Title: A Conformational Switch in Vinculin Drives Formation and Dynamics of a Talin-Vinculin Complex at Focal Adhesions
Abstract: 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. In vitro, purified vinculin and talin exhibit extremely weak interactions, which can be attributed to an autoinhibitory head-tail interaction (HTI) 2The abbreviations used are: HTI, head-tail interaction; Vhd1, vinculin head D1 domain (residues 1–258); Vt, vinculin tail domain (residues 884–1066); VBS, vinculin-binding site(s); Vh, vinculin head domain (residues 1–851); YFP, yellow fluorescent protein; HFFs, human foreskin fibroblasts; MES, 4-morpholineethanesulfonic acid; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein. in vinculin that allosterically blocks the talin-binding site (15Johnson R.P. Craig S.W. J. Biol. Chem. 1994; 269: 12611-12619Abstract Full Text PDF PubMed Google Scholar). These observations suggest the hypothesis that a conformational switch from an autoinhibited to an activated state of vinculin is required to form talin-vinculin complexes in cells. In vitro structural and biochemical studies have demonstrated that vinculin is tightly autoinhibited (Kd < 10–9) by the cooperative interaction of two low affinity intramolecular interfaces (vinculin head D1 domain (Vhd1)-vinculin tail domain (Vt) with Kd = ∼10–5 m and vinculin head D4 domain-Vt with Kd = ∼10–2 to 10–3 m) estimated from bimolecular interactions (16Bakolitsa C. Cohen D.M. Bankston L.A. Bobkov A.A. Cadwell G.W. Jennings L. Critchley D.R. Craig S.W. Liddington R.C. Nature. 2004; 430: 583-586Crossref PubMed Scopus (300) Google Scholar, 17Cohen D.M. Chen H. Johnson R.P. Choudhury B. Craig S.W. J. Biol. Chem. 2005; 280: 17109-17117Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). These data suggest a model in which two ligands act coordinately to disrupt the two head-tail interfaces and thereby promote vinculin activation. Alternatively, it has been suggested that activation of cryptic vinculin-binding sites (VBS) in talin is sufficient to disrupt the autoinhibitory HTI in vinculin (18Izard T. Evans G. Borgon R.A. Rush C.L. Bricogne G. Bois P.R.J. Nature. 2004; 427: 171-175Crossref PubMed Scopus (204) Google Scholar, 19Bois P.R.J. O'Hara B.P. Nietlispach D. Kirkpatrick J. Izard T. J. Biol. Chem. 2006; 281: 7228-7236Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Bass M.D. Patel B. Barsukov I.G. Fillingham I.J. Mason R. Smith B.J. Bagshaw C.R. Critchley D.R. Biochem. J. 2002; 362: 761-768Crossref PubMed Scopus (50) Google Scholar, 21Gingras A.R. Ziegler W.H. Frank R. Barsukov I.L. Roberts G.C. Critchley D.R. Emsley J. J. Biol. Chem. 2005; 280: 37217-37224Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Structural studies have revealed that binding of VBS peptides to Vhd1 (residues 1–258) induces a large conformational change that disrupts a major binding site for Vt (18Izard T. Evans G. Borgon R.A. Rush C.L. Bricogne G. Bois P.R.J. Nature. 2004; 427: 171-175Crossref PubMed Scopus (204) Google Scholar). Moreover, VBS3 peptide has been reported to bind with similar affinity to either Vhd1 or full-length vinculin (residues 1–1066) when these proteins are adsorbed onto a solid-phase substrate (22Bois P.R.J. Borgon R.A. Vonrhein C. Izard T. Mol. Cell. Biol. 2005; 14: 6112-6122Crossref Scopus (77) Google Scholar). In addition, VBS3 peptide alters the protease sensitivity of full-length vinculin in solution (22Bois P.R.J. Borgon R.A. Vonrhein C. Izard T. Mol. Cell. Biol. 2005; 14: 6112-6122Crossref Scopus (77) Google Scholar). These observations were interpreted to suggest that HTI makes no significant contribution to modulating the affinity of vinculin for the VBS sequence and therefore that the VBS sequences in talin and α-actinin constitute a sufficient mechanism for vinculin activation (19Bois P.R.J. O'Hara B.P. Nietlispach D. Kirkpatrick J. Izard T. J. Biol. Chem. 2006; 281: 7228-7236Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). VBS sequences are buried, however, in the context of intact talin (23Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (135) Google Scholar, 24Gingras A.R. Vogel K.P. Steinhoff H.J. Ziegler W.H. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Biochemistry. 