A Conformational Switch in Vinculin Drives Formation and Dynamics of a Talin-Vinculin Complex at Focal Adhesions

Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin–cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or α-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd ∼ 10–7 is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells. Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin–cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or α-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd ∼ 10–7 is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells. Integrins mediate transmembrane connections between the actin cytoskeleton and extracellular matrix (1Neff N.T. Lowrey C. Decker C. Tovar A. Damsky C. Buck C. Horwitz A.F. J. Cell Biol. 1982; 95: 654-666Crossref PubMed Scopus (195) Google Scholar, 2Damsky C.H. Knudsen K.A. Bradley D. Buck C.A. Horwitz A.F. J. Cell Biol. 1985; 100: 1528-1539Crossref PubMed Scopus (156) Google Scholar). These connections are organized into discrete clusters such as focal complexes (3Hotchin N.A. Hall A. J. Cell Biol. 1995; 131: 1857-1865Crossref PubMed Scopus (371) Google Scholar) and focal adhesions (3Hotchin N.A. Hall A. J. Cell Biol. 1995; 131: 1857-1865Crossref PubMed Scopus (371) Google Scholar, 4Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 793-805Crossref PubMed Scopus (1869) Google Scholar) in adherent cells. Focal adhesions serve dual, opposing functions in cell motility, acting both in transmission of traction forces to generate displacement of the cell body and as anchors that resist detachment from the substratum (5Palecek S.P. Loftus J.C. Ginsberg M.H. Lauffenburger D.A. Horwitz A.F. Nature. 1997; 385: 537-540Crossref PubMed Scopus (1202) Google Scholar). Thus, dynamic regulation of focal adhesion structure, and the integrin-cytoskeleton associations contained therein, plays a central role in balancing adhesive and migratory stimuli in the cell. Two key proteins implicated in the physical connections between integrins and F-actin are the actin-binding proteins talin and vinculin. In vitro, talin binds to β integrin tails through its FERM domain (6Garcia-Alvarez B. de Pereda J.M. Calderwood D.A. Ulmer T.S. Critchley D. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) and induces conformational changes in integrin associated with increased binding to the extracellular matrix (7Calderwood D.A. Zent R. Grant R. Rees D.J. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). In cells, exposure of activated epitopes on β1 and β3 integrins and fibronectin binding are strongly inhibited by knockdown of talin expression (8Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (997) Google Scholar). Moreover, talin-1 null cell lines are deficient in the formation of mechanical linkages between fibronectin, β3 integrin, and the cytoskeleton as shown by loss of a transient molecular slip bond that can sustain up to 2 piconewtons of force (9Jiang G. Giannone G. Critchley D.R. Fukumoto E. Sheetz M.P. Nature. 2003; 424: 334-337Crossref PubMed Scopus (368) Google Scholar). Interestingly, the seminal report that talin binds directly to α5β1 integrin also demonstrated that integrin, talin, and vinculin form a ternary complex in vitro (10Horwitz A. Duggan K. Buck C. Beckerle M.C. Burridge K. Nature. 1986; 320: 531-533Crossref PubMed Scopus (828) Google Scholar). This observation implicated vinculin as part of the integrin-cytoskeleton linkage. Evidence that vinculin participates in transmembrane connections in vivo is based on defects in fibronectin-based adhesion induced by genetic disruption of vinculin. Vinculin null cells show decreased strength of adhesion to fibronectin surfaces (11Xu W. Baribault H. Adamson E.D. Development (Camb.). 1998; 125: 327-337Crossref PubMed Google Scholar, 12Coll J.-L. Ben-Ze'ev A. Ezzell R.M. Rodriguez Fernandez J.L. Baribault H. Oshima R.G. Adamson E.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9161-9165Crossref PubMed Scopus (178) Google Scholar), and integrin-cytoskeleton linkages are more easily disrupted by the application of mechanical force in the absence of vinculin (13Goldmann W.H. Ingber D.E. Biochem. Biophys. Res. Commun. 2002; 290: 749-755Crossref PubMed Scopus (81) Google Scholar, 14Alenghat F.J. Fabry B. Tsai K.Y. Goldmann W.H. Ingber D.E. Biochem. Biophys. Res. Commun. 2000; 277: 93-99Crossref PubMed Scopus (182) Google Scholar). Despite these intriguing findings, there is no direct evidence for a ternary complex of talin-vinculin-integrin in cells, and it is not known how this putative complex is regulated. 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Full Text Full Text PDF PubMed Scopus Google Scholar), and more of talin associated with exposure in the of E. B. J. Critchley D.R. (Camb.). Full Text Full Text PDF PubMed Scopus Google Scholar). it as to or talin for talin or an induced by vinculin In conformational changes in vinculin directly and to exposure of its and in focal adhesions of cells H. J. Cell Biol. PubMed Scopus Google Scholar). In this we the of HTI to the formation of a talin-vinculin complex in cells by the extent to which HTI regulates the recruitment of vinculin to talin or clusters in cells. we the by HTI the dynamic of talin, and of which are of vinculin at focal adhesions. The data that HTI of vinculin binding of talin to vinculin in vivo and as a complexes of vinculin and talin are not in an ectopic recruitment HTI is HTI by to Kd ∼ 10–7 is sufficient to the talin-binding site in vinculin and to induce We conclude that HTI plays an role in the of vinculin for talin in cells. In the of this in talin is not for vinculin to with talin, and HTI is the barrier to formation of talin-vinculin In we that vinculin HTI regulates the of time that vinculin and talin remain associated with focal adhesions the of these proteins is whereas the dynamics of vinculin in focal adhesions are not We conclude that regulation of HTI can the lifetime of complexes the of a focal was by and H. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The mutation was into by H. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) was by to of in the The site in the was to and a site was into the site the vinculin the site was to and a site was to the vinculin in of a the vinculin domain and by and from expression into the to and was into the a site and a site the talin in of was by was a of was by as a of with a site and a In to the these also contained ectopic a to of the on vinculin a was by of the vinculin with and to a for and in a This was into and The was and in the to The mutation H. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) was into by The was also into a and in the site to a vinculin deficient in both HTI and the talin-binding domain G. G. Nature. PubMed Scopus Google Scholar), was by the in with a to the in was to a site was by The talin a was for in the to generate HTI of the HTI in vinculin H. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in are as and and of talin and and of as H. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). binding, or was at with talin for at in a and was on with for at fluorescence in the was on a was at and at was and and a of on with and in a at on with fibronectin to and with for in to Vinculin null cells by Adamson and on in a which cell The was to and to to and to In and to in of the in and to and Vinculin null cells on or for to and to by of the in in for in in and for that in this was or vinculin null cells in and in for at The for as integrin of and from from and in the or or was from after fluorescence with both and was at for and enhanced in at the of cells and of in a for to was a at at from to to loss of The was on the in the form of a in The was to the of the by a with a focal and a with a focal a and with a with a and an by a at and with exposure to in the was for a of and contained time an and a slow The domain and of to or of of the recovery to the data as as through the recovery was and by a by also for in fluorescence induced by in by to fluorescence in a of the cell. for the of in these to or discrete focal adhesions. the and a recovery the is the fluorescence at time is the fluorescence is the fluorescence to after and from the contained focal adhesion or or fluorescence recovery at of the of to a was by a and at time which from was for for the at this was for with at fluorescence in of the recovery and in to the for of the vinculin In these the data are in with the of the data the and the of the data the the and of the The is as a The lines from the the and of the data that an are or to in the was to the as for the HTI in a was to between In the of vinculin and HTI was to of at at such as for the adhesion a with was for of these at of Vinculin HTI in of a with β1 and in the role of vinculin conformation in assembly of a ternary complex of talin, and β1 integrin in cells, we these interactions in the of an ectopic recruitment proteins from adhesions to an ectopic to the interaction of talin, and integrin from focal adhesion Using the S. 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Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). is that talin is activated by the or that of talin on the is sufficient to and are in the cell and are to into with the Thus, the and integrin, or activated The of to integrin binding is in as it of it into the of formation of talin-vinculin-integrin complexes in cells. β1 integrin and talin talin is and at at the autoinhibited vinculin is not to this These data a for of HTI in vinculin to binding of talin in cells. these for talin, we the recruitment with vinculin. did not induce of talin to In the HTI-deficient vinculin talin to the of talin focal adhesion was with the for integrin binding in this assay, HTI-deficient vinculin induced the formation of integrin ternary complexes at the wild-type vinculin to form this ternary complex, and of the talin-binding site in disrupted recruitment of both talin and β1 integrin and these that in vitro of interactions between talin and vinculin and between talin and β1 integrin the formation of a ternary complex between these proteins in cells. in this of ternary complex of vinculin its recruitment to ectopic complexes in of Vinculin HTI in the of Vinculin in Focal of HTI at focal adhesions be an in the of vinculin for its from the decreased of to with for Thus, the of HTI be to slow the of vinculin complexes from focal adhesions. this we the of HTI on the dynamics of vinculin and its focal adhesion by of these we vinculin null cells with wild-type vinculin or HTI and the focal adhesion dynamics of vinculin and of its talin, and α-actinin. adhesion proteins of adhesion to fibronectin to the of focal adhesion or to the fluorescence these the cells did not and and on a time with the the to focal adhesions in to cells these HTI of vinculin not focal adhesion or in to the in which HTI are H. 