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• W1988822956 abstract "The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin β subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing αIIbβ3, linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984–2344), devoid of any known actin- or vinculin-binding sites, colocalized with β3-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin αIIbβ3 or with the recombinant wild type, but not the Y747A mutant β3 cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between β3-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984–2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin-binding site within the talin rod domain is important for β3-cytoskeletal interactions but does not participate in αIIbβ3 activation. The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin β subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing αIIbβ3, linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984–2344), devoid of any known actin- or vinculin-binding sites, colocalized with β3-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin αIIbβ3 or with the recombinant wild type, but not the Y747A mutant β3 cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between β3-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984–2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin-binding site within the talin rod domain is important for β3-cytoskeletal interactions but does not participate in αIIbβ3 activation. Integrin-mediated cell adhesion and signaling are crucial events for numerous biological processes such as morphogenesis, the immune response, hemostasis, cell growth, and differentiation as well as for cell survival (1Shimaoka M. Springer T.A. Nat. Rev. Drug Discov. 2003; 2: 703-716Crossref PubMed Scopus (296) Google Scholar). Integrins function as noncovalent αβ heterodimeric transmembrane receptors that link the extracellular matrix to the actin cytoskeleton; they are, however, unable to directly interact with actin filaments. A number of actin-binding proteins, including talin, α-actinin, filamin, myosin, and skelemin, have been identified that act as intermediates in connecting integrins to the actin cytoskeleton (2Liu S. Calderwood D.A. Ginsberg M.H. J. Cell Sci. 2000; 113: 3563-3571Crossref PubMed Google Scholar). Among these, talin was the first intracellular ligand shown to interact directly with integrin β-subunit cytoplasmic tails (3Horwitz A. Duggan K. Buck C. Beckerle M.C. Burridge K. Nature. 1986; 320: 531-533Crossref PubMed Scopus (826) Google Scholar). Talin is a multifunctional ∼270-kDa (2541 amino acids) cytoskeletal protein that forms antiparallel homodimers, which represent the biologically active form of the protein (4Goldmann W.H. Bremer A. Haner M. Aebi U. Isenberg G. J. Struct. Biol. 1994; 112: 3-10Crossref PubMed Scopus (62) Google Scholar). In vivo, talin is found in equilibrium between a membrane-bound and a cytoplasmic form (4Goldmann W.H. Bremer A. Haner M. Aebi U. Isenberg G. J. Struct. Biol. 1994; 112: 3-10Crossref PubMed Scopus (62) Google Scholar, 5Molony L. McCaslin D. Abernethy J. Paschal B. Burridge K. J. Biol. Chem. 1987; 262: 7790-7795Abstract Full Text PDF PubMed Google Scholar), and several studies have emphasized its role in regulating integrin-actin cytoskeleton complexes (6Calderwood D.A. Ginsberg M.H. Nat. Cell Biol. 2003; 5: 694-697Crossref PubMed Scopus (117) Google Scholar) and integrin activation (7Calderwood D.A. Yan B. de Pereda J.M. Alvarez B.G. Fujioka Y. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; 277: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 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 (987) Google Scholar). Talin colocalizes with integrins at sites of cell-matrix interactions and membrane ruffles of moving cells (9Burridge K. Connell L. J. Cell Biol. 1983; 97: 359-367Crossref PubMed Scopus (266) Google Scholar, 10DePasquale J.A. Izzard C.S. J. Cell Biol. 1991; 113: 1351-1359Crossref PubMed Scopus (95) Google Scholar), and it is necessary for the assembly of focal adhesions (FAs). 