2006; 45: 1805-1817Crossref PubMed Scopus (62) Google Scholar). Whereas weakly structured fragments of talin show enhanced affinity for Vhd1 compared with intact talin in vitro (25Patel B. Gingras A.R. Bobkov A.A. Fujimoto L.M. Zhang M. Liddington R.C. Mazzeo D. Emsley J. Roberts G.C.K. Barsukov I.L. Critchley D.R. J. Biol. Chem. 2006; 281: 7458-7467Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), well folded and measurably more stable domains of talin undergo structural rearrangement associated with VBS exposure only in the presence of Vhd1 (26Fillingham I. Gingras A.R. Papagrigoriou E. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Structure (Camb.). 2005; 13: 65-74Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). As such, it remains controversial as to whether VBS peptides or weakly structured talin fragments reflect bona fide conformations for talin or merely mimic an end state induced by vinculin binding. In contrast, conformational changes in vinculin have been directly visualized and linked to exposure of its actin- and vinexin-binding sites in focal adhesions of live cells (27Chen H. Cohen D.M. Choudhury D.M. Kioka N. Craig S.W. J. Cell Biol. 2005; 169: 459-470Crossref PubMed Scopus (140) Google Scholar). In this study, we have examined the functional relevance of HTI to the formation of a talin-vinculin complex in living cells by testing the extent to which HTI regulates the recruitment of vinculin to talin or talin-integrin clusters in cells. Furthermore, we have explored whether the biochemical constraints imposed by HTI affect the dynamic behavior of vinculin, talin, α-actinin, and paxillin, all of which are potential ligands of vinculin at focal adhesions. The data demonstrate that intramolecular HTI of vinculin outcompetes binding of talin to vinculin in vivo and that, as a consequence, complexes of vinculin and talin are not detectable in an ectopic recruitment assay unless HTI is partially attenuated. Reducing HTI by 100-fold to Kd ∼ 10–7 is sufficient to activate the talin-binding site in vinculin and to induce bimolecular complexes. We conclude that HTI plays an important role in modulating the affinity of vinculin for intact talin in cells. In the context of this system, prior change in talin structure is not required for vinculin to interact with talin, and HTI is the major barrier to formation of talin-vinculin complexes. In addition, we have found that vinculin HTI regulates the length of time that vinculin and talin remain associated with focal adhesions (i.e. the exchange rate of these proteins is decreased), whereas the dynamics of two other vinculin ligands in focal adhesions are not affected. We conclude that regulation of HTI can determine the lifetime of specific vinculin-based functional complexes within the longer lived architecture of a mature focal adhesion. Plasmids—pET30a/talin rod was provided by Dr. Stephen Lam. pET15b/YFP/V1–851 and pET15b/YFP/V1–258 were described previously (17Cohen D.M. Chen H. Johnson R.P. Choudhury B. Craig S.W. J. Biol. Chem. 2005; 280: 17109-17117Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The A50I mutation was introduced into specified constructs by QuikChange mutagenesis (Stratagene). pEGFPC1/vinculin (17Cohen D.M. Chen H. Johnson R.P. Choudhury B. Craig S.W. J. Biol. Chem. 2005; 280: 17109-17117Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) was modified by QuikChange PCR to facilitate cloning of cDNAs previously in the pET15b vector. The NdeI site present in the cytomegalovirus promoter region was removed (mutating adenine 234 to thymine), and a new NdeI site was introduced into the multiple cloning site immediately before the vinculin start codon. Additionally, the 5′-XhoI site was removed (mutating cytosine 1345 to adenine), and a 3′-XhoI site was introduced 3′ to the vinculin cDNA in lieu of a PstI site. cDNAs encoding the vinculin head domain (Vh; residues 1–851), Vhd1 (residues 1–258), and Vt (residues 811–1066), flanked by NdeI and XhoI sites, were subcloned from pET15b bacterial expression constructs into the modified pEGFPC1 vector to yield pEGFPC1/Vh, pEGFPC1/Vhd1, and pEGFPC1/Vt. Talin was cloned into the NdeI-compatible pEGFPC1 using a 5′-NdeI site and a 3′-SalI site flanking the talin cDNA in pET30a/talin (a gift of Dr. Stephen Lam). pEGFPC1/paxillin was kindly provided by Dr. Chris Turner. pEGFPN3/non-muscle α-actinin was a generous gift of Dr. Beatrice Haimovich. ActA Constructs—The ActA tag (residues 610–639) was cloned by PCR amplification using pGFP/NtLPP/ActA as a template plasmid (a gift of Dr. Roy Golsteyn) with PCR primers containing a 5′-KpnI site and a 