Establishment and Characterization of Cultured Epithelial Cells Lacking Expression of ZO-1

Abstract

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology. In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology. The tight junction (TJ) 1The abbreviations used are: TJs, tight junctions; AJs, adherens junctions; JAM, junctional adhesion molecule; mAb, monoclonal antibody; pAb, polyclonal antibody; MAGUKs, membrane-associated guanylate kinase-like homologues; GST, glutathione S-transferase; PBS, phosphate-buffered saline; Pt, paracellular tracer flux; P0, paracellular tracer flux at time 0; HA, hemagglutinin; TER, transepithelial electric resistance; FITC, fluorescein isothiocyanate.1The abbreviations used are: TJs, tight junctions; AJs, adherens junctions; JAM, junctional adhesion molecule; mAb, monoclonal antibody; pAb, polyclonal antibody; MAGUKs, membrane-associated guanylate kinase-like homologues; GST, glutathione S-transferase; PBS, phosphate-buffered saline; Pt, paracellular tracer flux; P0, paracellular tracer flux at time 0; HA, hemagglutinin; TER, transepithelial electric resistance; FITC, fluorescein isothiocyanate. is one type of cell-to-cell adhesion structure in epithelial cells. TJs constitute the epithelial junctional complex together with adherens junctions (AJs) and desmosomes and are located in the most apical part of the complex (1Farquhar M.G. Palade G.E. J. Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2078) Google Scholar). TJs seal cells to create a primary barrier to the diffusion of solutes across the cellular sheet and also function as a boundary between the apical and basolateral membrane domains to produce their polarization (2Schneeberger E.E. Lynch R.D. Am. J. Physiol. 1992; 262: L647-L661PubMed Google Scholar, 3Anderson J.M. Van Itallie C.M. Am. J. Physiol. 1995; 269: G467-G475Crossref PubMed Google Scholar, 4Balda M.S. Matter K. J. Cell Sci. 1998; 111: 541-547Crossref PubMed Google Scholar, 5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (1979) Google Scholar). Therefore, TJs are essential structures for epithelial cells to exert their physiological functions. On ultrathin-section electron microscopy, TJs appear as a series of discrete sites of apparent fusion involving the outer leaflets of the plasma membranes of adjacent cells (1Farquhar M.G. Palade G.E. J. Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2078) Google Scholar). On freeze-fracture electron microscopy, TJs appear as a set of continuous, anastomosing intramembranous particle strands (TJ strands) (6Staehelin L.A. Int. Rev. Cytol. 1974; 39: 191-283Crossref PubMed Scopus (1046) Google Scholar). TJ strands are mainly composed of linearly polymerized integral membrane proteins called claudins with molecular masses of ∼23 kDa, which comprise a multigene family consisting of >20 members (5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (1979) Google Scholar, 7Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1656) Google Scholar, 8Tsukita S. Furuse M. Trends Cell Biol. 1999; 9: 268-273Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). Claudin molecules bear four transmembrane domains with both NH2 and COOH termini located in the cytoplasm. In addition to claudins, two other types of integral membrane proteins have been reported to concentrate at TJs, occludin (9Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2073) Google Scholar), and JAM (10Martin-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. J. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1118) Google Scholar). One of the interesting questions regarding the molecular mechanism behind the morphogenesis of simple epithelial cells, i.e. epithelial polarization, is how claudins are polymerized exclusively at the most apical part of the junctional complex, i.e. more apically than AJs. In this connection, various scaffold proteins have been reported to be concentrated at the cytoplasmic surfaces of the junctional complex regions to determine the specialization and localization of junctions (5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (1979) Google Scholar, 11Kowalczyk A.P. Bomslaeger E.A. Norvell S.M. Palka H.L. Green K.J. Int. Rev. Cytol. 1999; 185: 237-302Crossref PubMed Google Scholar, 12Perez-Moreno M. Jamora C. Fuchs E. Cell. 2003; 112: 535-548Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar). At TJs, three closely related proteins called ZO-1, ZO-2, and ZO-3 were identified to constitute the plaque structures underlying plasma membranes (13Mitic L.L. Anderson J.M. Annu. Rev. Physiol. 