Deciphering the Roles of Nebulin in Cardiac Myofibril Assembly

描述（由申请人提供）：尽管心肌一直是激烈研究的焦点，但对于肌细胞如何调节肌动蛋白和相关蛋白的组装和组织，形成惊人精确长度的细丝，这一特性对生产性收缩至关重要，我们知之甚少。一种被认为可以作为细丝长度的分子标尺的分子是星云蛋白，它是一种巨大的（分子量500-900 kDa）模块化蛋白质，横跨细丝的整个长度。虽然心脏星云已经有了一些详细的生物化学定义，但对其功能特性的研究还很缺乏。我们有令人兴奋的初步数据显示，星云确实参与细丝长度的调节，也可能是多功能的。本提案的目的是描述星云的功能特性，并明确确定它是否确实可以作为调节心脏细丝长度的分子标尺。首先，我们将在内源性星云蛋白存在或不存在的情况下，通过在活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，但细丝结合超重复序列明显减少）来直接验证星云蛋白决定细丝长度的假设。我们提出心脏细丝的长度将对应于我们对迷你星云大小的分子操作。接下来，通过分析其与肌动蛋白丝盖蛋白、原调节蛋白和盖蛋白（CapZ）相互作用的意义，利用显性负技术结合siRNA策略，在确定的发育阶段解读朦胧蛋白在细丝组装中的作用的分子机制。最后，我们将开始破译星云的潜在的其他功能（包括z线组装和收缩活性），通过研究它与新的结合伙伴的相互作用，我们已经通过酵母双杂交筛选确定。生物化学、分子和细胞生物学方法的结合，包括活细胞成像，将与肌细胞原代培养结合使用，这是一种独特的小鼠胚胎干细胞培养系统，用于研究新生心肌纤维形成，并分析来自星云/-小鼠的心脏。我们假设星云蛋白确实是一个多功能巨人：它是细丝长度调节的分子标尺，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是通过确定导致各种人类肌病的星云突变来强调的，突出了它在正常肌肉发育和功能中的关键作用。此外，研究肌原纤维组装的机制至关重要，因为编码肌合成蛋白的bbbb10基因突变是家族性肥厚性心肌病（最常见的遗传性心血管疾病）的原因。这些发现意味着我们的研究将为了解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.