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

描述(申请人提供)：纽蛋白是一种普遍表达的蛋白质，存在于相邻细胞的交界处，细胞与细胞外基质接触。另一个通过选择性剪接产生的较大版本Metavinculin专门在心肌和平滑肌肉中表达，在那里它分别与vinculin共同定位在称为腔盘/间盘的粘连结构和致密的斑块/小体中。Metavinculin在其C末端肌动蛋白结合尾部插入68个氨基酸(Metavinculin Tail：MVT)。MVT插入的突变与间盘组织的破坏和扩张性心肌病有关，这表明这种纽蛋白亚型在心肌细胞中发挥着独特和重要的作用。在体外，Metavinculin将肌动蛋白细丝组织成精细的网状结构，而vinculin则将肌动蛋白捆绑在一起。最近，又观察到一种新的MVT活性，即肌动蛋白细丝断裂。这是相当惊人的，因为独立研究表明，Metavinculin的表达水平与肌肉长度相关。我的目标是深入了解MVT如何控制肌动蛋白组织的机制，以及与肌病相关的突变如何改变这种蛋白质的性质。我将表征MVT的切断活性，研究Metavinculin与vinculin的异构体二聚化以及两种异构体都存在的后续结构后果，就像在心肌和平滑肌中的情况一样。我将确定MVT引起的肌动蛋白蛋白间接触和动力学的变化，并获得肌动蛋白-Metavinculin复合体的高分辨率结构。这项工作将通过结合生化、生物物理和显微技术来完成。数据表明，Metavinculin对心肌细胞的机械转导很重要，这表明切断可能不是MVT插入的唯一结果。另一种但不是相互排斥的假设是，Metavinculin插入物招募更多的结合伙伴和/或改变Metavinculin对现有纽蛋白的亲和力 合作伙伴，这可能是其肌肉特定作用的关键。我建议研究绿维菌素与帕克西林的相互作用，帕克西林是纽维菌素的已知结合伙伴。我还将在大鼠心脏和平滑肌组织中寻找以前未知的Metavinculin结合伙伴，并使用商业心肌细胞系研究新发现的相互作用的功能重要性。我将结合生物化学、蛋白质组学和细胞生物学工具来实现我的目标。综上所述，本文提出的工作将确定Metavinculin切断肌动蛋白的机制，并鉴定独特的Metavinculin复合体，这两者对心肌和平滑肌细胞的正常功能都非常重要。

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.