The vinculin C-terminal hairpin mediates F-actin bundle formation, focal adhesion, and cell mechanical properties

Vinculin is an essential and highly conserved cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples integrins or cadherins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility, and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) contains determinants necessary for binding and bundling of actin filaments. Actin binding to Vt has been proposed to induce formation of a Vt dimer that is necessary for cross-linking actin filaments. Results from this study provide additional support for actin-induced Vt self-association. Moreover, the actin-induced Vt dimer appears distinct from the dimer formed in the absence of actin. To better characterize the role of the Vt strap and carboxyl terminus (CT) in actin binding, Vt self-association, and actin bundling, we employed smaller amino-terminal (NT) and CT deletions that do not perturb the structural integrity of Vt. Although both NT and CT deletions retain actin binding, removal of the CT hairpin (1061-1066) selectively impairs actin bundling in vitro. Moreover, expression of vinculin lacking the CT hairpin in vinculin knock-out murine embryonic fibroblasts affects the number of focal adhesions formed, cell spreading as well as cellular stiffening in response to mechanical force.     

Activation of vinculin induced by cholinergic stimulation regulates contraction of tracheal smooth muscle tissue

Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.     

The Interaction of Vinculin with Actin

Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion.     

Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein

The focal adhesion protein vinculin is an actin-binding protein involved in the mechanical coupling between the actin cytoskeleton and the extracellular matrix. An autoinhibitory interaction between the N-terminal head (Vh) and the C-terminal tail (Vt) of vinculin masks an actin filament side-binding domain in Vt. The binding of several proteins to Vh disrupts this intramolecular interaction and exposes the actin filament side-binding domain. Here, by combining kinetic assays and microscopy observations, we show that Vt inhibits actin polymerization by blocking the barbed ends of actin filaments. In low salt conditions, Vt nucleates actin filaments capped at their barbed ends. We determined that the interaction between vinculin and the barbed end is characterized by slow association and dissociation rate constants. This barbed end capping activity requires C-terminal amino acids of Vt that are dispensable for actin filament side binding. Like the side-binding domain, the capping domain of vinculin is masked by an autoinhibitory interaction between Vh and Vt. In contrast to the side-binding domain, the capping domain is not unmasked by the binding of a talin domain to Vh and requires the dissociation of an additional autoinhibitory interaction. Finally, we show that vinculin and the formin mDia1, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends.     

Cardiomyopathy Mutations in Metavinculin Disrupt Regulation of Vinculin-Induced F-Actin Assemblies

Debilitating heart conditions, notably dilated and hypertrophic cardiomyopathies (CMs), are associated with point mutations in metavinculin, a larger isoform of the essential cytoskeletal protein vinculin. Metavinculin is co-expressed with vinculin at sub-stoichiometric ratios in cardiac tissues. CM mutations in the metavinculin tail domain (MVt) occur within the extra 68-residue insert that differentiates it from the vinculin tail domain (Vt). Vt binds actin filaments (F-actin) and promotes vinculin dimerization to bundle F-actin into thick fibers. While MVt binds to F-actin in a similar manner to Vt, MVt is incapable of F-actin bundling and inhibits Vt-mediated F-actin bundling. We performed F-actin co-sedimentation and negative-stain EM experiments to dissect the coordinated roles of metavinculin and vinculin in actin fiber assembly and the effects of three known metavinculin CM mutations. These CM mutants were found to weakly induce the formation of disordered F-actin assemblies. Notably, they fail to inhibit Vt-mediated F-actin bundling and instead promote formation of large assemblies embedded with linear bundles. Computational models of MVt bound to F-actin suggest that MVt undergoes a conformational change licensing the formation of a protruding sub-domain incorporating the insert, which sterically prevents dimerization and bundling of F-actin by Vt. Sub-domain formation is destabilized by CM mutations, disrupting this inhibitory mechanism. These findings provide new mechanistic insights into the ability of metavinculin to tune actin organization by vinculin and suggest that dysregulation of this process by CM mutants could underlie their malfunction in disease.     

