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• W2040645020 abstract "The tight junction protein ZO-1 belongs to a family of multidomain proteins known as the membrane-associated guanylate kinase homologs (MAGUKs). ZO-1 has been demonstrated to interact with the transmembrane protein occludin, a second tight junction-specific MAGUK, ZO-2, and F-actin, although the nature and functional significance of these interactions is poorly understood. To further elucidate the role of ZO-1 within the epithelial tight junction, we have introduced epitope-tagged fragments of ZO-1 into cultured MDCK cells and identified domains critical for the interaction with ZO-2, occludin, and F-actin. A combination of in vitroand in vivo binding assays indicate that both ZO-2 and occludin interact with specific domains within the N-terminal (MAGUK-like) half of ZO-1, whereas the unique proline-rich C-terminal half of ZO-1 cosediments with F-actin. Consistent with these observations, we found that a construct encoding the N-terminal half of ZO-1 is specifically associated with tight junctions, whereas the unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures. In addition, we have identified a 244-amino acid domain within the N-terminal half of ZO-1, which is required for the stable incorporation of ZO-1 into the junctional complex of polarized MDCK cells. These observations suggest that one functional role of ZO-1 is to organize components of the tight junction and link them to the cortical actin cytoskeleton. The tight junction protein ZO-1 belongs to a family of multidomain proteins known as the membrane-associated guanylate kinase homologs (MAGUKs). ZO-1 has been demonstrated to interact with the transmembrane protein occludin, a second tight junction-specific MAGUK, ZO-2, and F-actin, although the nature and functional significance of these interactions is poorly understood. To further elucidate the role of ZO-1 within the epithelial tight junction, we have introduced epitope-tagged fragments of ZO-1 into cultured MDCK cells and identified domains critical for the interaction with ZO-2, occludin, and F-actin. A combination of in vitroand in vivo binding assays indicate that both ZO-2 and occludin interact with specific domains within the N-terminal (MAGUK-like) half of ZO-1, whereas the unique proline-rich C-terminal half of ZO-1 cosediments with F-actin. Consistent with these observations, we found that a construct encoding the N-terminal half of ZO-1 is specifically associated with tight junctions, whereas the unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures. In addition, we have identified a 244-amino acid domain within the N-terminal half of ZO-1, which is required for the stable incorporation of ZO-1 into the junctional complex of polarized MDCK cells. These observations suggest that one functional role of ZO-1 is to organize components of the tight junction and link them to the cortical actin cytoskeleton. amino acid(s) glutathione S-transferase membrane-associated guanylate kinase homolog Src homology 3 Madin-Darby canine kidney phosphate-buffered saline polyacrylamide gel electrophoresis PSD95/dlg/ZO-1. The tight junction forms the apical barrier to the paracellular movement of water, solutes and immune cells in polarized epithelia (1Cereijido, M. (ed) ull-92) Tight Junctions, pp. 1–13, CRC, Boca Raton, FLGoogle Scholar). Electron micrographs reveal that the paracellular seal is composed of a series of highly ordered membrane contact sites (2Farquhar M.G. Palade G.E. J. Cell Biol. 1963; 17: 412Crossref Scopus (2132) Google Scholar), which in freeze-fracture micrographs can be visualized as a series of interconnected fibrils within the plasma membrane (3Staehelin L.A. Int. Rev. Cytol. 