Metavinculin Induced Changes at the Actin Interprotomer Contacts and the Mechanism of Resulting Severing

Abstract

Vinculin is a ubiquitously expressed adhesion protein of cell-matrix and cell-cell junctions. A larger splice-isoform of vinculin, metavinculin, is specifically expressed in cardiac and smooth muscles where it co-localizes with vinculin. Metavinculin has 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 a distinct and important role for this isoform in cardiac cells. In vitro, metavinculin was shown to sever and organize actin filaments into fine meshworks while vinculin bundles them. Therefore, we focused on characterizing the structural changes MVT induces in F-actin and the severing activity of MVT. By using mutagenesis, cross-linking and site-directed labeling, we determined that MVT causes changes in the actin interprotomer contacts and dynamics. Specifically, the spectra of acrylodan attached to cysteine residues engineered in dynamic loops of actin -D-loop, W-loop, and C-terminus- are changed in the presence of MVT indicating a rearrangement of actin-actin contacts. Enhanced excimers fluorescence due to pyrenes attached to cysteines 265 and 374 shows that MVT binding affects mainly lateral actin contacts. By using a seeded actin elongation assay as a reporter of severing by MVT, we confirmed that MVT has a biphasic mode of actin severing. At partial decoration with MVT -as defined by co-pelleting of MVT-actin complexes- we observed increasing amounts of filament severing. At higher decoration, above 60%, MVT had a stabilizing effect on filaments. Our work enhances our understanding of MVT induced severing and changes in actin filaments structure, which appears to be important for the physiological role of this tissue-specific vinculin isoform.