Crystal structure of human vinculin

Alterations in the actin cytoskeleton following the formation of cell-matrix and cell-cell junctions are orchestrated by vinculin. Vinculin associates with a large number of cytoskeletal and signaling proteins, and this flexibility is thought to contribute to rapid dissociation and reassociations of adhesion complexes. Intramolecular interactions between vinculin's head (Vh) and tail (Vt) domains limit access of its binding sites for other adhesion proteins. While the crystal structures of the Vh and Vt domains are known, these domains represent less than half of the entire protein and are separated by a large central region of unknown structure and function. Here we report the crystal structure of human full-length vinculin to 2.85 A resolution. In its resting state, vinculin is a loosely packed collection of alpha-helical bundles held together by Vh-Vt interactions. The three new well ordered alpha-helical bundle domains are similar in their structure to either Vh (Vh2 and Vh3) or to Vt (Vt2) and their loose packing provides the necessary flexibility that allows vinculin to interact with its various protein partners at sites of cell adhesion.     

High-affinity interaction of vinculin with actin filaments in vitro

Immunofluorescence and microinjection experiments have shown that vinculin (molecular weight 130,000) is localized at adhesion plaques of fibroblasts spread on a solid substrate. We found that this protein affects actin filament assembly and interactions in vitro at substoichiometric levels. Vinculin inhibits the rate of actin polymerization under conditions that limit nuclei formation, indicating an effect on the filament elongation step of the reaction. Vinculin also reduces actin filament-filament interaction measured with a low-shear viscometer. Scatchard plot analysis of the binding of ³H-labeled vinculin to actin filaments showed that there is one high-affinity binding site (dissociation constant = 20 nM) for every 1,500–2,000 actin monomers. These results suggest that vinculin interacts with a specific site located at the growing ends of actin filaments in a cytochalasin-like manner, a property consistent with its proposed function as a linkage protein between filaments and the plasma membrane.     

Vinculin tail conformation and self-association is independent of pH and H906 protonation

Vinculin is a highly conserved cytoskeletal protein that localizes to sites of cell adhesion. The tail domain of vinculin (Vt) forms tight autoinhibitory interactions with the head domain and down-regulates vinculin function by obscuring ligand binding sites. Ligand binding is required for both vinculin activation and function, and one of vinculin's primary roles as a cell adhesion protein involves its ability to link the Actin cytoskeleton to the cell membrane. Vt can bind F-Actin and phosphoinositol 4,5-bisphosphate, and association with these ligands has been reported to cause a conformational change in Vt. Moreover, a single histidine residue, H906, was reported to be critical for both a pH dependent conformational change and pH dependent self-association. In this study, we investigate the role of pH on Vt structure and self-association. In contrast to earlier observations, our studies do not support a significant alteration in Vt conformation over this pH range. Moreover, while we identify a site of Vt dimerization, similar to that observed previously by X-ray crystallography, the weak K(d) (approximately 300 microM) determined for Vt self-association does not differ significantly between pH 5.5 and pH 7.5.     

The C-terminal tail domain of metavinculin, vinculin's splice variant, severs actin filaments

Vinculin and its splice variant, metavinculin (MV), are key elements of multiple protein assemblies linking the extracellular matrix to the actin cytoskeleton. Vinculin is expressed ubiquitously, whereas MV is mainly expressed in smooth and cardiac muscle tissue. The only difference in amino acid sequence between the isoforms is a 68-residue insert in the C-terminal tail domain of MV (MVt). Although the functional role of this insert remains elusive, its importance is exemplified by point mutations that are associated with dilated and hypertrophic cardiomyopathy. In vinculin, the actin binding site resides in the tail domain. In this paper, we show that MVt binds actin filaments similarly to the vinculin tail domain. Unlike its splice variant, MVt did not bundle actin filaments. Instead, MVt promoted severing of actin filaments, most efficiently at substoichiometric concentrations. This surprising and seemingly contradictory alteration of vinculin function by the 68-residue insert may be essential for modulating compliance of vinculin-induced actin bundles when exposed to rapidly increasing external forces.     