1974; 39: 191-282Crossref PubMed Scopus (1050) Google Scholar). Actin filaments terminate at these membrane contact sites (4Hirokawa N. Tilney L.G. J. Cell Biol. 1982; 95: 249-261Crossref PubMed Scopus (249) Google Scholar, 5Madara J.L. Pappenheimer J.R. J. Membr. Biol. 1987; 100: 149-164Crossref PubMed Scopus (426) Google Scholar), suggesting that the cytoskeleton is involved in the structural and functional organization of the tight junction. Although many of the molecular components of the tight junction have now been identified, little is known about how they interact to regulate the assembly and permeability of the paracellular barrier. At least one transmembrane component of the tight junction, occludin (6Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2129) Google Scholar), has been demonstrated to contribute to the paracellular seal in cultured cells (7Balda M.S. Whitney J.A. Flores C. Gonzalez S. Cereijido M. Matter K. J. Cell Biol. 1996; 134: 1031-1049Crossref PubMed Scopus (732) Google Scholar, 8McCarthy K.M. Skare I.B. Stankewich M.D. Furuse M. Tsukita S. Rogers R.A. Lynch R.D. Schneeberger E.E. J. Cell Sci. 1996; 109: 2287-2298Crossref PubMed Google Scholar, 9Chen Y. Merzdorf C. Paul D.L. Goodenough D.A. J. Cell Biol. 1997; 138: 891-899Crossref PubMed Scopus (254) Google Scholar, 10Wong V. Gumbiner B.M. J. Cell Biol. 1997; 136: 399-409Crossref PubMed Scopus (456) Google Scholar). More recently, investigators have identified two novel transmembrane proteins, claudin-1 and claudin-2, which are also presumably components of the paracellular seal (11Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1715) Google Scholar). Both occludin and claudins localize to the discrete membrane contact sites, or kisses, within the freeze-fracture fibrils (6Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2129) Google Scholar). These kisses are in turn intimately associated with the cytosolic plaque proteins ZO-1 (12Stevenson B.R. Siliciano J.D. Mooseker M. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1285) Google Scholar), ZO-2 (13Jesaitis L.A. Goodenough D.A. J. Cell Biol. 1994; 124: 949-961Crossref PubMed Scopus (387) Google Scholar), and ZO-3 (14Balda M.S. Gonzales-Mariscal L. Matter L. Contreras R.G. Cereijido M. Anderson J.M. J. Cell Biol. 1993; 123: 293-302Crossref PubMed Scopus (352) Google Scholar). These proteins can be co-immunoprecipitated as a complex from cultured epithelial cells (14Balda M.S. Gonzales-Mariscal L. Matter L. Contreras R.G. Cereijido M. Anderson J.M. J. Cell Biol. 1993; 123: 293-302Crossref PubMed Scopus (352) Google Scholar,15Gumbiner B. Lowenkopf T. Apatira D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3460-3464Crossref PubMed Scopus (430) Google Scholar), and have been demonstrated to bind directly to the C-terminal 146 amino acids (aa)1 of occludin (16Furuse M. Itoh M. Hirase T. Nagafuchi F. Yonemura S. Tsukita S. J. Cell Biol. 1994; 127: 1617-1626Crossref PubMed Scopus (808) Google Scholar). Other components of the cytosolic plaque include cingulin (17Citi S. Sabannay H. Jakes R. Geiger B. Kendrick-Jones J. Nature. 1988; 333: 272-275Crossref PubMed Scopus (404) Google Scholar), 7H6 (18Zhong Y. Saitoh T. Minase T. Sawada N. Enomoto K. Mori M. J. Cell Biol. 1993; 120: 477-483Crossref PubMed Scopus (244) Google Scholar), AF-6 (19Yamamoto T. Harada N. Kano K. Taya S. Canaani E. Matsuura Y. Mizoguchi A. Ide C. Kaibuchi K. J. Cell Biol. 1997; 139: 785-795Crossref PubMed Scopus (285) Google Scholar), and symplekin (20Keon B.H. Schafer S. Kuhn C. Grund C. Franke W.W. J. Cell Biol. 1996; 134: 1003-1018Crossref PubMed Scopus (267) Google Scholar). The plaque proteins ZO-1, ZO-2, and ZO-3 probably play a unique role in the organization/regulation of tight junctions. These proteins are members of a family of membrane-associated signaling proteins known as the membrane-associated guanylate kinase homologs (MAGUKs), which include the Drosophila tumor suppressor dlg (21Woods D.F. Bryant P.J. Dev. Biol. 1989; 134: 222-235Crossref PubMed Scopus (148) Google Scholar), theCaenorhabditis elegans signaling protein LIN2 (22Hoskins R. Hajnal A. Harp S. Kim S.K. Development. 1995; 122: 97-111Google Scholar), synaptic proteins hDlg and PSD95 (23Lue R. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 91: 9818-9822Crossref Scopus (344) Google Scholar, 24Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1006) Google Scholar, 25Kistner U. Wenzel B.M. Veh R.W. Cases-Langhoff C. Garner A.M. Appeltauer U. Voss B. Gundelfinger E.D. Garner C.C. J. Biol. Chem. 1993; 268: 4580-4583Abstract Full Text PDF PubMed Google Scholar), and the erythrocyte membrane protein p55 (26Ruff P. Speicher D.W. Husain-Chishti A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6595-6599Crossref PubMed Scopus (139) Google Scholar). Members of this family are distinguished by a core cassette of protein-binding domains, which include one or more PSD95/dlg/ZO-1 (PDZ) domains, an SH3 domain, and a region of homology to guanylate kinase (GuK) (27Fanning A.S. Lapierre L.A. Brecher A.R. Van Itallie C.M. Anderson J.M. Curr. Top. Membr. 1996; 43: 211-235Crossref Scopus (22) Google Scholar). All of these proteins are associated with cell-cell contact sites such as synapses, intercalated disks, and epithelial tight junctions. Mutations in the genes for Dlg and LIN2 cause severe alteration in cell growth and differentiation (21Woods D.F. Bryant P.J. Dev. Biol. 1989; 134: 222-235Crossref PubMed Scopus (148) Google Scholar, 22Hoskins R. Hajnal A. Harp S. Kim S.K. Development. 1995; 122: 97-111Google Scholar), suggesting that MAGUKs are involved in signal transduction pathways controlling growth and differentiation (27Fanning A.S. Lapierre L.A. Brecher A.R. Van Itallie C.M. Anderson J.M. Curr. Top. Membr. 1996; 43: 211-235Crossref Scopus (22) Google Scholar, 28Anderson J.M. Curr. Biol. 1996; 6: 326-329Abstract Full Text Full Text PDF Scopus (219) Google Scholar, 29Kornau H.C. Seeburg P.H. Kennedy M.B. Curr. Opin. Neurobiol. 1997; 7: 368-373Crossref PubMed Scopus (313) Google Scholar). The role of MAGUKs in signal transduction may be due in large part to the demonstrated ability of these proteins to organize protein complexes at the plasma membrane. There are several examples in which expression of a given MAGUK with its transmembrane binding partner results in clustering of both proteins within the plasma membrane (30Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng S. Nature. 1995; 378: 85-88Crossref PubMed Scopus (897) Google Scholar,31Kim E. Cho K.O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). This property appears to be due to the presence of multiple protein binding motifs within these proteins, as well as their ability to form heterodimers with other MAGUKs. This organizational capacity may also be enhanced by interactions with the cytoskeleton since hdlg, p55, and the human LIN2 homolog all bind to the actin-binding protein 4.1 via a conserved motif located between the SH3 and GUK domains (23Lue R. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 91: 9818-9822Crossref Scopus (344) Google Scholar,32Marfatia S.M. Lue R.A. Branton D. Chishti A.H. J. Biol. Chem. 1995; 270: 715-719Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 33Cohen A.R. Woods D.F. Marfatia S.M. Walther Z. Chishti A.H. Anderson J.M. J. Cell Biol. 1998; 142: 129-138Crossref PubMed Scopus (321) Google Scholar). Many members of this family have been demonstrated to interact directly with ion channels, transmembrane receptors, and known cytosolic signal transduction proteins (34Matsumine A. Ogai A. Senda T. Okumura N. Satoh K. Baeg G.H. Kawahara T. Kobayashi S. Okada M. Toyoshima K. Akiyama T. Science. 1996; 272: 1020-1023Crossref PubMed Scopus (409) Google Scholar, 35Hanada T. Lin L. Chandy K.G. Oh S.S. Chishti A.H. J. Biol. Chem. 1997; 272: 26899-26904Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 36Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1443) Google Scholar). These observations suggest that MAGUKs can act as a scaffold for signal transduction complexes, organizing cytosolic signaling molecules at the plasma membrane with transmembrane receptors and ion channels. It is likely that the tight junction MAGUKs like ZO-1 have organizational/functional roles analogous to other MAGUKs. ZO-1 binds to several other tight junction components (13Jesaitis L.A. Goodenough D.A. J. Cell Biol. 1994; 124: 949-961Crossref PubMed Scopus (387) Google Scholar, 16Furuse M. Itoh M. Hirase T. Nagafuchi F. Yonemura S. Tsukita S. J. Cell Biol. 1994; 127: 1617-1626Crossref PubMed Scopus (808) Google Scholar, 37Haskins J. Gu L. Wittchen E. Hibbard J. Stevenson B.R. J. Cell Biol. 1998; 141: 199-208Crossref PubMed Scopus (494) Google Scholar) and has also been demonstrated to interact with several known signaling proteins, such as the Ras substrate AF-6 (19Yamamoto T. Harada N. Kano K. Taya S. Canaani E. Matsuura Y. Mizoguchi A. Ide C. Kaibuchi K. J. Cell Biol. 1997; 139: 785-795Crossref PubMed Scopus (285) Google Scholar), heterotrimeric G-proteins (38Denker B.M. Saha C. Khawaja S. Nigam S.K. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), an unidentified serine kinase (39Balda M.S. Anderson J.M. Matter K. FEBS Lett. 1996; 399: 326-332Crossref PubMed Scopus (94) Google Scholar), and connexin 43 (40Toyofuku T. Yabuki M. Otsu K. Kuzuya T. Hori M. Tada M. J. Biol. Chem. 1998; 273: 12725-12731Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). ZO-1 has also been shown to bind directly to F-actin in vitro (41Itoh M. Nagafuchi A. Moroi S. Tsukita S. J. Cell Biol. 1997; 138: 181-192Crossref PubMed Scopus (570) Google Scholar). However, the mechanism of these interactions and their relevance to tight junction assembly is poorly understood. For example, it is not known whether the binding sites for other tight junction proteins reside within the MAGUK-like N-terminal half or within the C-terminal domain, which is unique to tight junction MAGUKs. In addition, although ZO-1 interacts with the cytoskeleton like other MAGUKs, it lacks the band 4.1 binding site found in other members of this family. To better understand the role of MAGUK proteins like ZO-1 in the tight junction, we have used epitope-tagged deletion constructs to map the binding sites for ZO-2, occludin, and F-actin on ZO-1. Furthermore, we have examined how these domains are involved in the assembly of ZO-1 into the tight junction complex. Our results suggest that ZO-1 may serve as a link between the proteins of the tight junction and the actin cytoskeleton. A diagram of the expression constructs used in this study is shown in Fig. 1. To insert an epitope tag on the C terminus of the full-length human ZO-1 (ZO1myc), a 203-base pair fragment of the C terminus was amplified from a ZO-1 cDNA using the sense primer 5′-GAAGATGGTCATACTGTGG-3′, which is complimentary to a sequence 5′ of a unique MscI site in ZO-1, and the antisense primer 5′-GGACTAGTTTACAAGTCCTCTTCAGATATCAGCTTTTGCTCGGCAAAGTGGTCAATAAGGACAG-3′. This antisense primer contained a 20-base pair region of homology to ZO-1 (bold) followed by a sequence encoding an alanine linker, an 11-amino acid epitope from the c-Myc protein, a stop codon, and aSpeI restriction enzyme site. This product was amplified using Taq polymerase (Promega, Madison, WI), digested withMscI and SpeI (New England Biolabs, Beverly, MA), and subcloned into a pSK+ Bluescript plasmid (Stratagene, La Jolla, CA) containing ZO-1, which had been digested with MscI and SpeI to remove the 203-base pair sequence encoding the native C terminus. All C-terminal deletion constructs were produced using a variation of this technique. N-terminal deletion constructs were produced by digesting ZO1myc with restriction enzymes that removed specific DNA sequences (for example amino acids 2–156 for del 2–156), and replacing those sequences with an annealed pair of complimentary oligonucleotides that reestablish the appropriate reading frame. Exact details of plasmid construction are available from the authors by request. The pSK+ Bluescript constructs described here were subsequently digested with KpnI and SpeI and subcloned into the KpnI and XbaI sites of the eukaryotic expression vector pCB6 (Karl Matter, University of Geneva, Geneva, Switzerland). MDCK cells were obtained from Michael Caplan (Yale University, New Haven, CT) and maintained as described previously (14Balda M.S. Gonzales-Mariscal L. Matter L. Contreras R.G. Cereijido M. Anderson J.M. J. Cell Biol. 1993; 123: 293-302Crossref PubMed Scopus (352) Google Scholar). Expression constructs were introduced into cells using a variation on standard calcium-phosphate mediated transfection (42Chen C.A. Okayama A. BioTechniques. 