1The abbreviations used are: FA, focal adhesion; FRET, fluorescence resonance energy transfer; SPR, surface plasmon resonance; CHO, Chinese hamster ovary; GST, glutathione S-transferase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; IMDM, Iscove's modified Dulbecco's medium; Pipes, 1,4-piperazinediethanesulfonic acid; TRITC, tetramethylrhodamine isothiocyanate; bis-Tris, 2-[bis-(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. The functional role of talin has been elucidated by antibody inhibition studies and genetic approaches. Microinjection of anti-talin antibodies into fibroblasts led to the disruption of FAs and associated stress fibers (11Nuckolls G.H. Romer L.H. Burridge K. J. Cell Sci. 1992; 102: 753-762Crossref PubMed Google Scholar), and down-regulation of talin by antisense mRNA or short hairpin RNAs reduced cell spreading and FA assembly in HeLa cells, integrin processing and transport to the cell surface, and energy-dependent integrin activation (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 (987) Google Scholar, 12Albiges-Rizo C. Frachet P. Block M.R. J. Cell Sci. 1995; 108: 3317-3329Crossref PubMed Google Scholar, 13Martel V. Racaud-Sultan C. Dupe S. Marie C. Paulhe F. Galmiche A. Block M.R. Albiges-Rizo C. J. Biol. Chem. 2001; 276: 21217-21227Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Disruption of the Talin1 gene by a knockout approach in mice revealed that undifferentiated Talin1 (-/-) embryonic stem cells exhibited defective cell adhesion and spreading and were unable to assemble FAs or stress fibers, whereas differentiated cells readily formed these adhesion complexes (14Monkley S.J. Zhou X.H. Kingston S.J. Giblett S.M. Hemmings L. Priddle H. Brown J.E. Pritchard C.A. Critchley D.R. Fassler R. Dev. Dyn. 2000; 219: 560-574Crossref PubMed Scopus (179) Google Scholar). The only partially inhibitory phenotype observed in Talin1 (-/-) mouse fibroblast-like cells may be explained by the presence of a second, recently identified TALIN2 gene in mammalian cells (15Monkley S.J. Pritchard C.A. Critchley D.R. Biochem. Biophys. Res. Commun. 2001; 286: 880-885Crossref PubMed Scopus (90) Google Scholar). Indeed, in Drosophila, disruption of the rhea locus, which corresponds to the unique talin gene, mimics the integrin knockout phenotype, underlining the important role of talin as an obligatory component of integrin-mediated adhesion (16Brown N.H. Gregory S.L. Rickoll W.L. Fessler L.I. Prout M. White R.A. Fristrom J.W. Dev. Cell. 2002; 3: 569-579Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). In living cells, talin can be cleaved into a globular 47-kDa N-terminal head domain and an elongated 190-kDa C-terminal rod domain by the calcium-dependent protease calpain II (17Rees D.J. Ades S.E. Singer S.J. Hynes R.O. Nature. 1990; 347: 685-689Crossref PubMed Scopus (244) Google Scholar). The talin head (residues 1–433), which includes a region (residues 86–410) homologous to the N-terminal FERM domain of the band 4.1, ezrin, radixin, moesin family of cytoskeletal proteins (17Rees D.J. Ades S.E. Singer S.J. Hynes R.O. Nature. 1990; 347: 685-689Crossref PubMed Scopus (244) Google Scholar), contains an actin-binding site (18Hemmings L. Rees D.J. Ohanian V. Bolton S.J. Gilmore A.P. Patel B. Priddle H. Trevithick J.E. Hynes R.O. Critchley D.R. J. Cell Sci. 1996; 109: 2715-2726Crossref PubMed Google Scholar), three potential membrane-association sites (19Niggli V. Kaufmann S. Goldmann W.H. Weber T. Isenberg G. Eur. J. Biochem. 1994; 224: 951-957Crossref PubMed Scopus (63) Google Scholar, 20Seelig A. Blatter X.L. Frentzel A. Isenberg G. J. Biol. Chem. 2000; 275: 17954-17961Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and binding sites for integrins, layilin, focal adhesion kinase, myosin (21Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (499) Google Scholar), and type 1γ phosphatidylinositol phosphate kinase (22Barsukov I.L. Prescot A. Bate N. Patel B. Floyd D.N. Bhanji N. Bagshaw C.R. Letinic K. Di Paolo G. De Camilli P. Roberts G.C. Critchley D.R. J. Biol. Chem. 2003; 278: 31202-31209Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The binding affinity of talin for integrins increases upon calpain cleavage or in the presence of phosphatidylinositol 4,5-biphosphate, suggesting that conformational changes within talin unmask cryptic integrin-binding sites (13Martel V. Racaud-Sultan C. Dupe S. Marie C. Paulhe F. Galmiche A. Block M.R. Albiges-Rizo C. J. Biol. Chem. 2001; 276: 21217-21227Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 23Yan B. Calderwood D.A. Yaspan B. Ginsberg M.H. J. Biol. Chem. 2001; 276: 28164-28170Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Recent biochemical (7Calderwood D.A. Yan B. de Pereda J.M. Alvarez B.G. Fujioka Y. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; 277: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 24Calderwood 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 (564) Google Scholar), crystallographic, and NMR studies (25Garcia-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 (421) Google Scholar, 26Ulmer T.S. Calderwood D.A. Ginsberg M.H. Campbell I.D. Biochemistry. 2003; 42: 8307-8312Crossref PubMed Scopus (73) Google Scholar) have mapped the integrin-binding site in the talin head fragment to a mainly hydrophobic area in the F3 subdomain of the FERM domain. Recombinant F3, which binds integrin tails with a similar affinity as the talin head domain (7Calderwood D.A. Yan B. de Pereda J.M. Alvarez B.G. Fujioka Y. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; 277: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar), functions as a phosphotyrosine-binding domain (25Garcia-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 (421) Google Scholar), recognizing the 744NPXY747 motif in the β3 cytoplasmic tail. Residue Tyr747 appears to be critical for this interaction, since the Y747A mutation abrogates the talin head binding to the β subunit (24Calderwood 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 (564) Google Scholar). Binding of the talin head domain to the cytoplasmic tail of integrin β subunits leads to integrin activation (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 (987) Google Scholar), and this was shown by overexpression of the F3 subdomain of the talin FERM domain in CHO αIIbβ3 cells, which results in increased binding of the ligand mimetic anti-αIIbβ3 antibody PAC-1 (7Calderwood D.A. Yan B. de Pereda J.M. Alvarez B.G. Fujioka Y. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; 277: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 24Calderwood 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 (564) Google Scholar). The talin rod domain is composed of multiple α-helical alanine-rich repeats (27McLachlan A.D. Stewart M. Hynes R.O. Rees D.J. J. Mol. Biol. 1994; 235: 1278-1290Crossref PubMed Scopus (43) Google Scholar) and contains a dimerization site (28Winkler J. Lunsdorf H. Jockusch B.M. Eur. J. Biochem. 1997; 243: 430-436Crossref PubMed Scopus (45) Google Scholar), an integrin-binding site (3Horwitz A. Duggan K. Buck C. Beckerle M.C. Burridge K. Nature. 1986; 320: 531-533Crossref PubMed Scopus (826) Google Scholar, 29Xing B. Jedsadayanmata A. Lam S.C. J. Biol. Chem. 2001; 276: 44373-44378Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), three vinculin-binding sites (30Gilmore A.P. Wood C. Ohanian V. Jackson P. Patel B. Rees D.J. Hynes R.O. Critchley D.R. J. Cell Biol. 1993; 122: 337-347Crossref PubMed Scopus (65) Google Scholar), a recently identified TES-binding site (31Coutts A.S. MacKenzie E. Griffith E. Black D.M. J. Cell Sci. 2002; 116: 897-906Crossref Scopus (79) Google Scholar), and two actin-binding sites, the C-terminal one of which is highly conserved among actin-binding proteins (32McCann R.O. Craig S.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5679-5684Crossref PubMed Scopus (148) Google Scholar, 33McCann R.O. Craig S.W. Biochem. Biophys. Res. Commun. 1999; 266: 135-140Crossref PubMed Scopus (44) Google Scholar). Using in vitro solid phase binding assays, the β3 integrin-binding site of the rod domain has recently been shown to be contained within a recombinant fragment encoding the 558 C-terminal residues of talin (29Xing B. Jedsadayanmata A. Lam S.C. J. Biol. Chem. 2001; 276: 44373-44378Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The functional significance of the interaction between the talin rod domain and the β-subunit of integrins in integrin-mediated cell adhesion and spreading remains largely unknown. In order to study this specific interaction, we have used a cell biology and a biochemical approach to map the integrin-binding site in the talin rod domain to a polypeptide of 130 amino acids, located between the C-terminal actin- and vinculin-binding sites. We also provide evidence that in FAs this fragment directly interacts with the cytoplasmic tail of β3 but that this interaction does not participate in integrin activation. Protein Expression and Affinity Purification—Talin cDNA fragments encoding amino acids 1–433 (fragment A), 430–1076 (fragment B), 1984–2541 (fragment F), and 1984–2344 (fragment G) were amplified by reverse transcriptase-PCR using human erythroleukemic HEL cell mRNA; cDNA fragments encoding amino acids 1984–2270 (fragment H), 1984–2113 (fragment J), and 