Three-dimensional structure of vinculin bound to actin filaments

Vinculin plays a pivotal role in cell adhesion and migration by providing the link between the actin cytoskeleton and the transmembrane receptors, integrin and cadherin. We used a combination of electron microscopy, computational docking, and biochemistry to provide an atomic model of how the vinculin tail binds actin filaments. The vinculin tail actin binding site comprises two distinct regions. One of these regions is exposed in the full-length autoinhibited conformation of vinculin, whereas the second site is sterically occluded by vinculin's N-terminal domain. The partial accessibility of the F-actin binding site in the autoinhibited full-length vinculin structure suggests that F-actin can act as part of a combinatorial input framework with other binding partners such as alpha-catenin or talin to induce vinculin head-tail dissociation, thus promoting vinculin activation. Furthermore, binding to F-actin potentiates a local rearrangement in the vinculin tail that in turn promotes vinculin dimerization and, hence, formation of actin bundles.     

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr¹⁰⁶⁵ on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr¹⁰⁶⁵ in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr¹⁰⁶⁵ (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr¹⁰⁶⁵ phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr¹⁰⁶⁵ phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.     

Evidence for direct binding of vinculin to actin filaments

The interaction of vinculin with actin filaments was investigated by methods which exclude interference by contaminating proteins which may occur in vinculin preparations. Vinculin which was blotted from SDS-polyacrylamide gels onto nitrocellulose, was stained specifically by fluorescently labeled polymeric actin (100 mM KCl, 2 mM MgCl2). Vinculin which was purified from alpha-actinin and an actin polymerization-inhibiting protein (HA1), was found to be cosedimented with polymeric actin. Maximally one vinculin molecule was cosedimented per one hundred actin filament subunits. Half maximal binding of vinculin was observed at about 0.25 microM free vinculin. Vinculin could be replaced from actin by the addition of tropomyosin.     

Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin

Vinculin, a major structural component of vertebrate cell-cell and cell- matrix adherens junctions, has been found to interact with several other junctional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli. In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin filaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893-1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction. In fixed and permeabilized cells (cell models), as well as after microinjection, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments. Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures.     

Vinculin Force-Sensitive Dynamics at Focal Adhesions Enable Effective Directed Cell Migration

Cell migration is a complex process, requiring coordination of many subcellular processes including membrane protrusion, adhesion, and contractility. For efficient cell migration, cells must concurrently control both transmission of large forces through adhesion structures and translocation of the cell body via adhesion turnover. Although mechanical regulation of protein dynamics has been proposed to play a major role in force transmission during cell migration, the key proteins and their exact roles are not completely understood. Vinculin is an adhesion protein that mediates force-sensitive processes, such as adhesion assembly under cytoskeletal load. Here, we elucidate the mechanical regulation of vinculin dynamics. Specifically, we paired measurements of vinculin loads using a Förster resonance energy transfer-based tension sensor and vinculin dynamics using fluorescence recovery after photobleaching to measure force-sensitive protein dynamics in living cells. We find that vinculin adopts a variety of mechanical states at adhesions, and the relationship between vinculin load and vinculin dynamics can be altered by the inhibition of vinculin binding to talin or actin or reduction of cytoskeletal contractility. Furthermore, the force-stabilized state of vinculin required for the stabilization of membrane protrusions is unnecessary for random migration, but is required for directional migration along a substrate-bound cue. These data show that the force-sensitive dynamics of vinculin impact force transmission and enable the mechanical integration of subcellular processes. These results suggest that the regulation of force-sensitive protein dynamics may have an underappreciated role in many cellular processes.     

