Two Stage Cadherin Kinetics Require Multiple Extracellular Domains but Not the Cytoplasmic Region
2007; Elsevier BV; Volume: 283; Issue: 4 Linguagem: Inglês
10.1074/jbc.m708044200
ISSN1083-351X
AutoresYuan-Hung Chien, Ning Jiang, Fang Li, Fang Zhang, Cheng Zhu, Deborah Leckband,
Tópico(s)Cell Adhesion Molecules Research
ResumoMicropipette manipulation measurements quantified the pre-steady state binding kinetics between cell pairs mediated by Xenopus cleavage stage cadherin. The time-dependence of the intercellular binding probability exhibits a fast forming, low probability binding state, which transitions to a slower forming, high probability state. The biphasic kinetics are independent of the cytoplasmic region, but the transition to the high probability state requires the third extracellular domain EC3. Deleting either EC3 or EC3–5, or substituting Trp2 for Ala reduces the binding curves to a simple, monophasic rise in binding probability to a limiting plateau, as predicted for a single site binding mechanism. The two stage cadherin binding process reported here directly parallels previous biophysical studies, and confirms that the cadherin ectodomain governs the initial intercellular adhesion dynamics. Micropipette manipulation measurements quantified the pre-steady state binding kinetics between cell pairs mediated by Xenopus cleavage stage cadherin. The time-dependence of the intercellular binding probability exhibits a fast forming, low probability binding state, which transitions to a slower forming, high probability state. The biphasic kinetics are independent of the cytoplasmic region, but the transition to the high probability state requires the third extracellular domain EC3. Deleting either EC3 or EC3–5, or substituting Trp2 for Ala reduces the binding curves to a simple, monophasic rise in binding probability to a limiting plateau, as predicted for a single site binding mechanism. The two stage cadherin binding process reported here directly parallels previous biophysical studies, and confirms that the cadherin ectodomain governs the initial intercellular adhesion dynamics. The cadherin family of adhesion proteins mediates cell-cell interactions in all solid tissues (1Gumbiner B.M. Nat. Rev. Mol. Cell. Biol. 2005; 6: 622-634Crossref PubMed Scopus (1223) Google Scholar). These calcium-dependent cell surface glycoproteins are critical for morphogenesis and for directing the segregation of cells into distinct tissues during development. In addition to their mechanical role as adhesion molecules, they are also signaling proteins that influence cytoskeletal reorganization, cell migration, and proliferation through interactions with other cadherins and possibly with other cell surface receptors. Classical cadherins are the most extensively studied of the cadherin superfamily. The proteins comprise an extracellular region, and single-pass transmembrane domain, and a cytoplasmic domain (2Yap A.S. Brieher W.M. Pruschy M. Gumbiner B.M. Curr. Biol. 1997; 7: 308-315Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The extracellular region embeds the adhesive and selectivity functions of the protein. It folds into five structurally homologous extracellular (EC) 4The abbreviations used are: ECextracellularBSAbovine serum albuminFACSfluorescent-activated cell sortingFITCfluorescein isothiocyanateRBCred blood cell. domains numbered 1–5 from the N-terminal domain (3Boggon T.J. Murray J. Chappuis-Flament S. Wong E. Gumbiner B.M. Shapiro L. Science. 2002; 296: 1308-1313Crossref PubMed Scopus (548) Google Scholar). The cytoplasmic domain mediates signaling through interactions with catenins (1Gumbiner B.M. Nat. Rev. Mol. Cell. Biol. 2005; 6: 622-634Crossref PubMed Scopus (1223) Google Scholar). extracellular bovine serum albumin fluorescent-activated cell sorting fluorescein isothiocyanate red blood cell. Several approaches have been used to investigate cadherin recognition, binding, and signal transduction. Sequence exchange and cell aggregation studies mapped the specificity-determining region to the first extracellular domain EC1 (4Nose A. Tsuji K. Takeichi M. Cell. 1990; 61: 147-155Abstract Full Text PDF PubMed Scopus (414) Google Scholar). For this reason, this domain has been the focus of the majority of mechanistic studies of cadherin adhesion and binding specificity. In the crystal structure of the soluble N-terminal domain (EC1) of neural cadherin, the Trp2 (W2) residue was docked in a hydrophobic pocket of the adjacent EC1 domain (5Shapiro L. Fannon A.M. Kwong P.D. Thompson A. Lehmann M.S. Grubel G. Legrand J.F. Als-Nielsen J. Colman D.R. Hendrickson W.A. Nature. 