2093–2344 (fragment K) were amplified by PCR from the talin G construct. The primers used for PCR amplification were designed to generate appropriate restriction sites allowing the cloning of the amplified cDNAs into the pGEX-4T-2 vector (Amersham Biosciences); for fragment A, SalI and EcoRI restriction sites were generated at the 5′- and 3′-ends, respectively; all remaining fragments were cloned using a single SalI site, and their orientation in the vector was determined by PCR. Fidelity of all cloned talin cDNA fragments with the human talin sequence (GenBank™ AF078828) was confirmed by automated sequencing (Applied Biosystems). Expression of GST fusion proteins in Escherichia coli BL21(DE3) was induced with 0.2 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h at 37 °C. Cells were lysed as previously described (34Woodside D.G. Obergfell A. Talapatra A. Calderwood D.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 2002; 277: 39401-39408Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Fusion proteins from the soluble fraction were affinity-purified on glutathione-Sepharose 4B beads according to the manufacturer's instructions (Amersham Biosciences) and finally dialyzed against PBS. Talin fragments used for surface plasmon resonance analysis were cleaved from the GST tag by thrombin digestion (10 units/mg of protein, Amersham Biosciences) for 1 h at room temperature. Thrombin was neutralized with phenylmethylsulfonyl fluoride (1 mm), and the fragments were dialyzed against PBS. cDNA fragments encoding the cytoplasmic tail of wild type β3 or the β3Y747A mutant (residues 716–762) were generated from the corresponding full-length constructs (35Schaffner-Reckinger E. Gouon V. Melchior C. Plançon S. Kieffer N. J. Biol. Chem. 1998; 273: 12623-12632Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) using primers introducing BamHI and SmaI restriction sites at the 5′- and 3′-ends, respectively. These fragments were ligated into the pGEX-4T-2 vector, and the corresponding GST-fusion proteins were expressed and purified as described above. For expression of talin fragments in CHO αIIbβ3-GFP cells, recombinant DsRed fusion proteins of human talin were generated by cloning the cDNA of talin fragment A into the SalI and SacI and the cDNAs of all the other talin fragments into the SalI and BamHI restriction sites of the pDsRed-N1 vector (Clontech), resulting in fragments covering talin residues 1–433 (fragment A), 430–1076 (fragment B), 1075–1623 (fragment C), 1622–2270 (fragment D), 2267–2541 (fragment E), 1984–2541 (fragment F), 1984–2344 (fragment G), 1984–2270 (fragment H), 2267–2344 (fragment I), 1984–2113 (fragment J), 2093–2344 (fragment K), and 2093–2270 (fragment L). For integrin activation studies by flow cytometry, GFP fusion proteins of talin fragments A, G, and J were generated by cloning the corresponding cDNAs into the pEGFP-N1 vector using SalI and BamHI restriction sites. Furthermore, the cDNA encoding talin residues 1977–2113 was also inserted into the pcDNA4/TO/myc-His vector (Invitrogen) using BamHI and EcoRI restriction sites, generating a recombinant protein with a C-terminal myc-His tag (Jmyc). Cell Transfection and Indirect Immunofluorescence—CHO cells, stably transfected with αIIbβ3-GFP (36Plançon S. Morel-Kopp M.C. Schaffner-Reckinger E. Chen P. Kieffer N. Biochem. J. 2001; 357: 529-536Crossref PubMed Scopus (27) Google Scholar), or HT-144 human melanoma cells (ATCC HTB 63) were cultured in IMDM supplemented with 10% fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin and were transfected as previously described (35Schaffner-Reckinger E. Gouon V. Melchior C. Plançon S. Kieffer N. J. Biol. Chem. 1998; 273: 12623-12632Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Briefly, for each cDNA construct, 1.5 × 106 adherent cells were transfected with 3–15 μg of cDNA using LipofectAMINE™ (Invitrogen) according to the manufacturer's instructions. Fetal calf serum (10% final concentration) was added after 24 h, and 48 h after transfection, cells were detached with EDTA buffer (50 mm Hepes, 126 mm NaCl, 5 mm KCl, 1 mm EDTA, pH 7.5), washed, resuspended in serum-free IMDM, and seeded onto glass coverslips coated with fibrinogen (20 μg/ml). After 2 h of adhesion at 37 °C, cells were fixed for 15 min at 4 °C in fixation buffer (PBS, pH 7.4, 3% paraformaldehyde, 2% sucrose) and washed four times in washing buffer (PBS, pH 7.4, 0.5% Triton X-100, 0.5% bovine serum albumin). For indirect