崩离析。

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. PUBLIC HEALTH RELEVANCE: Myofibril assembly exemplifies cellular engineering of exacting precision that produces an array of proteins dedicated to regulated force production in skeletal muscles. Although the component proteins are arranged in a precise structural framework, they are dynamic and capable of exchanging between the myofibril and a cellular pool of proteins. We think that mutated proteins associated with skeletal muscle diseases have altered dynamic and binding properties that lead to an instability of the myofibrils. Myofibrils containing these mutated proteins cannot support the force production, and thus the myofibrils become unstable and fall apart.