1995; 374: 327-337Crossref PubMed Scopus (978) Google Scholar). This reciprocal Trp2 exchange is referred to as a "strand dimer." The structure of the ectodomain of Xenopus cleavage stage cadherin (C-cadherin) similarly exhibited this Trp2 exchange, but between anti-parallel EC1 domains (3Boggon T.J. Murray J. Chappuis-Flament S. Wong E. Gumbiner B.M. Shapiro L. Science. 2002; 296: 1308-1313Crossref PubMed Scopus (548) Google Scholar). Electron tomography images of desmosomal cadherins in mouse epidermis also suggested that similar interactions form in tissue, although the images contain a wide variety of other configurations and possible interactions (6He W. Cowin P. Stokes D.L. Science. 2003; 302: 109-113Crossref PubMed Scopus (198) Google Scholar). Studies showing that W2A and W2G mutations eliminate cell adhesion, also suggest that the docked Trp2 side chain forms the sole adhesive interface (7Harrison O.J. Corps E.M. Berge T. Kilshaw P.J. J. Cell Sci. 2005; 118: 711-721Crossref PubMed Scopus (39) Google Scholar, 8Shan W.S. Tanaka H. Phillips G.R. Arndt K. Yoshida M. Colman D.R. Shapiro L. J. Cell Biol. 2000; 148: 579-590Crossref PubMed Scopus (166) Google Scholar). Other biophysical measurements, however, identified additional cadherin bonds, which involve other regions of the cadherin ectodomain than EC1. Surface force measurements first identified additional domain interactions (9Sivasankar S. Brieher W. Lavrik N. Gumbiner B. Leckband D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11820-11824Crossref PubMed Scopus (145) Google Scholar, 10Sivasankar S. Gumbiner B. Leckband D. Biophys. J. 2001; 80: 1758-1768Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In addition to adhesion between EC1 domains, Zhu et al. (11Zhu B. Chappuis-Flament S. Wong E. Jensen I.E. Gumbiner B.M. Leckband D. Biophys. J. 2003; 84: 4033-4042Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) mapped a second, stronger bond to the third EC domain (EC3). Other classical cadherins exhibit similar behavior (12Prakasam A.K. Maruthamuthu V. Leckband D.E. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15434-15439Crossref PubMed Scopus (90) Google Scholar). Cell adhesion studies using flow assays also implicated additional domains in adhesion (13Chappuis-Flament S. Wong E. Hicks L.D. Kay C.M. Gumbiner B.M. J. Cell Biol. 2001; 154: 231-243Crossref PubMed Scopus (188) Google Scholar). Similar to the surface force measurements, single bond rupture measurements demonstrated that the outer EC12 fragment forms two relatively weak bonds with fast dissociation kinetics. However, the full-length extracellular fragment EC1–5 forms two stronger bonds with slow dissociation kinetics, in addition to the weak, fast EC12 bonds (14Bayas M.V. Leung A. Evans E. Leckband D. Biophys. J. 2006; 90: 1385-1395Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Perret E. Leung A. Feracci H. Evans E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16472-16477Crossref PubMed Scopus (108) Google Scholar). The population of strong bonds also increases at the expense of the weak bonds with increasing protein contact times (15Perret E. Leung A. Feracci H. Evans E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16472-16477Crossref PubMed Scopus (108) Google Scholar). These findings are not consistent with a simple, one site binding mechanism. A recent proposal that Trp2 is an allosteric regulator of global cadherin adhesive activity may reconcile the multi-bond model with the Trp2 requirement for adhesion. Prakasam et al. (16Prakasam A. Chien Y.H. Maruthamuthu V. Leckband D.E. Biochemistry. 