Deciphering the Roles of Nebulin in Cardiac Myofibril Assembly

描述（由申请人提供）：虽然心肌一直是密集研究的焦点，但对肌细胞如何调节肌动蛋白和相关蛋白质的组装和组织成具有惊人精确长度的细丝知之甚少：这些特性对于生产性收缩至关重要。有一种分子被认为可以作为分子尺子来指定细丝的长度，这就是星云蛋白，一种巨大的（分子量500-900 kDa）模块化蛋白质，它跨越了细丝的整个长度。虽然心脏星云蛋白已被定义在一些详细的生物化学，研究解决其功能特性是缺乏的。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。这项提议的目的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一个分子标尺来调节心脏细丝的长度。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，星云蛋白通过在活的肌细胞中表达一种新的迷你星云蛋白（包含来自其所有独特区域的模块，但显着较少的薄的有害物质结合的超重复序列）来决定细丝长度。我们提出，心脏细丝的长度将对应于我们对迷你星云蛋白大小的分子操作。接下来，通过分析其与肌动蛋白丝帽蛋白，tropomodulin和帽蛋白（CapZ）的相互作用的意义，使用显性负性技术结合siRNA的策略在定义的发展阶段的分子机制，星云蛋白在细丝组装的功能将被破译。最后，我们将开始破译星云蛋白的潜在的其他功能（包括Z-线组装和收缩活动），通过研究它与新的结合伙伴，我们已经确定了酵母双杂交筛选的相互作用。生物化学、分子和细胞生物学方法（包括活细胞成像）的组合将与肌细胞的原代培养物、独特的小鼠胚胎干（ES）细胞培养系统（用于研究从头心脏肌原纤维发生）和来自星云蛋白-/-小鼠的心脏分析结合使用。我们假设，星云蛋白确实作为一个多功能的巨人：它是一个分子统治者的细丝长度调节，其独特的区域有独特的生理作用，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星云蛋白突变来强调，突出了其在正常肌肉发育和功能中的关键作用。此外，研究负责肌原纤维组装的机制是至关重要的，因为编码肌节蛋白的>10个基因中的突变负责家族性肥厚型心肌病，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.