Distinct roles for vinculin isoforms in cardiac and smooth muscle cell function

描述（由申请人提供）：白藜芦醇是一种在相邻细胞界面和细胞与细胞外基质接触处发现的普遍表达的蛋白质。通过选择性剪接产生的更大的版本，metavinculin，特异性地表达于心脏和平滑肌中，其中它分别在称为costameres/闰盘的粘附结构和致密斑块/体中与vinculin共定位。后黏着斑蛋白在其C-末端肌动蛋白结合尾（后黏着斑蛋白尾：MVT）处包括68个氨基酸的插入物。MVT插入物中的突变与破坏的闰盘组织和扩张的心肌病相关，这表明黏着斑蛋白同种型在心脏细胞中起着独特而重要的作用。在体外，后黏着斑蛋白将肌动蛋白丝组织成精细的网状结构，而黏着斑蛋白则将肌动蛋白束在一起。最近，观察到MVT的一种新的活动，肌动蛋白丝切断。这是相当惊人的，因为独立的研究表明，metavinculin表达水平与肌肉长度相关。我的目标是提供深入了解MVT如何控制肌动蛋白组织的机制，以及肌病相关突变如何改变这种蛋白质的特性。我将描述MVT的切断活性，检查与黏着斑蛋白的后黏着斑蛋白异源二聚化，以及随后存在两种亚型的结构后果，如在心脏和平滑肌中的情况。我将确定MVT诱导的肌动蛋白interprotomer接触和动力学的变化，并获得高分辨率的肌动蛋白-metavinculin复合物的结构。这项工作将通过结合生物化学，生物物理和显微镜技术来完成。证明后黏着斑蛋白对于心脏细胞中的机械转导是重要的数据表明，切断可能不是MVT插入物的唯一结果。另一种但不相互排斥的假设是后黏着斑蛋白插入物募集额外的结合配偶体和/或改变后黏着斑蛋白与现有黏着斑蛋白的亲和力 伙伴，这可能是至关重要的肌肉特定的作用。我建议检查metavinculin的相互作用与桩蛋白，一个已知的结合伙伴的黏着斑蛋白。我还将搜索以前未识别的结合伙伴的metavinculin在大鼠心脏和平滑肌组织和调查的功能重要性，新发现的相互作用，使用市售的心肌细胞系。我将结合联合收割机生物化学，蛋白质组学和细胞生物学工具来实现我的目标。总之，本文提出的工作将确定肌动蛋白切断的机制，并确定独特的后黏着斑蛋白复合物，这两者都可能是非常重要的心脏和平滑肌细胞的正常功能。

DESCRIPTION (provided by applicant): Vinculin is a ubiquitously expressed protein found at the interface of adjacent cells and where cells are in contact with the extracellular matrix. A larger version made by alternative splicing, metavinculin, is specifically expressed in cardiac and smooth muscles where it co-localizes with vinculin at adhesion structures called costameres/intercalated discs and in dense plaques/bodies, respectively. Metavinculin includes a 68 amino acid insert at its C-terminal actin binding tail (metavinculin tail: MVT). Mutations in the MVT insert are associated with disrupted intercalated disc organization and dilated cardiomyopathies suggesting that this vinculin isoform plays a unique and important role in cardiac cells. In vitro, metavinculin organizes actin filaments into fine meshworks in contrast to vinculin which bundles actin. Recently, a new activity of MVT was observed, actin filament severing. This was quite striking as independent studies reveal that metavinculin expression levels correlate with muscle length. My goal is to provide insight into the mechanism of how MVT controls actin organization and how myopathy related mutations alter the properties of this protein. I will characterize the severing activity of MVT, examine metavinculin heterodimerization with vinculin and the subsequent structural consequences of having both isoforms present, as is the case in cardiac and smooth muscle. I will identify MVT-induced changes in the actin interprotomer contacts and dynamics, and obtain a high resolution structure of the actin-metavinculin complex. This work will be accomplished by combining biochemical, biophysical, and microscopy techniques. Data demonstrating that metavinculin is important for mechano-transduction in cardiac cells suggest that severing may not be the sole consequence of the MVT insert. An alternative, but not mutually exclusive, hypothesis is that the metavinculin insert recruits additional binding partners and/or alters metavinculin's affinity to the existing vinculin partners, which may be critical to its muscle-specific role. I propose to examine metavinculin's interaction with paxillin, a known binding partner of vinculin. I will also search for previously unidentified binding partners of metavinculin in rat cardiac and smooth muscle tissue and investigate the functional importance of the newly identified interactions using commercially available cardiomyocyte cell lines. I will combine biochemistry, proteomics and cell biology tools to achieve my aims. In summary, the work proposed here will determine the mechanism of actin severing by metavinculin and identify unique metavinculin complexes, both of which could be very important to the proper function of cardiac and smooth muscle cells.