2006; 45: 6930-6939Crossref PubMed Scopus (41) Google Scholar) showed that the W2A mutation both abrogates the weak EC12-mediated bond and substantially attenuates the strong, EC3-dependent binding. Tsuji et al. (17Tsuiji H. Xu L. Schwartz K. Gumbiner B.M. J. Biol. Chem. 2007; 282: 12871-12882Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) similarly reported weak residual binding between ectodomain dimers with the W2A mutation, and showed that this mutation disrupts lateral C-cadherin dimers on the cell surface. Prakasam et al. (16Prakasam A. Chien Y.H. Maruthamuthu V. Leckband D.E. Biochemistry. 2006; 45: 6930-6939Crossref PubMed Scopus (41) Google Scholar) postulated that W2 mediates the EC1 bond and allosterically regulates the activity of other domains, e.g. EC3 in the extracellular segment. Nevertheless, the translation of these force measurements at the molecular level to adhesion at the cell level has not yet been demonstrated. Quantitative biophysical studies of cadherins have been based primarily on measurements of soluble, cadherin extracellular domains (3Boggon T.J. Murray J. Chappuis-Flament S. Wong E. Gumbiner B.M. Shapiro L. Science. 2002; 296: 1308-1313Crossref PubMed Scopus (548) Google Scholar, 4Nose A. Tsuji K. Takeichi M. Cell. 1990; 61: 147-155Abstract Full Text PDF PubMed Scopus (414) Google Scholar, 5Shapiro L. Fannon A.M. Kwong P.D. Thompson A. Lehmann M.S. Grubel G. Legrand J.F. Als-Nielsen J. Colman D.R. Hendrickson W.A. Nature. 1995; 374: 327-337Crossref PubMed Scopus (978) Google Scholar, 9Sivasankar S. Brieher W. Lavrik N. Gumbiner B. Leckband D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11820-11824Crossref PubMed Scopus (145) Google Scholar, 10Sivasankar S. Gumbiner B. Leckband D. Biophys. J. 2001; 80: 1758-1768Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15Perret E. Leung A. Feracci H. Evans E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16472-16477Crossref PubMed Scopus (108) Google Scholar, 16Prakasam A. Chien Y.H. Maruthamuthu V. Leckband D.E. Biochemistry. 2006; 45: 6930-6939Crossref PubMed Scopus (41) Google Scholar, 18Ahrens T. Pertz O. Haussinger D. Fauser C. Schulthess T. Engel J. J. Biol. Chem. 2002; 277: 19455-19460Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 19Koch A.W. Pokutta S. Lustig A. Engel J. Biochemistry. 1997; 36: 7697-7705Crossref PubMed Scopus (130) Google Scholar, 20Pertz O. Bozic D. Koch A.W. Fauser C. Brancaccio A. Engel J. EMBO J. 1999; 18: 1738-1747Crossref PubMed Scopus (344) Google Scholar, 21Pokutta S. Herrenknecht K. Kemler R. Engel J. Eur. J. Biochem. 1994; 223: 1019-1026Crossref PubMed Scopus (250) Google Scholar, 22Tomschy A. Fauser C. Landwehr R. Engel J. EMBO J. 1996; 15: 3507-3514Crossref PubMed Scopus (168) Google Scholar). The underlying assumption that the truncated, soluble ectodomain accurately models the full-length, membrane-bound protein is untested. In the case of integrins, for example, allosteric coupling between the cytoplasmic and extracellular regions underlies outside-in and inside-out signaling (23Hynes R.O. 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There is also evidence for inside-out signaling in the cadherin family. In Xenopus embryos, chemokines can activate cleavage stage cadherin, independent of changes in cadherin expression (28Brieher W.M. Gumbiner B.M. J. Cell Biol. 1994; 126: 519-527Crossref PubMed Scopus (139) Google Scholar, 29Zhong Y. Brieher W.M. Gumbiner B.M. J. Cell Biol. 1999; 144: 351-359Crossref PubMed Scopus (93) Google Scholar). Src kinase activation correlates with the disruption of (cadherin-mediated) cell-cell junctions (30Avizienyte E. Wyke A.W. Jones R.J. McLean G.W. Westhoff M.A. Brunton V.G. Frame M.C. Nat. Cell Biol. 2002; 4: 632-638Crossref PubMed Scopus (306) Google Scholar, 31Wang Y. Botvinick E.L. Zhao Y. Berns M.W. Usami S. Tsien R.Y. Chien S. Nature. 