3′-XmaI site. In addition to the restriction sites, these primers also contained ectopic sequence containing a peptide spacer (PRGSPPAAS) designed to prevent deleterious effects of the ActA tag on protein folding. Full-length vinculin cDNA (residues 1–1066) lacking a stop codon was generated by digestion of the vinculin cDNA with NcoI and ligation to a linker sequence coding for residues 1064–1066 and terminating in a SalI site. This cDNA was subcloned into pEGFPC1 using EcoRI and SalI sites. The ActA sequence was then introduced using KpnI and XmaI sites present in the pEGFPC1 vector to yield pEGFP/V1–1066/VDGT/spacer/ActA. The T12 mutation (17Cohen D.M. Chen H. Johnson R.P. Choudhury B. Craig S.W. J. Biol. Chem. 2005; 280: 17109-17117Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) was introduced into pEGFP/vinculin/ActA by QuikChange mutagenesis. The ActA tag was also subcloned into pEGFP/V1–851 (constructed without a stop codon) using KpnI and XmaI sites present in the multiple cloning site to yield pEGFP/V1–851/STV/spacer/ActA. pEGFP/V159–1066/T12/ActA, a vinculin construct deficient in both HTI sites and the talin-binding domain (18Izard T. Evans G. Borgon R.A. Rush C.L. Bricogne G. Bois P.R.J. Nature. 2004; 427: 171-175Crossref PubMed Scopus (204) Google Scholar), was generated by replacing the cDNA encoding Vh in pEGFP/V1–851/ActA with a PCR-amplified 2.75-kb product corresponding to T12 vinculin-(159–1066). To construct pEGFP/talin/ActA, the stop codon in pEGFP/talin was converted to a serine codon, anda3′-KpnI restriction site was introduced by QuikChange mutagenesis. The resulting NdeI/KpnI-flanked talin cDNA lacking a stop codon was then substituted for vinculin-(1–851) in the pEGFP/V1–851/ActA vector to generate pEGFP/talin/RSLV/spacer/ActA. HTI Mutations—Characterization of the HTI mutations in vinculin used herein has been reported (17Cohen D.M. Chen H. Johnson R.P. Choudhury B. Craig S.W. J. Biol. Chem. 2005; 280: 17109-17117Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The specific mutations present in each cluster are as follows: T8, K944A/R945A; T12, D974A/K975A/R976A/R978A; T19, K1047A/R1049A/D1051A; T20, R1057A/R1060A/K1061A; and T8/19, K944A/R945A/D1047A/K1049A/R1051A. Talin Rod Binding Assay—Expression and purification of His-tagged talin rod and His-tagged yellow fluorescent protein (YFP)-vinculin-(1–258) and thrombin digestion of His-tagged YFP-vinculin-(1–258) were carried out as described previously (17Cohen D.M. Chen H. Johnson R.P. Choudhury B. Craig S.W. J. Biol. Chem. 2005; 280: 17109-17117Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). To assay binding, YFP-vinculin-(1–258) (wild-type or A50I) was incubated at 0.2 μm with His-tagged talin rod (0–1.4 μm) for 1 h at room temperature in a 250-μl reaction containing phosphate-buffered saline, 0.1 mg/ml bovine serum albumin, 0.1% Triton X-100, 0.1% β-mercaptoethanol, and 10 mm imidazole (pH 7.8). Talin rod was recovered on nitrilotriacetic acid resin (Qiagen Inc.) with inversion for 30 min at room temperature. YFP fluorescence remaining in the supernatant was assayed on a Fluoromax-3 fluorometer (HORIBA Jobin Yvon). YFP was excited at 490 nm, and peak emission at 527 nm was observed using 3-mm excitation and 5-mm emission slit widths. Tissue Culture and Transfections—Human foreskin fibroblasts (HFFs) were a gift of Dr. Denis Wirtz. HFFs were propagated on 0.1% gelatin-coated 10-cm dishes with 90% high glucose Dulbecco's modified Eagle's medium (Mediatech, Inc.) and 10% fetal calf serum (Hyclone) in a 5% CO2 incubator at 37 °C. HFFs were cultured overnight on glass coverslips coated with fibronectin (20 μg/ml) prior to transfection. Transfections were carried out using Lipofectamine Plus (Invitrogen) and serum-free Iscove's medium (American Type Culture Collection). HFFs were labeled with 200 nm MitoTracker Red CM-H2XRos (catalog no. M-7513, Invitrogen) for 30 min in growth medium prior to fixation. Vinculin null cells (clone 54) were provided by Dr. Eileen Adamson and grown on 0.1% gelatin-coated coverslips in a custom filming medium, which improved live cell imaging. The high glucose Dulbecco's modified Eagle's medium formulation (Invitrogen) was modified to exclude phenol red and to reduce sodium bicarbonate to 0.37 g/liter and sodium chloride to 4.75 g/liter. In addition, calcium pantothenate, choline chloride, folic acid, myo-inositol, niacinamide, riboflavin, thiamine