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1.  The domain structure of talin: Residues 1815–1973 form a five-helix bundle containing a cryptic vinculin-binding site 
Febs Letters  2010;584(11):2237-2241.
Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. Its rod region consists of a series of helical bundles. Here we show that residues 1815–1973 form a 5-helix bundle, with a topology unique to talin which is optimally suited for formation of a long rod such as talin. This is much more stable than the 4-helix (1843–1973) domain described earlier and as a result its vinculin binding sequence is inaccessible to vinculin at room temperature, with implications for the overall mechanism of the talin-vinculin interaction.
Structured summary
MINT-7722300, MINT-7760951: Talin-1 (uniprotkb:P26039) and Vinculin (uniprotkb:P12003) bind (MI:0407) by molecular sieving (MI:0071)
doi:10.1016/j.febslet.2010.04.028
PMCID: PMC2887493  PMID: 20399778
HSQC, heteronuclear single quantum coherence; SCOP, structural classification of proteins; VBS, vinculin-binding site; Talin; Vinculin; NMR; Domain structure; Helical bundle
2.  RIAM and Vinculin Binding to Talin Are Mutually Exclusive and Regulate Adhesion Assembly and Turnover* 
The Journal of Biological Chemistry  2013;288(12):8238-8249.
Background: Talin mediates RIAM-dependent integrin activation and binds vinculin, which stabilizes adhesions.
Results: Structural and biochemical data show that vinculin inhibits RIAM binding to the compact N-terminal region of the talin rod, a region essential for focal adhesion assembly.
Conclusion: Talin·RIAM complexes activate integrins at the leading edge, whereas talin·vinculin promotes adhesion maturation.
Significance: Talin changes partners in response to force-induced conformational change.
Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1–R13) organized into a compact cluster of four-helix bundles (R2–R4) within a linear chain of five-helix bundles. Nine of the bundles contain vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit vinculin to stabilize the complex.
doi:10.1074/jbc.M112.438119
PMCID: PMC3605642  PMID: 23389036
Adhesion; Cell Biology; Integrins; Nuclear Magnetic Resonance; Structural Biology; RIAM; Focal Adhesions; Talin; Vinculin
3.  How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism 
PLoS Computational Biology  2008;4(2):e24.
Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds.
Author Summary
For cell survival, most eukaryotic cells need to be mechanically anchored to their environment. This is done by transmembrane proteins, including integrins, which externally bind to the extracellular matrix and on the cell interior to the contractile cytoskeleton via scaffolding proteins. One essential scaffolding protein is talin, which binds to integrins via its head and to the cytoskeletal filament f-actin via its rodlike tail. As cells apply tensile forces to newly formed adhesion sites, proteins that are part of such force-bearing networks get stretched and might change their structure and thus function. One of many proteins that are recruited to newly formed adhesions is vinculin, and vinculin recruitment is upregulated by tensile mechanical force—but how? Since talin's vinculin binding sites are buried in its native structure, we used steered molecular dynamics here to derive a high resolution structural model of how tensile mechanical forces might activate talin's vinculin binding sites. Once tensile forces break up the talin rod into helix subbundles, an event that we find here to constitute the main energy barrier, we propose how the strain-induced gradual exposure of the vinculin-binding helices finally allows for their activation and enables helix swapping with the vinculin head.
doi:10.1371/journal.pcbi.0040024
PMCID: PMC2242828  PMID: 18282082
4.  How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism 
PLoS Computational Biology  2008;4(2):e24.
Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds.
Author Summary
For cell survival, most eukaryotic cells need to be mechanically anchored to their environment. This is done by transmembrane proteins, including integrins, which externally bind to the extracellular matrix and on the cell interior to the contractile cytoskeleton via scaffolding proteins. One essential scaffolding protein is talin, which binds to integrins via its head and to the cytoskeletal filament f-actin via its rodlike tail. As cells apply tensile forces to newly formed adhesion sites, proteins that are part of such force-bearing networks get stretched and might change their structure and thus function. One of many proteins that are recruited to newly formed adhesions is vinculin, and vinculin recruitment is upregulated by tensile mechanical force—but how? Since talin's vinculin binding sites are buried in its native structure, we used steered molecular dynamics here to derive a high resolution structural model of how tensile mechanical forces might activate talin's vinculin binding sites. Once tensile forces break up the talin rod into helix subbundles, an event that we find here to constitute the main energy barrier, we propose how the strain-induced gradual exposure of the vinculin-binding helices finally allows for their activation and enables helix swapping with the vinculin head.
doi:10.1371/journal.pcbi.0040024
PMCID: PMC2242828  PMID: 18282082
5.  Vinculin controls focal adhesion formation by direct interactions with talin and actin 
The Journal of Cell Biology  2007;179(5):1043-1057.
Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.
doi:10.1083/jcb.200703036
PMCID: PMC2099183  PMID: 18056416
6.  Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation 
Scientific Reports  2014;4:4610.
The force-dependent interaction between talin and vinculin plays a crucial role in the initiation and growth of focal adhesions. Here we use magnetic tweezers to characterise the mechano-sensitive compact N-terminal region of the talin rod, and show that the three helical bundles R1–R3 in this region unfold in three distinct steps consistent with the domains unfolding independently. Mechanical stretching of talin R1–R3 enhances its binding to vinculin and vinculin binding inhibits talin refolding after force is released. Mutations that stabilize R3 identify it as the initial mechano-sensing domain in talin, unfolding at ∼5 pN, suggesting that 5 pN is the force threshold for vinculin binding and adhesion progression.
doi:10.1038/srep04610
PMCID: PMC3980218  PMID: 24714394
7.  Raver1 interactions with Vinculin and RNA Suggest a Feed-Forward Pathway in Directing mRNA to Focal Adhesions 
SUMMARY
The translational machinery of the cell re-localizes to focal adhesions following the activation of integrin receptors. This response allows for rapid, local production of components needed for adhesion complex assembly and signaling. Vinculin links focal adhesions to the actin cytoskeleton following its activation by integrin signaling, which severs intramolecular interactions of the vinculin head and tail (Vt) domains. Our vinculin:raver1 crystal structures and binding studies show that activated Vt selectively interacts with one of the three RNA recognition motifs (RRM) of raver1, that the vinculin:raver1 complex binds to F-actin, and that raver1 binds selectively to RNA, including a sequence found in vinculin mRNA. Further, mutation of residues that mediate interaction of raver1 with vinculin abolish their co-localization in cells. These findings suggest a feed-forward model where vinculin activation at focal adhesions provides a scaffold for recruitment of raver1 and its mRNA cargo to facilitate the production of components of adhesion complexes.
doi:10.1016/j.str.2009.04.010
PMCID: PMC2811071  PMID: 19523901
focal adhesion; actin cytoskeleton; crystallography; RNP motif; RNA binding
8.  Structures linking microfilament bundles to the membrane at focal contacts 
The Journal of Cell Biology  1993;122(2):485-496.
We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin- membrane interactions at focal contacts, including beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.
PMCID: PMC2119644  PMID: 7686554
9.  The C-terminal tail domain of metavinculin, vinculin’s splice variant, severs actin filaments 
The Journal of Cell Biology  2012;197(5):585-593.
Relative to vinculin, a unique 68-residue insert in the C-terminal tail of metavinculin results in a loss of actin filament-bundling activity but gain of actin filament-severing activity.
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.
doi:10.1083/jcb.201111046
PMCID: PMC3365496  PMID: 22613835
10.  Vinculin regulation of F-actin bundle formation 
Cell Adhesion & Migration  2013;7(2):219-225.
Vinculin is an essential cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples transmembrane proteins 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) has the ability to both bind and bundle actin filaments. Binding to actin induces a conformational change in Vt believed to promote formation of a Vt dimer that is able to crosslink actin filaments. We have recently provided additional evidence for the actin-induced Vt dimer and have shown that the vinculin carboxyl (C)-terminal hairpin is critical for both the formation of the Vt dimer and for bundling F-actin. We have also demonstrated the importance of the C-terminal hairpin in cells as deletion of this region impacts both adhesion properties and force transduction. Intriguingly, we have identified bundling deficient variants of vinculin that show different cellular phenotypes. These results suggest additional role(s) for the C-terminal hairpin, distinct from its bundling function. In this commentary, we will expand on our previous findings and further investigate these actin bundling deficient vinculin variants.