2005; 434: 1040-1045Crossref PubMed Scopus (583) Google Scholar). Apparent differences between adhesion by soluble ectodomains and membrane-bound cadherin also suggest some influence from the cytoplasmic domain (32Panorchan P. Thompson M.S. Davis K.J. Tseng Y. Konstantopoulos K. Wirtz D. J. Cell Sci. 2006; 119: 66-74Crossref PubMed Scopus (170) Google Scholar). There is, however, no direct comparison of the binding properties of soluble, recombinant, and membrane-bound cadherin. Only a few techniques allow quantitative comparisons between recombinant, soluble protein, and the cell surface forms. Comparing the trajectories of cells in flow chambers with those of protein-decorated beads could test this (33Kaplanski G. Farnarier C. Tissot O. Pierres A. Benoliel A.M. Alessi M.C. Kaplanski S. Bongrand P. Biophys. J. 1993; 64: 1922-1933Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 34Pierres A. Benoliel A.M. Bongrand P. J. Biol. Chem. 1995; 270: 26586-26592Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 35Pierres A. Benoliel A.M. Bongrand P. J. Immunol. Methods. 1996; 196: 105-120Crossref PubMed Scopus (40) Google Scholar). One could also compare the bond rupture forces between soluble ectodomains (15Perret E. Leung A. Feracci H. Evans E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16472-16477Crossref PubMed Scopus (108) Google Scholar) with those between soluble ectodomains and membrane-bound cadherin (32Panorchan P. Thompson M.S. Davis K.J. Tseng Y. Konstantopoulos K. Wirtz D. J. Cell Sci. 2006; 119: 66-74Crossref PubMed Scopus (170) Google Scholar). Such comparisons are possible with the micropipette manipulation technique, which quantifies binding between individual cell pairs bearing complementary receptors and ligands (36Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 37Evans E. Berk D. Leung A. Biophys. J. 1991; 59: 838-848Abstract Full Text PDF PubMed Scopus (275) Google Scholar). Live cells with surface-bound receptors and ligands are aspirated into opposite micropipettes, and brought into contact for a defined period. Typically, at least one of the cells is a red blood cell (RBC). Adhesion causes the RBC to distort during separation, and to then recoil at bond rupture. The RBC distortion gives the adhesion strength (37Evans E. Berk D. Leung A. Biophys. J. 1991; 59: 838-848Abstract Full Text PDF PubMed Scopus (275) Google Scholar). Alternatively, the kinetic rates and two-dimensional affinities of the receptor-ligand bonds are quantified from the dependence of the binding probability on the cell contact time (36Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). The binding probability is the number of detected binding events divided by the total number of cell-cell contacts. Because these micropipette measurements are also used to quantify the binding kinetics between soluble protein fragments immobilized on RBCs, they enable quantitative comparisons of the properties of recombinant proteins with their membrane bound forms. Micropipette measurements have been used to study interactions between Fcγ receptors, selectins, integrins, and CD8 with their respective receptors (36Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 38Zhang F. Marcus W.D. Goyal N.H. Selvaraj P. Springer T.A. Zhu C. J. Biol. Chem. 2005; 280: 42207-42218Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 39Huang J. Edwards L.J. Evavold B.D. Zhu C. J. Immunol. 2007; 179: 7653-7662Crossref PubMed Scopus (72) Google Scholar, 40Chesla S.E. Li P. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2000; 275: 10235-10246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 41Long M. Zhao H. Huang K.S. Zhu C. Ann. Biomed. Eng. 2001; 29: 935-946Crossref PubMed Scopus (58) Google Scholar, 42Williams T.E. Nagarajan S. Selvaraj P. Zhu C. Biophys. J. 2000; 79: 1867-1875Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 43Williams T.E. Selvaraj P. Zhu C. Biophys. J. 2000; 79: 1858-1866Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 44Williams T.E. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2001; 276: 13283-13288Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Here, we describe micropipette binding probability measurements of the pre-steady state kinetics of intercellular adhesion mediated by Xenopus cleavage stage cadherin. The binding probability curves exhibit complex kinetics characterized by a fast, low probability binding state and a slower forming, high probability binding state. This kinetic behavior contrasts with the simple, monophasic rise to a limiting binding probability, predicted by a single site binding model. Studies with isolated extracellular domains tested the impact of the cytoplasmic domain on the cadherin binding dynamics. In addition, by deleting either the EC3 or EC3–5 domains we identified protein segments required for this complex kinetic behavior. The biphasic kinetics exhibited by the full-length extracellular region directly parallels prior biophysical studies, and confirms that the nanomechanical properties of the recombinant cadherin ectodomain govern the initial dynamics of cell-cell contact formation. Cell Lines, Proteins, and Plasmids—The pEE14 plasmid containing the full-length Xenopus C-cadherin cDNA, the pEE14 plasmid containing the cDNA encoding the hexahistidinetagged C-cadherin extracellular domain with the W2A mutation, and the CHO cell line expressing the full-length C-cadherin with the W2A mutation were generously provided by B. Gumbiner (University of Virginia). To generate cell lines expressing the W2A mutant of soluble CEC1–5-His6 and the full-length C-cadherin, CHO-K1 cells were stably transfected with the plasmids encoding the different C-cadherin constructs. Cells were transfected using Lipofectamine 2000, according to the manufacturer's protocols (Invitrogen). The cells were cultured in Glasgow MEM medium supplemented with 10% dialyzed fetal calf serum, and selected with 25 μm methionine sulfoximine (MSX) as described (13Chappuis-Flament S. Wong E. Hicks L.D. Kay C.M. Gumbiner B.M. J. Cell Biol. 2001; 154: 231-243Crossref PubMed Scopus (188) Google Scholar). For the secreted proteins, the clone with highest protein production rate was identified by Western blot. The mouse monoclonal anti-His antibody (Upstate, Charlottesville, VA) and goat anti-mouse HRP conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were used to detect the protein. Prior to the micropipette experiment, the cells were detached from the flask with Hank's Balanced Salt Solution (HBSS) (Invitrogen, Carlsbad, CA), with 0.01% trypsin and 1 mm CaCl2 for 15 min (45Nose A. Nagafuchi A. Takeichi M. Cell. 1988; 54: 993-1001Abstract Full Text PDF PubMed Scopus (556) Google Scholar). The cells were incubated in phosphate buffered saline (PBS) with 5 mm EDTA and 1% bovine serum albumin (BSA) at 4 °C for at least 30 min, and then used in micropipette assays within 12 h. Prior to use, the EDTA was removed and replaced with 2 mm Ca2+. The production and purification of the soluble C-cadherin extracellular domain deletion mutants EC1245-Fc and EC12-Fc, CEC1–5-His6, and W2A CEC1–5-His6 are described elsewhere (11Zhu B. Chappuis-Flament S. Wong E. Jensen I.E. Gumbiner B.M. Leckband D. Biophys. J. 2003; 84: 4033-4042Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 13Chappuis-Flament S. Wong E. Hicks L.D. Kay C.M. Gumbiner B.M. J. Cell Biol. 2001; 154: 231-243Crossref PubMed Scopus (188) Google Scholar). The protein purity was assessed by SDS-PAGE. The aggregation activity of each protein was further characterized with bead aggregation assays (16Prakasam A. Chien Y.H. Maruthamuthu V. Leckband D.E. Biochemistry. 2006; 45: 6930-6939Crossref PubMed Scopus (41) Google Scholar). Sample Configuration and Preparation in the Micropipette Manipulation Assays—Fig. 1A exemplifies the configuration of the cells in the micropipette manipulation experiment. In this case, the CHO cell on the left expresses the full-length, wild-type C-cadherin (C-CHO) (Fig. 1B). The RBC on the right is modified with anti-hexahistidine antibody, which in turn captured soluble hexahistidine-tagged C-cadherin ectodomain fragments (CEC1–5His6). In other cases, the capture antibody was anti-human IgG, and the C-cadherin fragment was C-terminally fused to a human Fc tag (Fig. 1B). In a third configuration (not shown), both cells were modified RBCs. The monoclonal anti-hexahistidine (Upstate) or anti-human immunoglobin G Fc domain antibodies (Sigma-Aldrich), were used to capture cadherin on the RBC surface. The antibodies were attached chemically to red blood cell surfaces, using CrCl3 coupling chemistry (46Gold E.R. Fudenberg H.H. J. Immunol. 