1988; 6: 632-638Crossref PubMed Scopus (28) Google Scholar). For the isolation of stably transfected lines, cells were selected in 1.0 mg/ml G418 (Life Technologies, Inc.). Colonies were isolated using cloning loops and subsequently analyzed for transgene expression by immunofluorescence and immunoblotting with the anti-myc monoclonal antibody 9E10. In all assays, transgene expression was induced by supplementing the normal growth medium with 5.0 mm sodium butyrate. To examine the interactions between ZO-1 and ZO-2, two 60-mm dishes of transfected MDCK cells were placed on ice, washed twice with PBS, and incubated for 30 min in 1.0 ml of extraction/binding buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 2 mm EDTA, 1.0% Triton X-100, 0.05% SDS, 1.0 mg/ml bovine serum albumin, 1 mmdithiothreitol, 20 mm phenylmethylsulfonyl fluoride, 20 mm benzamidine, 1.0 μg/ml aprotinin, 1.0 μg/ml leupeptin, 1.0 μg/ml antipain). Cell lysates were transferred to a microcentrifuge tube and clarified for 30 min at 18,000 ×g. The supernatants were mixed with 40 μl of a 1:1 slurry of Protein A-Sepharose CL-4B (Pharmacia Biotech, Uppsala, Sweden), incubated for 2.0 h at 4 °C, and centrifuged for 1 min at 500 × g. Supernatants were then transferred to a new tube, mixed with 5 μl of the anti-myc monoclonal antibody 9E10, and incubated for 2–15.0 h at 4 °C. Following this incubation, 40 μl of a 1:1 slurry of Protein A-Sepharose were added and incubated for 2.0 h at 4 °C. The Sepharose beads were washed three times in extraction/binding buffer, once in extraction/binding buffer without Triton X-100 or SDS, and resuspended in 50 μl of 2× gel sample buffer (43Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1997Google Scholar). These immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose filters, and analyzed by Western blotting using standard techniques (43Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1997Google Scholar). Filters were incubated for 2.0 h in a 1:2500 dilution of the anti-ZO-2 polyclonal antiserum R9989, and developed using the enhanced chemiluminescence technique (Amersham Pharmacia Biotech). The same filters were then stripped (Amersham Pharmacia Biotech) and incubated with a 1:1000 dilution of the anti-myc ascites 9E10 or a 1:300 dilution of a culture supernatant from the anti-ZO-1 hybridoma R40.76. To assess the binding of different epitope-tagged proteins to occludin, confluent monolayers of transfected MDCK cells were washed twice with PBS+ (containing 1.0 mm CaCl2 and 1.0 mm MgCl2) and lysed in extraction/binding buffer as described for ZO-2 co-immunoprecipitations. The supernatants were then transferred to a new tube, mixed with 40 μl of a 1:1 slurry of GST beads, and incubated for 2.0 h at 4 °C. The GST beads were sedimented by a quick pulse in a microcentrifuge, and the supernatant was transferred to a new tube. A GST fusion protein encoding aa 358–504 of chicken occludin (GST-occ) (44Fallon M.B. Brecher A. Balda M.S. Matter K. Anderson J.M. Am. J. Physiol. 1995; 260: C1057-C1062Crossref Google Scholar) was prepared as described by Ausubel et al. (43Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1997Google Scholar), and 40 μl of a 1:1 slurry of GST-occ was added to the supernatant and incubated overnight (12–18 h) at 4 °C. The GST-occ beads were then washed, resolved by SDS-PAGE, and analyzed by Western blotting with the 9E10 sera. Cells were plated into 100-mm dishes at confluent density, grown for 2 days, and induced for 20 h with 5.0 mm sodium butyrate. For each construct, two dishes of cells were washed twice with PBS+ and scraped on ice into 1.0 ml of a hypotonic carbonate buffer consisting of 10 mm sodium carbonate (pH 11.0), 1.0 mm K-EGTA, 5.0 mm MgCl2, 0.2 mmdithiothreitol, and protease inhibitors. Cells were homogenized using a Dounce homogenizer (∼50 strokes), and the resulting lysate was clarified at 100,000 × g for 60 min at 4°/C. The pH of the resulting supernatant was adjusted to 7.0 by the addition of 9.0 μl of 1.0 m HCl and 20 μl of 1.0 m Tris (pH 7.0) before being used in cosedimentation assays. To polymerize F-actin, G-actin stocks (Cytoskeleton Inc., Denver, CO) were diluted to 2.5 mg/ml in binding buffer (10 mmimidazole, pH 7.2, 75 mm KCl, 5.0 mmMgCl2, 0.5 mm dithiothreitol), incubated for 30 min on ice, and stabilized by adjusting to 25 μg/ml phalloidin (Molecular Probes, Eugene, OR). F-actin, at a final concentration of 22 μm, was mixed with 60 μl of the cell lysate in binding buffer in a final volume of 200 μl and incubated for 20 min at room temperature. Samples were subsequently spun at 100,000 ×g for 20 min. Binding was tested in the presence or absence of 2.0 mm ATP (Boehringer Mannheim) and/or 5.0 μm myosin subfragment-1 (S1) (Sigma) as indicated in the legend of Fig. 7. Gel samples were prepared as described previously (45Fanning A.S. Wolenski J.S. Mooseker M.S. Izant J.G. Cell Motil. Cytoskel. 