immunofluorescence staining of the myc-tagged fragment J, the fixed cells were incubated for 40 min with a monoclonal mouse anti-myc antibody (9B11, 0.5 μg/ml; Cell Signaling) and for 30 min with a rhodamine (TRITC)-conjugated goat anti-mouse IgG antibody (7.5 μg/ml; Jackson ImmunoResearch Laboratories, Inc.). After each incubation step, the coverslips were washed three times for 5 min in washing buffer. Finally, the coverslips were mounted on microscopy slides in Mowiol/DABCO (Sigma). Fluorescence Microscopy and Fluorescence Resonance Energy Transfer (FRET) Analysis—For fluorescence analysis, GFP- and DsRed-conjugated proteins were visualized in paraformaldehyde-fixed cells adherent on fibrinogen-coated glass coverslips. Single images were collected under a conventional fluorescence microscope (LEICA Leitz DMRB) with a 63× oil immersion objective and a LEICA DC 300F camera using the LEICA IM1000 1.20 software. Images were processed digitally with Photoshop 6.0 (Adobe Systems). FRET experiments were performed on a confocal microscope (Bio-Rad 1024, krypton-argon laser 488 nm, 568 nm; Nikon Eclipse TE300, 40× oil immersion CFI Plan-Fluor numerical aperture 1.3 objective), using the GFP/DsRed couple as a donor/acceptor pair. A spectral parameter indicating the ability of donor/acceptor fluorescent molecules to exhibit efficient FRET is R0, the Förster distance equal to the donor/acceptor separation at which FRET is 50% efficient. For the GFP/DsRed couple, this value is 5.7 nm (37Erickson M.G. Biophys. J. 2003; 85: 599-611Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), comparable with that reported for cyan fluorescent protein/yellow fluorescent protein, the leading pair for FRET experiments (38Miyawaki A. Llopis J. Heim R. J.M. M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2650) Google Scholar, 39Patterson G.H. Piston D.W. Barisas B.G. Anal. Biochem. 2000; 284: 438-440Crossref PubMed Scopus (324) Google Scholar). Fluorescent images of the donor were acquired by exciting GFP with the 488-nm line of the krypton-argon laser and detected using a 522 ± 35-nm filter. Images of the acceptor were acquired by exciting DsRed with the 568-nm line of the krypton-argon laser and detected with a 585-nm long pass filter. FRET was determined by the acceptor photobleaching method (40Bastiaens P.I. Jovin T.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8407-8412Crossref PubMed Scopus (153) Google Scholar, 41Wouters F.S. Bastiaens P.I. Wirtz K.W. Jovin T.M. EMBO J. 1998; 17: 7179-7189Crossref PubMed Scopus (180) Google Scholar). Briefly, after acquisition of an acceptor image (A1), a first Z-series of the donor (GFP) was acquired by scanning repeatedly with the 488-nm light of the confocal system (prebleach; D1). Then a region of interest of the cell was photobleached at 568 nm with 100% laser power until all acceptor (DsRed) was photodestroyed (A2), and a second Z-series of the donor was acquired (postbleach; D2). After correction for image acquisition using the Amira software (TGS), maximum intensity projections from the Z-series of GFP images were generated. The FRET-dependent increase in fluorescence of the donor in the photobleached region was visualized by subtracting the prebleach GFP image from the postbleach GFP image (D2 - D1). For each experiment, FRET efficiencies in FA sites (EFA) were calculated from the pre- and postbleach donor images according to the following equation: EFA = 1 - (IFA, prebleach/IFA, postbleach), where I is the average intensity measured for the corresponding FA (41Wouters F.S. Bastiaens P.I. Wirtz K.W. Jovin T.M. EMBO J. 1998; 17: 7179-7189Crossref PubMed Scopus (180) Google Scholar). For each cell analyzed, EFA values were calculated for 4–8 FA sites in the photobleached region as well as in the control (nonbleached) region, and the results were expressed as means ± S.E. Pull-down Assays—Platelets were isolated from freshly drawn whole blood as described previously (42Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and lysed by sonication on ice in lysis buffer (50 mm NaCl, 150 mm sucrose, 10 mm Pipes, 1% Triton X-100, 0.5% deoxycholate, 1 mm EDTA, 1 mm Na3VO4, 50 mm NaF, 5 μg/ml aprotinin, 2.5 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride, pH 6.8). Lysates were clarified by centrifugation at 4 °C for 30 min at 13,000 rpm, and protein concentration was determined using the Bio-Rad protein assay according to the manufacturer's instructions. For direct protein-protein