1998; 60: 121-142Crossref PubMed Scopus (616) Google Scholar, 14González-Mariscal L. Betanzos A. Avila-Flores A. Semin. Cell Dev. Biol. PubMed Scopus Google together with various proteins including cingulin, complex, and S. J. PubMed Scopus Google Scholar, S. C. C. J. Cell Biol. PubMed Scopus Google Scholar, M.S. Matter K. J. PubMed Scopus Google Scholar, S. Cell Biol. 2001; PubMed Scopus Google Scholar, A. C. J. M. M.S. Matter K. J. Cell Biol. 2003; PubMed Scopus Google Scholar). ZO-1 with a molecular of was first identified as for a monoclonal a from the M.S. J. Cell Biol. PubMed Scopus Google Scholar). ZO-2 was identified as a that was with ZO-1 from cell T. D. Sci. S. A. PubMed Scopus Google Scholar). was also in the ZO-1 M.S. L. Matter K. M. Anderson J.M. J. Cell Biol. 1993; 123: PubMed Scopus Google and is called The and of these molecules that have three domains one and one guanylate kinase-like in this from their NH2 that ZO-1, ZO-2, and ZO-3 are membrane-associated guanylate kinase-like M. Nagafuchi A. Yonemura S. T. Tsukita S. Tsukita S. J. Cell Biol. 1993; PubMed Scopus Google Scholar, E. M.S. Van Itallie C. Anderson J.M. Sci. S. A. 1993; PubMed Scopus Google Scholar, L.A. J. Cell Biol. PubMed Scopus Google Scholar, J. L. J. J. Cell Biol. 1998; 141: PubMed Scopus Google Scholar). these three of TJs, ZO-1 is well In well polarized epithelial cells, ZO-1 is exclusively concentrated at TJs to claudins M. Furuse M. K. K. M. Tsukita S. J. Cell Biol. 1999; PubMed Scopus Google Scholar), occludin M. Itoh M. Hirase T. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. PubMed Scopus Google Scholar, L.A. Anderson J.M. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and JAM M. S. Dejana E. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Furuse M. T. Tsukita S. J. Cell Biol. 2001; PubMed Scopus Google Scholar), in cells lacking TJs as and cells as well as in epithelial cells at the initial phase of epithelial polarization, is with with a M. Nagafuchi A. Yonemura S. T. Tsukita S. Tsukita S. J. Cell Biol. 1993; PubMed Scopus Google Scholar). ZO-1 with ZO-2 and ZO-3 J. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google and to at L.A. Anderson J.M. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Nagafuchi A. S. Tsukita S. J. Cell Biol. PubMed Scopus Google Scholar). together with ZO-2 ZO-1 a in the of not TJs but also epithelial have been to clarify the of ZO-1, for exogenous expression of various of ZO-1 L.A. Anderson J.M. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Nagafuchi A. S. Tsukita S. J. Cell Biol. PubMed Scopus Google Scholar, D. Mol. Biol. Cell. PubMed Scopus Google but been to the physiological of were this was thought to be to a between ZO-1 and ZO-2 also One of the most to this would be to the expression of ZO-1 in epithelial cells and to in these cells. this we to the of Sci. S. A. PubMed Scopus Google to cultured mouse epithelial cells but to the expression of ZO-1 the of we to the of of junctions in the expression of be and in cultured epithelial cells. In this we that of cultured mouse epithelial cells were successfully generated by homologous and in from the of junction that the gene knock-out at the of cultured epithelial cells is for a of the molecular behind the junction formation and epithelial and epithelial cells were in with cells were a from E. E. J. Cell Biol. PubMed Scopus Google Scholar). M. Nagafuchi A. Yonemura S. T. Tsukita S. Tsukita S. J. Cell Biol. 1993; PubMed Scopus Google Scholar), A. Itoh M. A. M. Furuse M. Tsukita S. 2003; PubMed Scopus Google Scholar), and M. Furuse M. T. Tsukita S. J. Cell Biol. 2001; PubMed Scopus Google were and pAb, and were by M. for S. and pAb, pAb, and were from and were generated the fusion of mouse cingulin as of mouse was a mouse ZO-1 as a As in and a were to the was to both of the ZO-1 The first and a and gene in the the first gene the was at a located at the of the and the cells were with of a were in with for and by at a of At of the were and by of with the clones were identified by a in addition to the of the the gene the was at a located at the of the and was as were cultured in the with for and and were at a of to both and were by of with the and clones were identified by a in addition to the of the first were cultured in for ZO-1, ZO-2, and cells were with for K. Yonemura S. T. Tsukita S. Tsukita S. J. Cell Sci. 1999; 112: PubMed Google Scholar). cells were with in for at cells were with and with in for cingulin, and