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. 1989 Sep 1;109(3):1047–1056. doi: 10.1083/jcb.109.3.1047

ZO-1 mRNA and protein expression during tight junction assembly in Caco- 2 cells

PMCID: PMC2115763  PMID: 2670954

Abstract

We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of approximately 7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++- free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at approximately 10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase- isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.

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Selected References

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  1. Anderson J. M., Stevenson B. R., Jesaitis L. A., Goodenough D. A., Mooseker M. S. Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J Cell Biol. 1988 Apr;106(4):1141–1149. doi: 10.1083/jcb.106.4.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balcarova-Ständer J., Pfeiffer S. E., Fuller S. D., Simons K. Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line. EMBO J. 1984 Nov;3(11):2687–2694. doi: 10.1002/j.1460-2075.1984.tb02194.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bendori R., Salomon D., Geiger B. Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes. EMBO J. 1987 Oct;6(10):2897–2905. doi: 10.1002/j.1460-2075.1987.tb02593.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benecke B. J., Ben-Ze'ev A., Penman S. The control of mRNA production, translation and turnover in suspended and reattached anchorage-dependent fibroblasts. Cell. 1978 Aug;14(4):931–939. doi: 10.1016/0092-8674(78)90347-1. [DOI] [PubMed] [Google Scholar]
  5. Bertolotti R., Rutishauser U., Edelman G. M. A cell surface molecule involved in aggregation of embryonic liver cells. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4831–4835. doi: 10.1073/pnas.77.8.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boller K., Vestweber D., Kemler R. Cell-adhesion molecule uvomorulin is localized in the intermediate junctions of adult intestinal epithelial cells. J Cell Biol. 1985 Jan;100(1):327–332. doi: 10.1083/jcb.100.1.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Cioe L., Curtis P. Detection and characterization of a mouse alpha-spectrin cDNA clone by its expression in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1367–1371. doi: 10.1073/pnas.82.5.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Citi S., Sabanay H., Jakes R., Geiger B., Kendrick-Jones J. Cingulin, a new peripheral component of tight junctions. Nature. 1988 May 19;333(6170):272–276. doi: 10.1038/333272a0. [DOI] [PubMed] [Google Scholar]
  11. Danielson P. E., Forss-Petter S., Brow M. A., Calavetta L., Douglass J., Milner R. J., Sutcliffe J. G. p1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA. 1988 May;7(4):261–267. doi: 10.1089/dna.1988.7.261. [DOI] [PubMed] [Google Scholar]
  12. Diamond J. M. Twenty-first Bowditch lecture. The epithelial junction: bridge, gate, and fence. Physiologist. 1977 Feb;20(1):10–18. [PubMed] [Google Scholar]
  13. FARQUHAR M. G., PALADE G. E. Junctional complexes in various epithelia. J Cell Biol. 1963 May;17:375–412. doi: 10.1083/jcb.17.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Friend D. S., Gilula N. B. Variations in tight and gap junctions in mammalian tissues. J Cell Biol. 1972 Jun;53(3):758–776. doi: 10.1083/jcb.53.3.758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grasset E., Pinto M., Dussaulx E., Zweibaum A., Desjeux J. F. Epithelial properties of human colonic carcinoma cell line Caco-2: electrical parameters. Am J Physiol. 1984 Sep;247(3 Pt 1):C260–C267. doi: 10.1152/ajpcell.1984.247.3.C260. [DOI] [PubMed] [Google Scholar]
  16. Gumbiner B., Stevenson B., Grimaldi A. The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex. J Cell Biol. 1988 Oct;107(4):1575–1587. doi: 10.1083/jcb.107.4.1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gumbiner B. Structure, biochemistry, and assembly of epithelial tight junctions. Am J Physiol. 1987 Dec;253(6 Pt 1):C749–C758. doi: 10.1152/ajpcell.1987.253.6.C749. [DOI] [PubMed] [Google Scholar]
  18. Hauri H. P., Sterchi E. E., Bienz D., Fransen J. A., Marxer A. Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells. J Cell Biol. 1985 Sep;101(3):838–851. doi: 10.1083/jcb.101.3.838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  20. Hunziker W., Spiess M., Semenza G., Lodish H. F. The sucrase-isomaltase complex: primary structure, membrane-orientation, and evolution of a stalked, intrinsic brush border protein. Cell. 1986 Jul 18;46(2):227–234. doi: 10.1016/0092-8674(86)90739-7. [DOI] [PubMed] [Google Scholar]
  21. Jones B., Carding S., Kyes S., Mjolsness S., Janeway C., Jr, Hayday A. Molecular analysis of T cell receptor gamma gene expression in allo-activated splenic T cells of adult mice. Eur J Immunol. 1988 Dec;18(12):1907–1915. doi: 10.1002/eji.1830181207. [DOI] [PubMed] [Google Scholar]
  22. Labarca C., Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem. 1980 Mar 1;102(2):344–352. doi: 10.1016/0003-2697(80)90165-7. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Madara J. L., Dharmsathaphorn K. Occluding junction structure-function relationships in a cultured epithelial monolayer. J Cell Biol. 1985 Dec;101(6):2124–2133. doi: 10.1083/jcb.101.6.2124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marcial M. A., Carlson S. L., Madara J. L. Partitioning of paracellular conductance along the ileal crypt-villus axis: a hypothesis based on structural analysis with detailed consideration of tight junction structure-function relationships. J Membr Biol. 1984;80(1):59–70. doi: 10.1007/BF01868690. [DOI] [PubMed] [Google Scholar]
  26. Nelson W. J., Veshnock P. J. Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells. J Cell Biol. 1986 Nov;103(5):1751–1765. doi: 10.1083/jcb.103.5.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nelson W. J., Veshnock P. J. Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells. J Cell Biol. 1987 Jun;104(6):1527–1537. doi: 10.1083/jcb.104.6.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pasdar M., Nelson W. J. Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. II. Morphological analysis. J Cell Biol. 1988 Mar;106(3):687–695. doi: 10.1083/jcb.106.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Powell D. W. Barrier function of epithelia. Am J Physiol. 1981 Oct;241(4):G275–G288. doi: 10.1152/ajpgi.1981.241.4.G275. [DOI] [PubMed] [Google Scholar]
  30. Siliciano J. D., Goodenough D. A. Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells. J Cell Biol. 1988 Dec;107(6 Pt 1):2389–2399. doi: 10.1083/jcb.107.6.2389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Snyder M., Elledge S., Sweetser D., Young R. A., Davis R. W. Lambda gt 11: gene isolation with antibody probes and other applications. Methods Enzymol. 1987;154:107–128. doi: 10.1016/0076-6879(87)54073-3. [DOI] [PubMed] [Google Scholar]
  32. Stevenson B. R., Anderson J. M., Bullivant S. The epithelial tight junction: structure, function and preliminary biochemical characterization. Mol Cell Biochem. 1988 Oct;83(2):129–145. doi: 10.1007/BF00226141. [DOI] [PubMed] [Google Scholar]
  33. Stevenson B. R., Siliciano J. D., Mooseker M. S., Goodenough D. A. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol. 1986 Sep;103(3):755–766. doi: 10.1083/jcb.103.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. van Meer G., Simons K. The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells. EMBO J. 1986 Jul;5(7):1455–1464. doi: 10.1002/j.1460-2075.1986.tb04382.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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