Identification of an Actin Binding Surface on Vinculin that Mediates Mechanical Cell and Focal Adhesion Properties

Abstract

SUMMARY Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin, and developed an alternative model to better define this novel actin-binding surface, using negative-stain EM, discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this new actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes which promote actin bundling.

Vinculin promotes nuclear localization of TAZ to inhibit ECM stiffness-dependent differentiation into adipocytes

“…Interestingly, vinculin directly binds to Factin (Jockusch and Isenberg, 1981;Menkel et al, 1994) and has F-actin-bundling and -capping activities (Le Clainche et al, 2010;Wen et al, 2009). Here, we demonstrated that actin-bindingdeficient vinculin I997A (Thompson et al, 2014) lacks the ability to promote the change in the YAP/TAZ nuclear localization that is dependent on ECM stiffness. Taken together, these observations indicate that vinculin regulates YAP/TAZ at least partly through organization of actin cytoskeleton.…”

“…To further gain insight into the relation between actin organization and vinculin, we used the vinculin mutants I997A (denoted IA, deficient in actin binding) and T12 (constitutively active) (Thompson et al, 2014;Cohen et al, 2005). GFP-fused vinculin mutants were expressed in ST2 vinculin-depleted cells (Fig.…”

Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity

“…The adapter protein vinculin is an important protein involved in the transduction of mechanical signals (Carisey et al, 2013;Humphries et al, 2007;Thompson et al, 2014). To test the role of vinculin in LIPUS sensing, we first examined whether LIPUS can activate Rac1 in vinculin knockout MEFs (vin -/-MEFs).…”

“…The link between vinculin and actin has been shown to be critical for the function of vinculin as a transducer of mechanical stimuli (Carisey et al, 2013;Thompson et al, 2014). To test whether this link is also critical for sensing LIPUS, we performed rescue experiments with vin -/-MEFs expressing vinFL…”

Polymers in Tissue Engineering

“…Integrins are biochemically linked to a number of cell signaling pathways, but physically they are attached to α-actinin or talin and/ or vinculin, which then links it to the cell's thin filament actin which is a critical regulator of cell shape. This serves as a biochemical and physical link to the scaffold on the outside [64], and cell with mutations causing deficits in the binding capabilities between actin and vinculin demonstrate cell spreading deficits [65].…”