JAM cells were with for at a with PBS, cells were in in and with the primary for were with and with or for were with and in Ca2+ were at in and in the with for were with and cultured in a Ca2+ with and that been with the Ca2+ switch, the Ca2+ in both the and was with the Ca2+ with and of and of cells were in for cell and the was a was in a epithelial The were by the of and by by the of the paracellular tracer flux at and the Ca2+ switch, with a molecular of was to the in the apical at a of a at time a of the was from the and the paracellular tracer flux was as the of in the with a The paracellular tracer flux at time was a Ca2+ in the Ca2+ was as the Cell were in and in the Ca2+ When with and at for most of the cells from the as cells. were and in Ca2+ with at a of of this were in or a at to cell the were with a The extent of cell was by the and are the of and cells and Quantitative of the ZO-1/ZO-2 of ZO-1 and the of ZO-2 were the and expression fusion proteins were in and as M. Itoh M. Hirase T. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. PubMed Scopus Google Scholar). The of the fusion in was with a as a of cell and various of ZO-1 or ZO-2 were by the and with or pAb, respectively, as M. Itoh M. Hirase T. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. PubMed Scopus Google Scholar). the of of ZO-1 or ZO-2 in cell with that of fusion proteins of the ZO-1 in the function of ZO-1 in epithelial cells, we to the ZO-1 gene in cultured epithelial cells. In to the for the gene knock-out between the cultured cell and levels in we for epithelial cell for gene the of the well polarized epithelial as well as the we the cell from mouse E. J. Cell Biol. PubMed Scopus Google Scholar). The structure of the mouse ZO-1 gene was as in most of the were with the that homologous between the and the ZO-1 gene would in a of of In the first and the ZO-1 a of with the the the clones clones a homologous of these clones clones and were and to the When the was by the of with the was to a As in of clones from and two of clones from and the and in which the and not be in clones was by with a and clones were also for by with a or gene not the of ZO-1 expression in clones and at the that both the and of As in both were in the as well as two clones and but in clones and of ZO-1 was their cell were we the of ZO-1 or ZO-2 clones and and expression was by clones and the as as was the from the exogenous ZO-1 and are and of cultured cells (ZO-1-/- a confluent that these cells appeared to completely the epithelial a a and was between and ZO-1-/- cells in the of cells and the of the at the ultrathin-section electron ZO-1-/- cells were indistinguishable from ZO-1+/+ cells. appeared to be well polarized with apical membranes and In good agreement, that immunofluorescence with did not appear to between ZO-1+/+ and ZO-1-/- cells not The of the junctional complex did not appear to be by the deficiency of ZO-1, was to TJs from both in ZO-1+/+ and in ZO-1-/- cells and the of TJs of ZO-1-/- cells and ZO-1+/+ cells by freeze-fracture electron microscopy. As in the TJs of ZO-1+/+ cells were by a well of intramembranous particle strands and the of ZO-1 did not these and the and of ZO-1-/- cells with those of ZO-1+/+ cells. As in simple cell that ZO-1-/- cells the as ZO-1+/+ cells. the with the confluent that ZO-1-/- cells the as as ZO-1+/+ cells of TJs in confluent cultures, ZO-1-/- cells were well polarized and the TJs of the molecular of TJs in these cells by immunofluorescence microscopy. As we used ZO-1+/+ and ZO-1-/- cells the of ZO-1 cells were cultured highly confluent and with for the of the plaque structures of TJs ZO-1 was at TJs of ZO-1-/- cells and the of ZO-1 at TJs in cells appeared to be with that in ZO-1+/+ cells. the ZO-2 was significantly at TJs in ZO-1-/- cells as with ZO-1+/+ cells and this of the ZO-2 at TJs was to the ZO-1+/+ cell by exogenous expression of ZO-3 was in ZO-1+/+ cells by immunofluorescence microscopy, which was with A. Itoh M. A. M. Furuse M. Tsukita S. 2003; PubMed Scopus Google Scholar). ZO-3 did not in ZO-1-/- cells. of the TJ plaque cingulin was in in ZO-1-/- cells. from TJs in ZO-1-/- cells but was at TJs in cells. In ZO-1-/- cells, were in the of other TJ plaque as and not for the of TJ integral membrane claudins, and JAM, in ZO-1-/- cells At four of claudins, and were in ZO-1+/+ cells. As these four claudins in the in of the and as the of claudins did not appear to be by ZO-1 the of is as in this highly confluent and JAM were concentrated at TJs