Metavinculin Tunes the Flexibility and the Architecture of Vinculin-Induced Bundles of Actin Filaments

Abstract

Vinculin is an abundant protein found at cell-cell and cell-extracellular matrix junctions. In muscles, a longer splice-isoform of vinculin, metavinculin, is also expressed. The metavinculin-specific insert is part of the C-terminal tail domain, the actin-binding site of both isoforms. Mutations in the metavinculin-specific insert are linked to heart disease such as dilated cardiomyopathies. Vinculin tail domain (VT) both binds and bundles actin filaments. Metavinculin tail domain (MVT) binds actin filaments in a similar orientation but does not bundle filaments. Recently, MVT was reported to sever actin filaments. In this work, we asked how MVT influences F-actin alone or in combination with VT. Cosedimentation and limited proteolysis experiments indicated a similar actin binding affinity and mode for both VT and MVT. In real time TIRF microscopy experiments MVT’s severing activity was negligible. Instead, we found that MVT binding caused a two-fold reduction in F-actin’s bending persistence length and increased susceptibility to breakage. Perhaps MVT allows the load of muscle contraction to act as a signal to reorganize actin filaments. Using mutagenesis and site-directed labeling with fluorescence probes, we determined that MVT alters actin interprotomer contacts and dynamics, which presumably reflect the observed changes in bending persistence length. Finally, we found that MVT decreases the density and thickness of actin filament bundles generated by VT. Altogether, our data suggest that MVT alters actin filament flexibility and tunes filament organization in the presence of VT. Both of these activities are potentially important for muscle cell function.

The Structural Basis of Actin Organization by Vinculin and Metavinculin

“…At sub-nanometer resolution, this reconstruction is essentially indistinguishable from the VtΔC5 reconstruction, with minor differences in connectivity between the helical densities. In contrast to a previous negative stain reconstruction, where extra density was observed protruding from MVt when compared with Vt 37 , rigid-body docking of the MVt crystal structure into our sub-nanometer resolution density map demonstrates that helix H1’ is also displaced from the helical bundle when MVt binds actin, in agreement with the similar proteolysis susceptibility profile reported for MVt H1’ and Vt H1 upon actin binding 38 . In addition to the different resolutions of the studies, an explanation for this discrepancy may lie in the different constructs used for structural analysis, as the previous reconstruction excluded residues 858–879 and included residues 1130–1134.…”

“…MVt undergoes a similar structural transition, displacing helix H1’ to form an interaction with actin that is indistinguishable from Vt at the resolution of our reconstructions. Additionally, we find that the presence of sub-stoichiometric MVt suppresses the actin-bundling activity of Vt, in support of a recent study 38 . We interpret these results in a conceptual model wherein vinculin and metavinculin undergo partial unfolding transitions commensurate with actin engagement, which can either lead to the promotion or suppression of actin bundling depending on the presence of the MVt insertion.…”

“…1, Supplementary Movie 1, 2). This model suggests that helix 1 disengages from the helical bundle upon actin binding, supported by recent biochemical evidence that this segment becomes susceptible to proteolytic cleavage upon actin binding by Vt 38 .…”

“…The sequence comprising H1 was not visible in the crystallographic electron density maps, presumably becoming an unstructured element along with the rest of the linker connecting the head and tail domains. Although investigations of metavinculin have been limited, previous studies suggest that the metavinculin tail domain (MVt) possesses similar actin binding to Vt but diminished actin bundling activity 32; 37; 38 , as well as the intriguing property of suppressing actin bundling by Vt 38 . Disease mutations in MVt produce higher-order actin assemblies with aberrant architectures in vitro 32 , suggesting that a detailed mechanistic understanding of Vt- and MVt-actin interactions will provide insight into the molecular basis of these pathologies.…”

Mechanisms and Functions of Vinculin Interactions with Phospholipids at Cell Adhesion Sites

“…From data derived from a variety of in vivo and in vitro techniques, it was concluded that MVt "tunes" actin filament bundles by altering the architecture and flexibility (73). Using high resolution cryo-electron microscopy, a sub-nanometer three-dimensional reconstruction of the vinculin tailactin complex was obtained (74).…”