doi:10.4161/cam.23184
PMCID: PMC3954036  PMID: 23307141
vinculin; dimerization; F-actin bundling; focal adhesion; scaffold
11.  Vinculin Motion Modes Analysis with Elastic Network Model 
Vinculin is an important protein for the linkage between adhesion molecules and the actin cytoskeleton. The activation mechanism of vinculin is still controversial. In order to provide useful information for a better understanding of its activation, we analyze the motion mode of vinculin with elastic network model in this work. The results show that, to some extent, the five domains will present structural rigidity in the motion process. The differences between the structure fluctuations of these domains are significant. When vinculin interacted with other partners, the central long alpha-helix of the first domain becomes bent. This bending deformation can weaken the interaction between the first domain and the tail domain. This motion mode of the first domain is in good agreement with the information extracted from some realistic complex structures. With the aid of the anisotropy elastic network mode, we analyze the motion directions of these domains. The fourth domain has a rotational motion. This rotation is favorable for the releasing of the tail domain from the pincer-like clamp, which is formed by the first and the third domain. All these motion modes are an inherent feature of the structure, and these modes mainly depend on the topology character of the structure.
doi:10.3390/ijms13010208
PMCID: PMC3269682  PMID: 22312248
vinculin; motion mode; elastic network model; activation mechanism
12.  Mechano-Coupling and Regulation of Contractility by the Vinculin Tail Domain 
Biophysical Journal  2007;94(2):661-670.
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.
doi:10.1529/biophysj.107.108472
PMCID: PMC2481521  PMID: 17890382
13.  Identification of a talin binding site in the cytoskeletal protein vinculin 
The Journal of Cell Biology  1989;109(6):2917-2927.
Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746- 867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.
PMCID: PMC2115903  PMID: 2512301
14.  Molecular Mechanics of the α-Actinin Rod Domain: Bending, Torsional, and Extensional Behavior 
PLoS Computational Biology  2009;5(5):e1000389.
α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain.
Author Summary
The cell interacts with its environment in both biochemical and mechanical ways. In this study we explore one of the ways in which the cell interacts mechanically with its environment. α-Actinin is a cytoskeletal crosslinker: it functions to scaffold the cytoskeletal actin filaments that provide mechanical reinforcement to the cell. In its functional environment α-actinin is exposed to a multitude of mechanical stresses as it attaches itself to a dynamic network of actin filaments. The actin filaments extend, rotate, and bend the α-actinin crosslinkers. In this study we employ molecular dynamics techniques to understand the structural characteristics of α-actinin that underlie its ability to provide a scaffold in such a stressed environment. We analyzed the natural frequencies of α-actinin and simulated force-induced bending, extension, and twisting. Our results suggest that α-actinin has structural flexibility facilitating crosslinking in a dynamic environment and also structural rigidity stabilizing the linkage in the stressed environment. We have discovered novel natural bending movements of the rod domain that enhance its function as a crosslinker. We have also demonstrated the specific structural characteristics of α-actinin that give it the previously suggested property of having partial flexibility. Our results enhance the understanding of structural mechanics in the cytoskeletal molecules.
doi:10.1371/journal.pcbi.1000389
PMCID: PMC2676514  PMID: 19436721
15.  The Interaction of Vinculin with Actin 
PLoS Computational Biology  2013;9(4):e1002995.
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.
Author Summary
The interface between a cell and its substrate is strengthened by the formation of focal adhesions. In this study molecular dynamics simulations are used to explore the connectivity of one focal adhesion forming protein, vinculin, and the cytoskeletal filament, F-actin. The simulations demonstrate: (1) that vinculin can link along F-actin at these focal adhesions when it adopts an open conformation, (2) that the vinculin tail (Vt) can bind F-actin at its barbed-end preventing actin polymerization, (3) that vinculin can adopt two open conformations, and (4) that the second open conformation is necessary for vinculin to cap the actin filament. The results suggest that vinculin can act as a variable switch, changing its shape and the nature of its interaction with F-actin depending on the level of stress seen at a focal adhesion. Under the highest stress vinculin would adopt the open II conformation and link anywhere on F-actin, even its barbed-end. Under less stress vinculin could adopt the open I conformation and bind along F-actin. And under minimal stress vinculin could adopt its closed conformation. This variability allows for vinculin to truly function as the cell's mechanical reinforcing agent.
doi:10.1371/journal.pcbi.1002995
PMCID: PMC3635976  PMID: 23633939
16.  Actomyosin-dependent formation of the mechanosensitive talin–vinculin complex reinforces actin anchoring 
Nature Communications  2014;5:3095.