in ZO-1-/- cells, in the and at TJs as with ZO-1+/+ and cells. In ZO-1-/- cells, the of proteins as and also appeared to be and the of did not appear to be In of the molecular of TJs and AJs, were in ZO-2 and cingulin was in ZO-1-/- cells. the of ZO-2 more various clones by the of ZO-1+/+ cells were and with ZO-1-/- cells and to immunofluorescence with and As the ZO-2 from TJs more in ZO-1-/- cells than in ZO-1+/+ cells. in ZO-1-/- cells, the ZO-2 from the was significantly as with ZO-1+/+ cells. that the ZO-1 deficiency the of ZO-2 from the to TJs in ZO-1-/- cells. The of ZO-1-/- and cells that this localization of ZO-2 was with the exogenous expression of ZO-1 a of the of As cingulin from TJs in ZO-1-/- cells and the was completely rescued by exogenous expression of ZO-1 this was not by the exogenous of in of the recruitment of cingulin to TJs, ZO-2 was not functionally for ZO-1 were two and in ZO-1-/- cells, the expression of or of of cingulin was not that the ZO-1 deficiency the recruitment of cingulin to TJs and of and in the of junction i.e. epithelial polarization and cells. this the cells were cultured in a Ca2+ Ca2+ confluent and their polarization was initiated by to a Ca2+ The of TJ formation first was by immunofluorescence with or As in ZO-1+/+ cells to TJs at and appeared to have the at to the Ca2+ in the ZO-1-/- cell a in the Ca2+ the TJs were This of TJs in ZO-1-/- cells was by in the Ca2+ to the ZO-1+/+ cell This ZO-1 retardation of TJ formation was completely rescued by exogenous expression of ZO-1 ZO-1 in the TJ formation was by with pAb, at the Ca2+ switch, ZO-2 was concentrated TJs in ZO-1+/+ cells, in ZO-1-/- cells, ZO-2 from junctions were but a to that of The of TJ formation was we the and cells highly confluent As in was in these cells which were than cells and these Therefore, was to the of TJ formation the Ca2+ by we the paracellular tracer flux or cells were cultured in the Ca2+ at a confluent and epithelial polarization was initiated by the in both the apical and from a to Ca2+ At and the Ca2+ switch, with a molecular of was to the in the apical a at time the in the was and the paracellular tracer flux was as the of in the with a As in the Ca2+ switch, the of paracellular was significantly in ZO-1-/- cell as with ZO-1+/+ and cell however, at of in the Ca2+ the paracellular tracer flux of ZO-1-/- cell the as that of ZO-1+/+ and cell The is this retardation of TJ formation in ZO-1-/- cells be rescued by exogenous of In of the paracellular exogenous of ZO-2 did not the ZO-1 deficiency The by which TJs in ZO-1-/- cells was with that in ZO-1-/- and cells by immunofluorescence microscopy. As in exogenous ZO-1 expression the of TJ i.e. recruitment of occludin and to in ZO-1-/- cells to the exogenous ZO-2 expression in the of TJ ZO-2 was not functionally Finally, the Ca2+ the of AJ formation was by immunofluorescence with In to TJ AJ i.e. the of at did not appear to be by ZO-1 the Ca2+ switch, in ZO-1-/- cells, was recruited to a time to ZO-1+/+ and cells. the cell that adhesion was not by ZO-1 deficiency and that the of was with a time in ZO-1-/- cells by the Ca2+ of ZO-1 and ZO-2 in the the expression levels of ZO-1 and ZO-2 were in at both the and in ZO-1-/- cells and the expression of ZO-2 was not with that in ZO-1+/+ cells In good with the immunofluorescence ZO-3 was or in a by not in ZO-1+/+ cells but also in ZO-1-/- cells not in this cell is to ZO-3 the TJ the expression levels of ZO-1 and ZO-2 in four ZO-1+/+ ZO-1-/- cells and ZO-1-/- cells and this we first fusion proteins with a part of ZO-1 and ZO-2 in E. in and the of ZO-1 and ZO-2 in cell with that of of various of fusion In ZO-1+/+ cells, ZO-1 and ZO-2 were respectively, to be and In ZO-1-/- cells, the of ZO-2 was not and in cells, ZO-1 was to some extent at in ZO-1-/- cells the of ZO-2 and was to be which was with the of ZO-1 and ZO-2 in ZO-1+/+ cells levels of ZO-1 and ZO-1 in a the formation of junctions in simple epithelial cells, ZO-1 in a In the initial phase of the ZO-1 is with in spotlike at the of cellular S. Itoh M. Nagafuchi A. Tsukita S. J. Cell Sci. 1995; PubMed Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, T. T. K. K. M. T. 1999; PubMed Scopus Google Scholar). spotlike to at which time claudins/occludin to be recruited to these junctional to be polymerized as TJ strands Yonemura S. Itoh M. Furuse M. Tsukita S. J. Cell. Physiol. 