hydrochloride, and pyridoxine hydrochloride were reduced to levels present in L-15 medium (25% of the amounts in Dulbecco's modified Eagle's medium). HEPES and fetal calf serum were added to 15 mm and 10% final concentrations, respectively. Vinculin null cells were plated on 20 μg/ml fibronectin-coated coverslips or Delta T dishes (Bioptechs Inc.) for 18 h prior to transfection. Cells were transfected using Lipofectamine Plus and allowed to recover overnight. Transfection particles were removed by extensive washing of the dishes. Immunofluorescence—HFFs were fixed in ice-cold 4% paraformaldehyde in phosphate-buffered saline for 20 min. For experiments examining intracellular epitopes, HFFs were permeabilized in ice-cold 0.1% Triton X-100 dissolved in cytoskeletal buffer (10 mm MES, 138 mm KCl, 3 mm MgCl2, and 4 mm EGTA (pH 6.1)) for 3 min. Note that in experiments involving dual antibody 12G10/MitoTracker labeling, this permeabilization step was omitted. HFFs or digitonin-permeabilized vinculin null cells were blocked in 2% bovine serum albumin and 2% normal donkey serum in phosphate-buffered saline for 30 min at room temperature. The primary antibodies used for immunofluorescence were as follows: anti-β1 integrin antibody 12G10 (a generous gift of Dr. Steven Akiyama), monoclonal anti-FLAG M2 and anti-vinculin hVin1 antibodies (purchased from Sigma), anti-tensin monoclonal antibody 13820 (obtained from BD Transduction Laboratories), and rabbit anti-talin polyclonal antibody (TnC22 serum; generated in the Craig laboratory). Rhodamine Red-X- or Cy5-conjugated donkey anti-rabbit or anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. Fluorescence Recovery after Photobleaching (FRAP) Imaging—A Zeiss Axiovert 135TV fluorescence microscope equipped with both stage and objective heaters (Bioptechs Inc.) was equilibrated at 37 °C for 1 h before photobleaching experiments began. Dark-field and flat-field images (1 μm enhanced green fluorescent protein in filming medium) were collected at the beginning of each experiment. Bioptechs dishes containing transfected cells and 1.4 ml of filming medium were equilibrated in a normal atmospheric environment for 30 min prior to imaging. Photobleaching was achieved using a Stabilite 2017 argon/krypton 488 nm laser (Spectra-Physics) at 100 milliwatts at exposures ranging from 200 to 400 ms to achieve 40–80% loss of initial green fluorescent protein (GFP) fluorescence. The laser was focused on the specimen in the form of a diffraction-limited slit, measuring 0.5 μm in width. The laser beam was brought to the epi-illuminator of the microscope by beam-shaping lenses, including a spherical lens with a 100-mm focal length and a cylindrical lens with a 300-mm focal length; a beam steerer; two mirrors; and three aperture diaphragms. For photobleach recovery, time-lapse GFP epifluorescence images were collected with a Zeiss Plan Neofluar objective (×100) using mercury lamp illumination with a UV-blocking GG455 filter (Schott) and an infrared-blocking BG38 filter (Schott). Digital images captured by a CoolSNAPHQ camera (Photometrics) were binned at 4 × 4 pixels and with exposure times optimized to yield pixel intensity values in the linear range (300–2000 counts). Each photobleach sequence was collected for a period of 4–8 min and contained 25–37 frames representing three sequential time domains (a fast domain, 5–7-s intervals; an intermediate domain, 10–15-s intervals; and a slow domain, 15–30-s intervals). The domain intervals and number of frames/interval were adjusted to avoid over- or undersampling of early versus late phases of the recovery curve (i.e. conditions were chosen to disperse the data as evenly as possible through the recovery curve). FRAP Analysis—Each FRAP sequence was dark-field- and flat-field-corrected using IPLab software (Scanalytics, Inc.) driven by a LabVIEW (National Instruments) script, “FrapApp” (written by Brett Kutscher). FRAP sequences were also corrected for global fluctuations in fluorescence induced by variations in lamp output by normalizing to background fluorescence levels observed in a region of interest selected outside the cell. Photobleached regions were then selected for analysis using the IPLab ROI tool. Region of interests used in these analyses were limited to one or two discrete focal adhesions. Recovery curves were fit using the Levenberg-Marquardt algorithm and a single exponential recovery model using the