1994; 29: 29-45Crossref PubMed Scopus (83) Google Scholar), and examined by Western blotting with the 9E10 antisera. To analyze the distribution of the epitope-tagged proteins in immature and mature cell-cell contacts, clonal cell lines expressing these constructs were plated at 80% confluence (106 cells/ml) onto acid-washed 12-mm circular coverslips and induced for 12 h with 5.0 mm sodium butyrate starting 24–72 h after plating, as indicated in figure legend. Alternatively, 106 cells were plated onto 12-mm Transwell filter inserts (Corning Costar Corp., Cambridge, MA), incubated for 10 days, and induced for 24 h with 5.0 mm butyrate. Coverslips or filter inserts were then washed briefly with PBS+ and fixed in freshly prepared 1.0% paraformaldehyde for 20 min. Subsequently, coverslips were permeabilized for 15 min with a solution of 0.2% (w/v) Triton X-100, 2.0% donkey serum in PBS, incubated with 2.0% donkey serum (Life Technologies, Inc.) in PBS for 60 min, and finally incubated for 2.0 h with an undiluted cell supernatant from the 9E10 hybridoma supplemented with Texas Red-conjugated phalloidin (Molecular Probes, 1:40 dilution), a rat monoclonal antibody against ZO-1 (R40.76 cell supernatant, 1:10 dilution), or a rabbit polyclonal against human occludin (Zymed Laboratories Inc. Laboratories, South San Francisco, CA). Following incubation with the appropriate secondary antibody (Jackson Immunoresearch, West Grove, PA), coverslips were mounted on glass slides (Corning) in Vectashield antifade solution (Vector Laboratories, Burlingame, CA). Slides were viewed on a Nikon Microfot FX microscope using a 60× PlanApo lens and photographed using Kodak TMAX 400 film. Alternatively, slides were viewed using a Bio-Rad MRC1024 confocal microscope on a Zeiss Axiovert using a 63× PlanApo lens. Film images were digitized using a Sprintscan slide scanner (Polaroid, Cambridge, MA). Figures were assembled using Adobe Photoshop (Adobe Systems Inc., Mountain View, CA). ZO-1 can be divided into an N-terminal half, which constitutes the region of homology to other members of the MAGUK family, and a unique proline-rich C-terminal half. Each of these halves contains several previously identified protein domains whose specific roles are unknown in ZO-1 (46Willott E. Balda M.S. Fanning A.S. Jameson B. Van Itallie C. Anderson J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7834-7838Crossref PubMed Scopus (425) Google Scholar). To address the functional role of these domains in protein binding and tight junction assembly, we created a panel of myc epitope-tagged expression constructs which encode overlapping fragments of human ZO-1 (Fig. 1; see “Experimental Procedures”) and introduced these constructs into cultured MDCK epithelial cells. One of the first molecular interactions to be defined at the tight junction was that between ZO-1 and ZO-2. These proteins can be coimmunoprecipitated from epithelial cell extracts (Fig. 2) under relatively stringent conditions, suggesting a tight and possibly direct interaction (13Jesaitis L.A. Goodenough D.A. J. Cell Biol. 1994; 124: 949-961Crossref PubMed Scopus (387) Google Scholar, 15Gumbiner B. Lowenkopf T. Apatira D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3460-3464Crossref PubMed Scopus (430) Google Scholar). However, if ZO-1 does bind directly to ZO-2, then the striking homology between these two proteins (53% identity between human ZO-1 and ZO-2) suggests that these proteins might also form homodimers in vivo. To address these questions and further elucidate the interaction between ZO-1 and ZO-2, the epitope-tagged fusion proteins were immunoprecipitated from lysates of transfected MDCK cells. The immunoprecipitates were then resolved by SDS-PAGE and analyzed by Western blotting with antibodies