ROLE OF METAVINCULIN IN ACTIN REORGANIZATION AND FORCE TRANSMISSION

Vinculin is an essential cytoskeletal protein that acts as a scaffold to link transmembrane receptors to actin filaments, thereby playing a crucial role in cell adhesion, motility, and force transmission between cells. While vinculin is ubiquitously expressed, metavinculin, a larger isoform of vinculin, is selectively expressed in smooth and cardiac muscle cells. Metavinculin contains an additional exon that encodes a 68-residue insert. Point mutations in the 68-residue insert have been associated with altered actin organization and heart disease, notably dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). As metavinculin expression is higher in muscle cells that require greater force transmission, we postulate that metavinculin plays an important role in force generation and transmission through its interaction with the actin cytoskeleton. Results from cryo- and negative-stain electron microscopy (EM), in conjunction with actin co-sedimentation experiments, indicate that the tail domain of vinculin (Vt) and metavinculin (MVt) possess similar F-actin binding interactions yet are distinct in their ability to remodel actin filaments. Whereas actin binding of Vt promotes actin filament bundling in vitro, the MVt domain does not. Intriguingly, addition of MVt to Vt interferes with Vt-induced actin filament bundling. In contrast, cardiomyopathy-associated MVt mutants do not antagonize Vt-induced actin bundling. Based on these findings, we hypothesize that metavinculin plays a role in negatively regulating vinculin-mediated actin bundling in contractile tissues. Hence, we next examined the roles of vinculin and metavinculin within cells. To investigate functional differences between metavinculin and vinculin in cells, we additionally stably expressed either vinculin or metavinculin in vinculin-null mouse embryonic fibroblasts. While both metavinculin and vinculin were observed at focal adhesions (FA), metavinculin-expressing cells had larger but fewer FAs per cell compared to vinculin-expressing cells. Metavinculin-expressing cells migrated faster and exhibited greater persistence compared to vinculin-expressing cells, even though vinculin-containing FAs assembled and disassembled faster. Magnetic tweezer measurements on vinculin-expressing cells show a typical cell stiffening phenotype in response to externally applied force; however, this was absent in vinculin-null and metavinculin-expressing cells. Our findings that metavinculin expression leads to larger but fewer number of FAs per cell, in conjunction with the inability of metavinculin to bundle F-actin in vitro and rescue cell stiffening response, are consistent with our previous findings of actin bundling deficient vinculin variants, suggesting that deficient actin-bundling may account for some of the differences between vinculin and metavinculin.Doctor of Philosoph