The force generated by the actomyosin cytoskeleton controls focal adhesion dynamics during cell migration. This process is thought to involve the mechanical unfolding of talin to expose cryptic vinculin-binding sites. However, the ability of the actomyosin cytoskeleton to directly control the formation of a talin–vinculin complex and the resulting activity of the complex are not known. Here we develop a microscopy assay with pure proteins in which the self-assembly of actomyosin cables controls the association of vinculin to a talin-micropatterned surface in a reversible manner. Quantifications indicate that talin refolding is limited by vinculin dissociation and modulated by the actomyosin network stability. Finally, we show that the activation of vinculin by stretched talin induces a positive feedback that reinforces the actin–talin–vinculin association. This in vitro reconstitution reveals the mechanism by which a key molecular switch senses and controls the connection between adhesion complexes and the actomyosin cytoskeleton.
The interaction between focal adhesion proteins vinculin and talin is stimulated by mechanical stretching. Here the authors reconstitute actomyosin-dependent stretching of talin in vitro, and show that the resulting activation of vinculin reinforces anchoring of the adhesion complex to actin.
doi:10.1038/ncomms4095
PMCID: PMC3916842  PMID: 24452080
17.  Vinculin interaction with permeabilized cells: disruption and reconstitution of a binding site 
The Journal of Cell Biology  1986;103(2):641-648.
Fluorescently labeled vinculin binds to focal contact areas in permeabilized cells independent of actin (Avnur, Z., J. V. Small, and B. Geiger, 1983, J. Cell Biol., 96:1622-1630), but the nature of the binding site is unknown. In this study we have examined the interaction of vinculin with these sites in permeabilized L6 myoblasts to define conditions that perturb the binding and subsequently to reconstitute it. Mild treatment with low concentrations of protease prevents vinculin incorporation without gross changes in the cytoskeleton or extensive protein breakdown. Exposure to buffers of moderate ionic strength also reduces subsequent vinculin binding without large morphological effects. These extraction conditions were used to obtain a fraction from gizzard which was able to restore the vinculin localization. Talin, actin, and vinculin itself were able to alter the binding of labeled vinculin to permeabilized cells and each also interacted with vinculin in gel overlays; however, they were unable to reconstitute the binding site in treated permeabilized cells. The results show a requirement for an as yet unidentified protein to capacitate vinculin binding to focal contact sites and suggest that this protein is peripheral and interacts directly with the binding site.
PMCID: PMC2113843  PMID: 3090053
18.  CAS directly interacts with vinculin to control mechanosensing and focal adhesion dynamics 
Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS–vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS–vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS–vinculin interaction in mechanosensing and focal adhesion dynamics.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-013-1450-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00018-013-1450-x
PMCID: PMC3901934  PMID: 23974298
CAS; Focal adhesions; Mechanosensing; Vinculin; Src; Traction forces
19.  The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin 
The Journal of Cell Biology  1995;129(4):1155-1164.
The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L- selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L- selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha- actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L- selectin cytoplasmic domain. In contrast, a deletion mutant of L- selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L- selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L- selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.
PMCID: PMC2120488  PMID: 7538138
20.  The Phosphorylation of Vinculin on Tyrosine Residues 100 and 1065, Mediated by Src Kinases, Affects Cell Spreading 
Molecular Biology of the Cell  2004;15(9):4234-4247.
Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin is tyrosine phosphorylated in platelets spread on fibrinogen and that the phosphorylation is Src kinases dependent. The phosphorylation of vinculin on tyrosine was reconstituted in vanadate treated COS-7 cells coexpressing c-Src. The tyrosine phosphorylation sites in vinculin were mapped to residues 100 and 1065. A phosphorylation-specific antibody directed against tyrosine residue 1065 reacted with phosphorylated platelet vinculin but failed to react with vinculin from unstimulated platelet lysates. Tyrosine residue 1065 located in the vinculin tail domain was phosphorylated by c-Src in vitro. When phosphorylated, the vinculin tail exhibited significantly less binding to the vinculin head domain than the unphosphorylated tail. In contrast, the phosphorylation did not affect the binding of vinculin to actin in vitro. A double vinculin mutant protein Y100F/Y1065F localized to focal adhesion plaques. Wild-type vinculin and single tyrosine phosphorylation mutant proteins Y100F and Y1065F were significantly more effective at rescuing the spreading defect of vinculin null cells than the double mutant Y100F/Y1065F. The phosphorylation of vinculin by Src kinases may be one mechanism by which these kinases regulate actin filament assembly and cell spreading.
doi:10.1091/mbc.E04-03-0264
PMCID: PMC515355  PMID: 15229287
21.  The metavinculin tail domain directs constitutive interactions with raver1 and vinculin RNA 
Journal of molecular biology  2012;422(5):10.1016/j.jmb.2012.06.015.
Vinculin is a key regulator of the attachment of the actin cytoskeleton to the cell membrane at cellular adhesion sites that is crucial for processes like cell motility and migration, development, survival, and wound healing. Vinculin loss results in embryonic lethality, cardiovascular diseases, and cancer. Its tail domain, Vt, is crucial for vinculin activation and focal adhesion turnover and binds to the actin cytoskeleton and acidic phospholipids upon which it unfurls. The RNA binding protein raver1 regulates the assembly of focal adhesions transcriptionally by binding to vinculin. The muscle-specific splice form, metavinculin, is characterized by a 68 residue insert in the tail domain (MVt) and correlates with hereditary idiopathic dilated cardiomyopathy. Here we report that metavinculin can bind to raver1 in its inactive state. Our crystal structure explains this permissivity, where an extended coil unique to MVt is unfurled in the MVtΔ954:raver1 complex structure. Our binding assays show that raver1 forms a ternary complex with MVt and vinculin mRNA. These findings suggest that the metavinculin:raver1:RNA complex is constitutively recruited to adhesion complexes.
doi:10.1016/j.jmb.2012.06.015
PMCID: PMC3835166  PMID: 22709580
adherens junction; cardiomyopathy; focal adhesion; RRM domain; RNA binding
22.  The Crystal Structures of Dystrophin and Utrophin Spectrin Repeats: Implications for Domain Boundaries 
PLoS ONE  2012;7(7):e40066.
Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.
doi:10.1371/journal.pone.0040066
PMCID: PMC3401230  PMID: 22911693
23.  Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin 
The Journal of Cell Biology  1994;126(5):1231-1240.
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.
PMCID: PMC2120156  PMID: 8063860
24.  An interaction between alpha-actinin and the beta 1 integrin subunit in vitro 
The Journal of Cell Biology  1990;111(2):721-729.
A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1- integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha- actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha- actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.
PMCID: PMC2116186  PMID: 2116421
25.  A Helix Replacement Mechanism Directs Metavinculin Functions 
PLoS ONE  2010;5(5):e10679.
Cells require distinct adhesion complexes to form contacts with their neighbors or the extracellular matrix, and vinculin links these complexes to the actin cytoskeleton. Metavinculin, an isoform of vinculin that harbors a unique 68-residue insert in its tail domain, has distinct actin bundling and oligomerization properties and plays essential roles in muscle development and homeostasis. Moreover, patients with sporadic or familial mutations in the metavinculin-specific insert invariably develop fatal cardiomyopathies. Here we report the high resolution crystal structure of the metavinculin tail domain, as well as the crystal structures of full-length human native metavinculin (1,134 residues) and of the full-length cardiomyopathy-associated ΔLeu954 metavinculin deletion mutant. These structures reveal that an α-helix (H1′) and extended coil of the metavinculin insert replace α-helix H1 and its preceding extended coil found in the N-terminal region of the vinculin tail domain to form a new five-helix bundle tail domain. Further, biochemical analyses demonstrate that this helix replacement directs the distinct actin bundling and oligomerization properties of metavinculin. Finally, the cardiomyopathy associated ΔLeu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1′ α-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions.
doi:10.1371/journal.pone.0010679
PMCID: PMC2873289  PMID: 20502710

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