1999; PubMed Scopus Google Scholar). TJs are from in well polarized epithelial cells, ZO-1 is completely from to be concentrated at TJs M.S. J. Cell Biol. PubMed Scopus Google Scholar, M. Nagafuchi A. Yonemura S. T. Tsukita S. Tsukita S. J. Cell Biol. 1993; PubMed Scopus Google Scholar). have led to that ZO-1 a in the formation of TJs as well as the epithelial Therefore, was a to we confluent cells lacking the expression of ZO-1 (ZO-1-/- cells) were highly polarized with well organized junctional at the and freeze-fracture electron ZO-1 L. Betanzos A. Avila-Flores A. Semin. Cell Dev. Biol. PubMed Scopus Google Scholar, M.S. Matter K. J. PubMed Scopus Google Scholar, A. C. J. M. M.S. Matter K. J. Cell Biol. 2003; PubMed Scopus Google Scholar), was also that ZO-1-/- cells did not in their and that ZO-1-/- cells three from cells of ZO-2 was markedly at TJs of ZO-1-/- cells. In ZO-1-/- cells, or expression of ZO-2 was not and that the ZO-2 from the was significantly in ZO-1-/- cells. Therefore, we that the ZO-1 deficiency the of ZO-2 from to TJs, not more sites for at TJs. This that ZO-2 to TJs a complex with ZO-1 J. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). been thought that the cytoplasmic of TJ strands as a consisting of and cytoplasmic of claudins, which to and that TJ strands various proteins as ZO-1 and ZO-2 (5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (1979) Google Scholar). In ZO-1-/- cells, a ZO-1 deficiency more for ZO-2 the cytoplasmic of TJ strands to be recruited from the cytoplasm. The in ZO-1-/- cells was that cingulin at TJs. This was by the of two been identified and as a of the TJ plaque structure S. J. PubMed Scopus Google Scholar, S. Mol. Biol. PubMed Scopus Google Scholar), but of physiological is In ZO-1-/- cells, the or of cingulin did not appear to be that ZO-1 is for cingulin to be recruited to TJs. This is with that cingulin to ZO-1 in M. E. J. D. S. J. Cell Biol. 1999; PubMed Scopus Google Scholar, S. J. Biol. Full Text Full Text PDF PubMed Scopus Google but to be to a that cingulin lacking to TJs S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), is that is another cingulin or that is another molecular between cingulin and ZO-1 some ZO-2 was also reported to with cingulin in M. E. J. D. S. J. Cell Biol. 1999; PubMed Scopus Google Scholar), but the that ZO-2 not cingulin to TJs cells. be to the of cingulin in would be in the ZO-1-/- cells be with epithelial cells lacking the expression of The in which would be the most from the of epithelial polarization, was that the TJ formation was significantly the epithelial polarization was initiated by the Ca2+ from the immunofluorescence and cell in the formation of at the initial phase of epithelial polarization did not appear to be The of TJ i.e. the recruitment of claudins/occludin to the AJs, appeared to significantly time in ZO-1-/- cells than in ZO-1+/+ cells. The retardation of the of the TJ barrier was by the tracer be ZO-1-/- cells were cultured for a time confluent they highly polarized with well organized which were and functionally indistinguishable from those in ZO-1+/+ cells. The as to is the molecular mechanism behind this of ZO-1-/- cells, i.e. the retardation of TJ formation epithelial ZO-1, ZO-2, and ZO-3 have been to be functionally was not in in cells, we did not to that the cytoplasmic ZO-2 was recruited to TJs of ZO-1 in ZO-1-/- cells, the mechanism ZO-2 the function as ZO-1, but in ZO-1-/- cells to of ZO-2, for the ZO-1 in a retardation of TJ that the between ZO-1 and ZO-2 was not simple ZO-1+/+ cells ZO-1 and In ZO-1-/- cells, the expression of ZO-2 was not When ZO-1 was in ZO-1-/- cells, the formation of TJs was completely in ZO-1-/- cells ZO-2 the of exogenous and ZO-2 was to the of ZO-1 and ZO-2 in ZO-1+/+ cells, but the TJ was markedly retarded. Therefore, ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization, but be that they are not functionally identical. In this by homologous we have for the first time cultured epithelial cells in which the expression of ZO-1 was completely and is to in cultured epithelial cells, but knock-out cells are they are not for the of but also for as molecules are to be in junction formation/epithelial polarization in a S. Cell Biol. 2001; PubMed Scopus Google Scholar, Annu. Rev. 2001; PubMed Scopus Google Scholar, Am. J. Physiol. 2003; PubMed Scopus Google Scholar, Physiol. Sci. 2003; Google Scholar), of cells lacking the expression of various be cells lacking the expression of ZO-2 or both ZO-1 and ZO-2 would the of ZO-1/ZO-2 in with ZO-1-/- cells. The of ZO-1-/- cells in this is a first are this of the members of of Cell of for