1967; 99: 859-866PubMed Google Scholar, 47Kofler R. Wick G. J. Immunol. Methods. 1977; 16: 201-209Crossref PubMed Scopus (63) Google Scholar). RBCs were obtained from the peripheral blood of a healthy donor, which was collected in sterile Vacutainers (BD Biosciences, San Jose, CA) containing EDTA, using a protocol approved by the Institutional Review Board of Georgia Institute of Technology (38Zhang F. Marcus W.D. Goyal N.H. Selvaraj P. Springer T.A. Zhu C. J. Biol. Chem. 2005; 280: 42207-42218Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). After centrifugation, the RBCs were collected and washed with 0.9% NaCl, and then resuspended in red blood cell storage solution EAS 45 (48Dumaswala U.J. Wilson M.J. Jose T. Daleke D.L. Blood. 1996; 88: 697-704Crossref PubMed Google Scholar). About 108 red blood cells (in 100 μl of EAS 45) were collected for the CrCl3 coupling reaction. The cells were resuspended in 250 μl saline (0.85% NaCl, w/v) containing 1 μg capture antibody. To these cells were added 250 μl of CrCl3 solution (in 0.02 m acetate and 0.85% NaCl, w/v). However, to vary the surface density of capture antibodies, we varied the final concentrations of CrCl3 between 10–6 and 10–4 % (w/v). After 5 min, the reaction was quenched with 500 μl of PBS containing 5 mm EDTA and 1% BSA. The thus modified RBCs were stored in EAS 45 buffer at 4 °C. The RBCs can be thus stored for up to 3 weeks without significant hemolysis. To couple the different Xenopus C-cadherin extracellular fragments to the antibody-functionalized RBC surface, 1 μgof hexahistidine or human Fc-tagged C-cadherin fragments were incubated with 2 × 105 antibody-labeled RBCs at 4 °C for 1 h in 100 μl of phosphate-buffered saline, supplemented with 5 mm EDTA and 1% BSA. The cells were then pelleted and rinsed, in order to remove unbound cadherin. The thus modified cells were used for micropipette measurements. Quantifying the Cadherin Surface Density on RBCs and CHO Cells—The cadherin density on the cells was determined by fluorescence-activated cell sorting (FACS). Calibrated fluorescein isothiocyanate (FITC)-labeled standard beads (Bangs Laboratories, Fishers, IN) were used as a reference. Monoclonal anti-C-cadherin antibody (1 μg) (Santa Cruz Biotechnology) was used to stain the C-cadherin-labeled cells, which were stored in phosphate-buffered saline with 0.5 mm EDTA and 1% BSA at 4 °C for 30 min. Cells were then stained with 1 μgof FITC-conjugated anti-goat IgG antibody in phosphate-buffered saline with 0.5 mm EDTA and 1% BSA at 4 °C for 30 min. The fluorescence intensity of the labeled cells was quantified with a BD LSR flow cytometer (BD Biosciences, San Jose, CA) as described (38Zhang F. Marcus W.D. Goyal N.H. Selvaraj P. Springer T.A. Zhu C. J. Biol. Chem. 2005; 280: 42207-42218Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The fluorescence intensity for each population of the five standard beads was used to determine the fluorescence calibration curve. The protein density on the cells was determined by dividing the number of fluorophores on the cells by the estimated cell surface area. The fluorophore density on the cells was quantified by comparing the total fluorescence intensity on the cells against a calibration curve generated with the FITC standard beads. Micropipette Measurement of Cell Adhesion Dynamics—Adhesion probability measurements with the micropipette were conducted as described previously (36Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Both the C-cadherinexpressing CHO K1 cells (C-CHO) and modified RBCs were incubated in the sample chamber mounted on the microscope stage. The chamber was filled with L-15 medium (Invitrogen) containing 1.26 mm CaCl2, and supplemented with 1% BSA. Cells were aspirated into each of the two micropipettes, which were then used to position the cells