interaction assays, 200 μl of crude bacterial lysates containing the GST fusion proteins talin A, B, F, G, H, J, or K or GST alone were each mixed with 20 μl of glutathione-Sepharose beads for1hat4 °C and subsequently washed four times with PBS plus 0.05% Tween 20. Sepharose beads coated with the different talin fragments were then incubated with 1 mg of platelet lysate for 2.5 h at 4 °C. Unbound proteins were washed off three times with PBS. Protein complexes were extracted in 25 μl of SDS-PAGE loading buffer (5 min, 100 °C) and separated on a 4–12% NuPAGE Bis-Tris gradient gel (Invitrogen). Western blot analysis of the transferred proteins was performed with a monoclonal anti-β3 cytoplasmic tail antibody (C3a). Surface Plasmon Resonance (SPR)—Measurements were performed on a Biacore3000 instrument (Biacore, Uppsala, Sweden). Goat anti-GST antibodies (Amersham Biosciences) were immobilized on a CM5 sensor chip according to the manufacturer's instructions (Biacore). Briefly, after activation of the carboxylate groups on the chip by a mixture of N-ethyl-N′-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide (Biacore), a solution of 35 μg/ml goat anti-GST antibody in 10 mm acetate buffer (pH 4.9) was fluxed over the four channels of the chip for 7 min at a flow rate of 5 μl/min. The residual activated carboxylate groups were neutralized with 1 m ethanolamine (pH 8.5); for regeneration, 10 mm HCl was fluxed over the channels for 1 min. For kinetic analysis, two channels were pretreated for 2 min at 5 μl/min with a 400 nm solution of recombinant GST (control channel) or GST-β3 integrin constructs in a solution containing 150 mm NaCl, 10 mm Hepes, and 0.05% NP20 (HBS-N; Biacore). Subsequently, the purified recombinant talin fragment A or G, at a concentration ranging from 0.25 to 2 μm in HBS-N, was allowed to react with the two channels at a flow rate of 20 μl/min for 5 min, followed by a dissociation phase of 7.5 min. After each run, the channels were completely regenerated with 10 mm HCl. The obtained sensorgrams were analyzed with Biaeval 3.1 software (Biacore). Sensorgrams of the control channel were subtracted from the sensorgrams on the experimental channel and analyzed using nonlinear regression statistics to fit a simple Langmuir 1:1 binding model, taking into account the drifting base line, which is caused by the differential dissociation rate of GST alone and GST-β3 from the anti-GST antibodies. PAC-1 Binding Experiments and Flow Cytometry—Binding of the mouse antibody PAC-1, which is specific for the activated form of αIIbβ3 integrin, was assessed as previously described (36Plançon S. Morel-Kopp M.C. Schaffner-Reckinger E. Chen P. Kieffer N. Biochem. J. 2001; 357: 529-536Crossref PubMed Scopus (27) Google Scholar, 43O′Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (581) Google Scholar). Briefly, ∼5 × 105 cells in 50 μl of IMDM were preincubated for 20 min at room temperature in the presence or absence of the αIIbβ3-activating mouse antibody D3GP3 (3 μg). For control experiments, nonspecific binding of PAC-1 was assessed by incubating the cells in the presence of 1 mm RGDS peptide. PAC-1 antibody (3.5 μg in 50 μl of IMDM) was then directly added to the suspension, and cells were further incubated for 45 min at room temperature. Cells were washed in cold IMDM and incubated for 30 min on ice with a R-phycoerythrin-conjugated anti-mouse IgM antibody (Jackson ImmunoResearch Laboratories, Inc.), diluted in IMDM. Finally, cells were washed and resuspended in an appropriate buffer (137 mm NaCl, 5 mm KCl, 50 mm Hepes, 1 mg/ml glucose, pH 7.4). Flow cytometry was performed using a Coulter EPICS XL flow cytometer (Coulter, Hialeah, FL). After electronic compensation of the FL1 and FL2 fluorescence channels, PAC-1 binding (FL2) was analyzed on cells, which expressed the relevant GFP fusion proteins (FL1). Subcellular Localization of Recombinant Talin Fragments Covering the Entire Amino Acid Sequence of Human talin1—We have previously shown that fusion of GFP to the cytoplasmic tail of the β3 integrin subunit allowed surface expression of a fully functional αIIbβ3-GFP receptor (36Plançon S. Morel-Kopp M.C. Schaffner-R" @default.
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• W1988822956 title "A Fluorescence Cell Biology Approach to Map the Second Integrin-binding Site of Talin to a 130-Amino Acid Sequence within the Rod Domain" @default.
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