Deciphering the Roles of Nebulin in Cardiac Myofibril Assembly

描述(申请人提供)：尽管心肌一直是紧张研究的焦点，但人们对心肌细胞如何调节肌动蛋白和相关蛋白质的组装和组织成长度惊人的细丝知之甚少：这些特性对生产性收缩至关重要。有一种分子被认为是用来指定细丝长度的分子标尺，它是一种巨大的(分子量500-900 kDa)的模块化蛋白质，横跨细丝的整个长度。虽然从生物化学的角度对心脏星云蛋白进行了较为详细的定义，但缺乏对其功能特性的研究。我们有令人兴奋的初步数据显示，星云蛋白确实参与了细丝长度的调节，也可能是多功能的。这项提议的目标是描绘星云蛋白的功能特性，并最终确定它是否真的可以作为分子标尺来调节心脏细丝长度。首先，我们将直接测试这一假设，即在存在或不存在内源性星云蛋白的情况下，通过在活的肌细胞中表达一种新型的迷你星云蛋白(包含其所有独特区域的模块，但细纤维结合超重复序列明显较少)，星云蛋白决定细丝长度。我们认为心脏细丝的长度将与我们对迷你星云大小的分子操作相对应。接下来，将通过分析星云蛋白与肌动蛋白细丝覆盖蛋白、原调节蛋白和覆盖蛋白(CAPZ)相互作用的意义，结合显性-负性技术结合siRNA策略，在特定的发育阶段破译星云蛋白在细丝组装中的分子机制。最后，我们将通过研究它与我们通过酵母双杂交筛选确定的新的结合伙伴的相互作用，开始破译NeBulin的潜在其他功能(包括Z线组装和收缩活性)。包括活细胞成像在内的生化、分子和细胞生物学方法的组合将与心肌细胞的原代培养结合使用，这是一种独特的小鼠胚胎干细胞培养系统，用于研究从头开始的心肌纤维形成，以及分析来自neBulin-/-鼠的心脏。我们假设，星云蛋白确实扮演着多功能巨人的角色：它是调节细丝长度的分子标尺，其独特的区域具有独特的生理作用，对横纹肌的正常功能至关重要。这些研究的临床相关性通过鉴定导致各种人类肌病的雾化蛋白突变而得到强调，突出了其在正常肌肉发育和功能中的关键作用。此外，研究肌原纤维聚集的机制是至关重要的，因为编码肌节蛋白的&gt；10基因突变与家族性肥厚型心肌病有关，这是最常见的遗传性心血管疾病。这些发现意味着我们的研究将为各种肌肉疾病的分子基础提供有价值的见解。

DESCRIPTION (provided by applicant): Although cardiac muscle has been the focus of intense research, little is known about how myocytes regulate the assembly and organization of actin and associated proteins into thin filaments of strikingly precise lengths: properties that are critical for productive contraction. One molecule that has been proposed to function as a molecular ruler to specify thin filament lengths is nebulin, a giant (MW 500-900 kDa), modular protein that spans the entire length of the thin filaments. Although cardiac nebulin has been defined in some detail biochemically, studies addressing its functional properties are lacking. We have exciting preliminary data revealing that nebulin indeed is involved in thin filament length regulation and may also be multifunctional. The goal of this proposal is to delineate nebulin's functional properties and definitively determine whether it can indeed function as a molecular ruler to regulate cardiac thin filament lengths. First, we will directly test the hypothesis that nebulin determines thin filament length by expressing a novel mini-nebulin in living myocytes (containing modules from all of its unique regions but significantly fewer thin filament-binding super-repeats), in the presence or absence of endogenous nebulin. We propose that the lengths of the cardiac thin filaments will correspond to our molecular manipulation of mini-nebulin's size. Next, the molecular mechanisms by which nebulin functions in thin filament assembly will be deciphered by analyzing the significance of its interactions with the actin filament capping proteins, tropomodulin and capping protein (CapZ), using dominant-negative techniques combined with siRNA strategies during defined stages of development. Finally, we will begin to decipher nebulin's potential other functions (including Z-line assembly and contractile activity), by investigating its interactions with novel binding partners that we have identified by yeast two-hybrid screens. A combination of biochemical, molecular and cellular biological approaches including live-cell imaging will be used in conjunction with primary cultures of myocytes, a unique murine embryonic stem (ES) cell culture system to study de novo cardiac myofibrillogenesis, and analysis of hearts from nebulin -/- mice. We hypothesize that nebulin indeed acts as a multifunctional giant: it is a molecular ruler for thin filament length regulation, and its distinct regions have unique physiological roles critical for the proper functioning of striated muscle. The clinical relevance of these investigations is underscored by the identification of nebulin mutations resulting in various human myopathies, highlighting its pivotal role in normal muscle development and function. Additionally, studying mechanisms responsible for myofibril assembly is critical since mutations in >10 genes encoding sarcomeric proteins are responsible for familial hypertrophic cardiomyopathy, the most common heritable cardiovascular disease. These findings imply that our studies will provide valuable insight into the molecular bases of various muscle diseases.