adjacent to each other. The contact area was adjusted to ∼3 μm2 (∼2 μm diameter). One of the micropipette manipulators is interfaced to the computer via a piezoelectric actuator, which controls the cell contact time and changes the micropipette position by moving cells in and out of contact at a speed of 0.1 μm/s. The intercellular contact time is operator-programmed. The RBC deformations during cell contact and retraction are visualized in real time with a CCD camera and TV monitor. An adhesion event is identified from the elongation of the RBC during micropipette retraction. Adhesion is scored as 1, while a non-adhesion event is scored as 0. Each cell pair was subjected to 50–100 contact-retraction cycles, after which we determined the binding probability. The binding probability is defined as the number of binding events detected per total number of cell contact-retraction cycles attempted. For each cell-cell contact time, we measured three to five pairs of cells (n > 150). Data are represented as the mean ± S.D. from the mean. Cell pairs consisted of (i) a transfected CHO cell and a cadherin-modified RBC or (ii) two cadherin-modified RBCs. The RBCs were modified with either hexahistidine-tagged or Human Fc-tagged soluble ectodomain fragments. The CHO cells expressed either the wild-type C-cadherin or the W2A mutant of the full-length C-cadherin. The three different control measurements consisted of (i) an antibody-coated RBC (no cadherin) interacting with a C-CHO. The second set of control measurements were done with 5 mm EDTA in the chamber medium, and, in the third control, we first incubated the cadherin-coated RBC with 4 μg of anti-C-cadherin, polyclonal blocking antibody for 30 min. Adhesion between CHO Cells Expressing Wild-type C-Cadherin and RBCs Coated with CEC1–5-His6 Exhibits Biphasic Kinetics—Fig. 2A shows a representative time course of the binding probability obtained with the experimental configuration in Fig. 1. The cadherin densities on the CHO cell and RBC were ∼7 and 3/μm2, respectively. Instead of a simple rise to a limiting plateau, as observed with other receptors (36Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 38Zhang F. Marcus W.D. Goyal N.H. Selvaraj P. Springer T.A. Zhu C. J. Biol. Chem. 2005; 280: 42207-42218Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 39Huang J. Edwards L.J. Evavold B.D. Zhu C. J. Immunol. 2007; 179: 7653-7662Crossref PubMed Scopus (72) Google Scholar, 40Chesla S.E. Li P. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2000; 275: 10235-10246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 41Long M. Zhao H. Huang K.S. Zhu C. Ann. Biomed. Eng. 2001; 29: 935-946Crossref PubMed Scopus (58) Google Scholar, 42Williams T.E. Nagarajan S. Selvaraj P. Zhu C. Biophys. J. 2000; 79: 1867-1875Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 43Williams T.E. Selvaraj P. Zhu C. Biophys. J. 2000; 79: 1858-1866Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 44Williams T.E. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2001; 276: 13283-13288Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), this time course is biphasic, and exhibits two different, consecutive kinetic stages. Within the first 2 s, the background-corrected binding probability increases rapidly to the first plateau at ∼0.2; that is, ∼20% of cell-cell contacts result in adhesion. This low probability state is followed by a 2–5-s lag phase, and subsequent transition to a high probability binding state and second plateau. At these cadherin densities (Fig. 2A), the upper plateau is at 0.87 ± 0.05. There was no further change in the binding probability after 20 s. Several control measurements assessed the background adhesion. These included measurements between C-CHO and antibody-modified RBCs without the cadherin ectodomains (blank). A second control was conducted with 5 mm EDTA in the medium. In the third control, measurements were done in the presence of polyclonal anti-C-cadherin antibody, which blocks cadherin adhesion (blocking AB). These controls
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