following equation: Ft = Ff – (Ff – Fi)·e(–kt), where Ft is the fluorescence at time t, Ff is the final fluorescence (plateau), Fi is the initial fluorescence (extrapolating to t = 0 after photobleaching), and k = ln 2/t½. Movies were excluded from the analysis if they contained significant focus drift, showed focal adhesion growth or decay, or exhibited fluorescence recovery at <20% of the initial fluorescence. Goodness of fit to a single exponential model was assessed by a semilog plot (ln(Ff – Ft) versus time), and sequences were truncated at time points following which significant deviation from linearity was observed. Tolerance values for mean squared error for the nonlinear Levenberg-Marquardt fit were set at 250 (i.e. analyses exceeding this value were rejected). Typical mean squared error was below 100 for movies with at least 450 fluorescence units/pixel (mean value in region of interest determining the recovery rate)/frame. Data Processing and Statistical Analyses—Box-plot distributions generated in KaleidaGraph (Synergy Software) were used to summarize the t½ measurements for each of the vinculin mutants. In these plots, the data are represented in quartiles, with the upper quartile (25% of the data points above the median) and the lower quartile (25% of the data points below the median) representing the top and bottom of the each box, respectively. The median is depicted as a horizontal line within each box. The thin lines extending from the boxes mark the minimum and maximum values of the data set that fall within an acceptable range. Open circles denote outliers, points whose values are either >upper quartile + 1.5 × interquartile distance or <lower quartile – 1.5 × interquartile distance. Appropriate statistical methodology to analyze differences in the mean FRAP t½ values was used to evaluate the box-plot analyses. For non-normal distributions, as observed for the HTI mutants in Fig. 3C, a two-tailed non-parametric Mann-Whitney test was applied to compare means between two samples. In addition, the entire set of full-length vinculin (wild-type, T20, and HTI mutant) half-time measurements was subjected to analysis of variance using resources at available at www.physics.csbsju.edu/stats/anova.html. For normal distributions, such as those obtained for the GFP adhesion markers, a two-tailed Student's t test with unequal variance was utilized. p values reported for all of these tests were computed at 95% confidence limits. Role of Vinculin HTI in Formation of a Ternary Complex with β1 Integrin and Talin in Cells—To investigate the role of vinculin conformation in assembly of a ternary complex of talin, vinculin, and β1 integrin in cells, we examined these protein-protein interactions in the context of an ectopic recruitment assay. Targeting proteins away from cell-substrate adhesions to an ectopic location enabled us to evaluate the interaction potential of talin, vinculin, and integrin independently from other focal adhesion proteins. Using the C-terminal ActA tag (28Pistor S. Chakraborty T. Niebuhr K. Domann E. Wehland J. EMBO J. 1994; 13: 758-763Crossref PubMed Scopus (156) Google Scholar), we mislocalized either vinculin or talin to mitochondria as seen by co-distribution with a mitochondrion-specific dye, MitoTracker (Figs. 1A and 2A).FIGURE 2HTI regulates the ability of vinculin to direct assembly of talin-integrin complexes. HFFs expressing GFP-vinculin-ActA (wild-type or T12 mutant) were labeled with MitoTracker and/or immunostained for talin and β1 integrin and visualized by confocal fluorescence microscopy. The boxed regions are enlarged for visualization of marker co-localization. A, T12 vinculin-ActA (T12VincActA) targets to mitochondria as assessed by MitoTracker dye and induces ectopic assembly of integrin. B, T12 vinculin-ActA induces assembly of a ternary complex containing both talin and β1 integrin. C, wild-type vinculin-ActA (WtVincActA) fails to induce ternary complex assembly, as little correlation is observed between GFP staining and organization of talin or β1 integrin. White squiggles are fiduciary markers outlining edges of several mitochondria. These marks were superimposed onto the antibody hVin1 counterstain. D, a T12 deletion mutant, vinculin-(159–1066) (V159–1066 T12ActA), which lacks the talin-binding site, fails to direct assembly of ectopic mitochondrial talin-vinculin-integrin complexes.View Large Image Figure ViewerDownload Hi-res