The lack of interaction between vinculin and actin

Vinculin was purified from chicken gizzard by a modification of the method of Feramisco and Burridge [1980; J Biol Chem 255:1194]. Vinculin did not alter the viscosity of actin as measured in an Ostwald viscometer, nor did it affect actin polymerization as measured with the fluorescent NBD-actin assay. Sedimentation experiments demonstrated that vinculin did not bind to actin, and electron microscopy of negatively stained specimens indicated that vinculin did not aggregate actin filaments into bundles. These results suggest that vinculin, by itself, does not interact with actin at least under commonly used conditions to assay actin-protein interactions in vitro.     

Backbone 1H, 13C, and 15N NMR assignments of the tail domain of vinculin

Vinculin is an essential protein involved in linking the actin cytoskeleton to sites of cell-cell and cell-matrix adhesion. Here we report the majority of the backbone ¹H_N, ¹⁵N, ¹³C_α, ¹³CO, and side chain ¹³C_β NMR resonance assignments of the actin binding tail domain of vinculin (Vt).     

The vinculin binding sites of talin and alpha-actinin are sufficient to activate vinculin

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.     

Mechano-coupling and regulation of contractility by the vinculin tail domain

Vinculin binds to multiple focal adhesion and cytoskeletal proteins and has been implicated in transmitting mechanical forces between the actin cytoskeleton and integrins or cadherins. It remains unclear to what extent the mechano-coupling function of vinculin also involves signaling mechanisms. We report the effect of vinculin and its head and tail domains on force transfer across cell adhesions and the generation of contractile forces. The creep modulus and the adhesion forces of F9 mouse embryonic carcinoma cells (wild-type), vinculin knock-out cells (vinculin −/−), and vinculin −/− cells expressing either the vinculin head domain, tail domain, or full-length vinculin (rescue) were measured using magnetic tweezers on fibronectin-coated super-paramagnetic beads. Forces of up to 10 nN were applied to the beads. Vinculin −/− cells and tail cells showed a slightly higher incidence of bead detachment at large forces. Compared to wild-type, cell stiffness was reduced in vinculin −/− and head cells and was restored in tail and rescue cells. In all cell lines, the cell stiffness increased by a factor of 1.3 for each doubling in force. The power-law exponent of the creep modulus was force-independent and did not differ between cell lines. Importantly, cell tractions due to contractile forces were suppressed markedly in vinculin −/− and head cells, whereas tail cells generated tractions similar to the wild-type and rescue cells. These data demonstrate that vinculin contributes to the mechanical stability under large external forces by regulating contractile stress generation. Furthermore, the regulatory function resides in the tail domain of vinculin containing the paxillin-binding site.     

A Molecular Dynamics Investigation of Vinculin Activation

Vinculin activation plays a critical role in focal adhesion initiation and formation. In its native state, vinculin is in an autoinhibitory conformation in which domain 1 prevents interaction of the vinculin tail domain with actin by steric hindrance. Once activated, vinculin is able to interact with both actin and talin. Several hypotheses have been put forth addressing the mechanisms of vinculin activation. One set of studies suggests that vinculin interaction with talin is sufficient to cause activation, whereas another set of studies suggests that a simultaneous interaction with several binding partners is necessary to achieve vinculin activation. Using molecular-dynamics (MD) simulations, we investigate the mechanisms of vinculin activation and suggest both a trajectory of conformational changes leading to vinculin activation, and key structural features that are likely involved in stabilizing the autoinhibited conformation. Assuming that the simultaneous interaction of vinculin with both actin and talin causes a stretching force on vinculin, and that vinculin activation results from a removal of steric hindrance blocking the actin-binding sites, we simulate with MD the stretching and activation of vinculin. The MD simulations are further confirmed by normal-mode analysis and simulation after residue modification. Taken together, the results of these simulations suggest that bending of the vinculin-binding-site region in vinculin away from the vinculin tail is the likely trajectory of vinculin activation.