recognizing the endogenous canine ZO-1 and ZO-2 to identify which constructs coprecipitated either of these proteins (Fig. 2). We found that all of the constructs used expressed a polypeptide of the appropriate apparent molecular weight (Fig. 2,myc). In addition, the full-length epitope-tagged construct, ZO1myc, was targeted to the tight junction (discussed below), indicating that the myc epitope does not interfere with these properties of ZO-1. Analysis of the epitope tag immunoprecipitates with an antibody (R40.76) that recognizes only the endogenous canine ZO-1 isoform (Fig. 2, ZO-1), and not the transfected human polypeptide, indicates that the endogenous protein does not coimmunoprecipitate with the full-length epitope-tagged ZO-1 construct under these conditions, nor does it interact with any of the deletion constructs. This result strongly suggests that ZO-1 does not form homodimers in vivo, but instead forms a simple “αβ” heterodimer with ZO-2. When the same blots were probed with a polyclonal antisera against ZO-2, we found that only deletion constructs which still contained the second PDZ domain of ZO-1 (PDZ2) could coprecipitate the endogenous ZO-2 polypeptide (Fig. 2, ZO-2). Furthermore, a construct in which only PDZ2 was specifically deleted (del 159–252) also fails to coprecipitate ZO-2. These results, summarized in Fig. 1, indicate that the PDZ2 domain mediates the interaction with ZO-2. Furuse et al. (16Furuse M. Itoh M. Hirase T. Nagafuchi F. Yonemura S. Tsukita S. J. Cell Biol. 1994; 127: 1617-1626Crossref PubMed Scopus (808) Google Scholar) have identified a ZO-1 binding site within the C-terminal aa 358–504 of chicken occludin, and have established that this domain binds directly to the full-length ZO-1 polypeptide. To identify the reciprocal occludin binding site on ZO-1, we performed binding assays between the epitope-tagged ZO-1 constructs and a GST fusion protein encoding this 146-aa domain. Total lysates from MDCK cells transfected with the epitope-tagged cDNA constructs (Fig. 3 A, total) were incubated with the immobilized GST-occludin (GST-occ) fusion protein, and the myc-tagged proteins bound to the GST fusion protein were identified by Western blotting with the anti-myc antisera (Fig. 3 A, bound). The relative amount of protein binding to GST-occ was assessed by comparing the amount of myc-tagged protein in the bound fraction to that in the total lysate or unbound fraction. In all cases, the expression of deletion constructs was identical to or greater than that of the full-length construct ZO1myc. An initial comparison of tagged constructs encoding the N- and C-terminal halves of ZO-1 (z1–876 and del 67–1033, respectively) indicates that only the N-terminal half of ZO-1 associates with the GST-occ fusion protein (Fig. 3 A). Progressive C-terminal deletions beyond aa 876 severely diminish the ability to associate with GST-occ (Fig. 3 B). For example, no binding is detected with the constructs z1–412, z1–549, and z1–572. These observations suggest that sequences within the 305-aa domain between aa 572 and 876 mediate a strong interaction with occludin (Fig. 1). The binding site can be further refined from the observation that the construct del 294–633 also binds to the GST-occ fusion protein. Interactions with PDZ1 (del 2–156), PDZ2 (del 159–252, del 1–245), PDZ3 (del 294–633), and the SH3 domain (del 550–603) do not appear to be required for binding to occludin (Fig. 3). These results suggest that the primary occludin binding site resides within the 244-aa domain between aa 633 and 876 (Fig. 1). To determine the contribution of the N-terminal half, which interacts with ZO-2 and occludin, and the unique C-terminal half of ZO-1 t" @default.
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• W2040645020 date "1998-11-01" @default.
• W2040645020 modified "2023-10-10" @default.
• W2040645020 title "The Tight Junction Protein ZO-1 Establishes a Link between the Transmembrane Protein Occludin and the Actin Cytoskeleton" @default.
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• W2040645020 doi "https://doi.org/10.1074/jbc.273.45.29745" @default.
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