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

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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.