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

描述（由申请人提供）：Vinculin是一种普遍表达的蛋白，发现于邻近细胞的界面和细胞与细胞外基质接触的地方。另一种由选择性剪接产生的更大版本，后血管蛋白，在心脏和平滑肌中特异性表达，它分别与血管蛋白共同定位于称为肋肌/嵌入盘的粘附结构和致密斑块/体中。Metavinculin在其c端肌动蛋白结合尾部（Metavinculin tail: MVT）包含68个氨基酸插入。MVT插入物的突变与嵌入盘组织破坏和扩张型心肌病有关，这表明这种血管蛋白异构体在心肌细胞中起着独特而重要的作用。在体外，与成束肌动蛋白的血管蛋白不同，转血管蛋白将肌动蛋白丝组织成精细的网状结构。最近发现了一种新的MVT活性，即肌动蛋白丝切断。独立研究表明，metavinculin表达水平与肌肉长度相关，这是相当惊人的。我的目标是深入了解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该蛋白的特性。我将描述MVT的切断活性，检查后血管蛋白与血管蛋白的异二聚化以及两种同种异构体同时存在的后续结构后果，就像在心脏和平滑肌中的情况一样。我将确定mvt诱导的肌动蛋白间蛋白体接触和动力学的变化，并获得肌动蛋白-元血管蛋白复合物的高分辨率结构。这项工作将通过结合生化、生物物理和显微镜技术来完成。数据表明，metavinculin对心脏细胞的机械转导很重要，这表明切断可能不是MVT插入的唯一后果。另一种但并非相互排斥的假设是，metavinculin插入物招募了额外的结合伙伴和/或改变了metavinculin对现有血管蛋白的亲和力

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.