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PubMed Scopus Google Scholar), and we show that a in the for and In the mutation in vinculin talin binding of talin binding correlated with of the dynamics of vinculin at focal adhesions vinculin a recovery of which was the recovery of wild-type vinculin. with an role of exposure of in activated of the mutation rescued the slow of and the vinculin a recovery of of the for and vinculin a recovery of of the for the slow of both HTI-deficient vinculin and which the of vinculin dynamics in vinculin was to be by a conformational the autoinhibited the of the recovery of wild-type and on the mutation that vinculin from focal adhesion is by This for in focal adhesion residency time conformational between vinculin these proteins interactions in the focal Vinculin HTI the of but or in Focal vinculin is proposed to as a the increased residency time of vinculin be the dynamic of its focal adhesion is also HTI-deficient vinculin in the of focal adhesion we vinculin null cells with vinculin or and or In the of reduction of vinculin HTI no on paxillin residency time In the of wild-type or a of that paxillin not directly vinculin in this or that induce paxillin dynamics of vinculin In the of a in was in the of this was not talin in the of wild-type more its residency from to in Vinculin of Vinculin into with or and this we tested the extent to which vinculin conformation regulates the binding of vinculin and talin in cells. We that of vinculin HTI by Kd ∼ was sufficient to induce the formation of talin-vinculin-integrin complexes at ectopic in cells. In of talin to wild-type talin associated with β1 In the it therefore that talin conformation the of not a vinculin binding, whereas HTI constitutes an barrier for complex formation. the talin-vinculin complex that in ectopic not the that can between these we that it is that the interaction of HTI-deficient vinculin and talin sufficient to direct recruitment of as be for an interaction in the The from the ectopic recruitment an to a that ectopic clusters of integrin and talin by of integrins also to vinculin to of C. J. B. J. Cell. Biol. PubMed Scopus Google Scholar). with these show that is not a de for vinculin recruitment or vinculin of both is that not or mechanical that a role in and of talin for the autoinhibited conformation of vinculin. we this we remain that a conformation of talin of or to with In to the the ectopic recruitment evidence for the that HTI regulates the of vinculin for talin in Thus, mechanisms of HTI disruption such as a role in the of vinculin in cells, as C. Critchley D.R. Liddington R.C. Nature. PubMed Scopus Google Scholar, H. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HTI Vinculin Focal a for of vinculin at focal adhesions to be the that conformational of vinculin at of adhesion is a fluorescence of vinculin direct evidence that vinculin an actin-binding conformation at focal adhesions H. J. Cell Biol. PubMed Scopus Google Scholar). the conformation of vinculin is more in more dynamic adhesions such as or adhesions H. J. Cell Biol. 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PubMed Scopus Google Scholar), focal adhesion are also modulated by a between decreased of focal adhesion proteins and loss of mechanical of integrin-cytoskeleton linkages in null cells G. B. Sheetz M.P. J. 2003; PubMed Scopus Google Scholar). Thus, the of the adhesion, the of focal adhesion are to be to adhesion We tested the of HTI on the of vinculin and its to HTI is to regulation of vinculin of focal adhesion data the that a conformational from an to is directly for the of vinculin from the interactions that it to the focal adhesion This is on in the residency time of vinculin in focal adhesions is to the strength of exposure of the the talin-binding domain or the in focal adhesion residency in HTI-deficient vinculin. of binding in largely the focal adhesion dynamics of HTI-deficient vinculin. Thus, of interactions between and its focal adhesion through an mutation or a with is the force for vinculin dynamics in focal adhesions. of HTI is a for of vinculin from focal as the strength of HTI is to known binding of or Moreover, can from a such as a complex in vitro H. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of in Focal by the of HTI is to the of vinculin for its the HTI of vinculin increased the focal adhesion residency of talin, whereas paxillin and the of an of HTI-deficient vinculin on of or paxillin not vinculin binding it that vinculin binding is not to the of these proteins from the adhesion complex the of these Thus, a of the adhesion of paxillin or binds activated or interactions with vinculin are transient to adhesion In talin with activated its focal adhesion residency that talin the to with a more in the focal adhesion or a conformational that its to This is intriguing that binding induces conformational changes in talin that cryptic B. Liddington R.C. D. J. Critchley D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. B. J. Critchley D.R. (Camb.). 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Cell Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). are to be to how and the actin cytoskeleton with the focal adhesion and how it with to the focal adhesion to generate of the cell. We Adamson for the vinculin null cells, and for on the and for