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Journal Article
Tight junctions are membrane microdomains
A. Nusrat, C.A. Parkos, P. Verkade, C.S. Foley, T.W. Liang, W. Innis-Whitehouse, K.K. Eastburn, J.L. Madara
Journal of Cell Science 2000 113: 1771-1781;

Summary

Tight junctions (TJ) of polarized epithelial cells regulate barrier function at mucosal surfaces. Structural proteins of TJs include hyperphosphorylated occludin (HO) and the peripheral membrane protein, ZO-1. Since TJs are dynamically regulated, and lipid-modified signal transduction proteins localize to TJs, we considered the possibility that the TJ itself is composed of microdomains with unique structure. Differential detergent extraction and isopycnic sucrose density gradients were utilized to isolate TJ-enriched membranes from a polarized intestinal epithelial cell line, T84. Here we report that major pools of hyperphosphorylated occludin (HO) and ZO-1 are found in raft-like membrane microdomains with characteristics of the previously described detergent-insoluble glycolipid rafts (DIGs). Properties of such gradient fractions included Triton X-100 (TX-100) insolubility, light scattering at 600 nm, buoyant density of approximately 1.08 g/cm(3) and increased cholesterol content compared to high density fractions. Similar results were obtained using natural epithelium. Unlike the TJ proteins HO and ZO-1, other basolateral transmembrane proteins including E-cadherin, c-met and β 1 integrin were not increased in DIG-like fractions. Immunoprecipitation studies revealed coprecipitation of a pool of occludin with caveolin-1, a scaffolding protein abundant in DIGs. Coprecipitation results were supported by immunofluorescence and immunogold labeling studies demonstrating caveolin-1 localization in the apical membrane and focal colocalization with occludin in TJs. TJ disassembly by calcium chelation resulted in displacement of TJ proteins from the ‘raft-like’ compartment. Our findings suggest that raft-like compartments play an important role in the spatial organization of TJs and probably in regulation of paracellular permeability in epithelial cells.

REFERENCES

    1. Allain, C. C.,
    2. Poon, L. S.,
    3. Chan, C. S.,
    4. Richmond, W. and
    5. Fu, P. C.
    (1974). Enzymatic determination of total serum cholesterol. Clin. Chem 20, 470–5
    OpenUrlAbstract
    1. Anderson, J. M.,
    2. Stevenson, B. R.,
    3. Jesaitis, L. A.,
    4. Goodenough, D. A. and
    5. Mooseker, M. S.
    (1988). Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J. Cell Biol 106, 1141–9
    OpenUrlAbstract/FREE Full Text
    1. Anderson, J. M. and
    2. Van Itallie, C. M.
    ) (1995). Tight junctions and the molecular basis for regulation of paracellular permeability. Am. J. Physiol 269, 467–75
    OpenUrl
    1. Anderson, R. G.
    (1993). Caveolae: where incoming and outgoing messengers meet. Proc. Natl. Acad. Sci. USA 90, 10909–13
    OpenUrlAbstract/FREE Full Text
    1. Brown, D. A. and
    2. Rose, J. K.
    (1992). Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533–44
    OpenUrlCrossRefPubMedWeb of Science
    1. Cereijido, M.,
    2. Gonzalez-Mariscal, L. and
    3. Borboa, L.
    (1983). Occluding junctions and paracellular pathways studied in monolayers of MDCK cells. J. Exp. Biol 106, 205–15
    OpenUrlAbstract/FREE Full Text
    1. Fanning, A. S.,
    2. Jameson, B. J.,
    3. Jesaitis, L. A. and
    4. Anderson, J. M.
    (1998). The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J. Biol. Chem 273, 29745–53
    OpenUrlAbstract/FREE Full Text
    1. Farquhar, M. G. and
    2. Palade, G. E.
    (1963). Junctional complexes in various epithelia. J. Cell Biol 17, 375–412
    OpenUrlAbstract/FREE Full Text
    1. Feltkamp, C. A. and
    2. Van der Waerden, A. W.
    ) (1983). Junction formation between cultured normal rat hepatocytes. An ultrastructural study on the presence of cholesterol and the structure of developing tight-junction strands. J. Cell Sci 63, 271–86
    OpenUrlAbstract/FREE Full Text
    1. Friedrichson, T. and
    2. Kurzchalia, T. V.
    (1998). Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature 394, 802–5
    OpenUrlCrossRefPubMedWeb of Science
    1. Furuse, M.,
    2. Fujita, K.,
    3. Hiiragi, T.,
    4. Fujimoto, K. and
    5. Tsukita, S.
    (1998). Claudin-1 and-2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J. Cell Biol 141, 1539–50
    OpenUrlAbstract/FREE Full Text
    1. Furuse, M.,
    2. Hirase, T.,
    3. Itoh, M.,
    4. Nagafuchi, A.,
    5. Yonemura, S.,
    6. Tsukita, S. and
    7. Tsukita, S.
    (1993). Occludin: a novel integral membrane protein localizing at tight junctions [see comments]. J. Cell Biol 123, 1777–88
    OpenUrlAbstract/FREE Full Text
    1. Gumbiner, B.
    (1987). Structure, biochemistry, and assembly of epithelial tight junctions. Am. J. Physiol 253, 749–58
    OpenUrl
    1. Gumbiner, B. M.
    (1993). Breaking through the tight junction barrier [comment]. J. Cell Biol 123, 1631–3
    OpenUrlFREE Full Text
    1. Harder, T. and
    2. Simons, K.
    (1997). Caveolae, DIGs, and the dynamics ofsphingolipid-cholesterol microdomains. Curr. Opin. Cell Biol 9, 534–42
    OpenUrlCrossRefPubMedWeb of Science
    1. Jesaitis, L. A. and
    2. Goodenough, D. A.
    (1994). Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein. J. Cell Biol 124, 949–61
    OpenUrlAbstract/FREE Full Text
    1. Kachar, B. and
    2. Reese, T. S.
    (1982). Evidence for the lipidic nature of tight junction strands. Nature 296, 464–6
    OpenUrlCrossRefPubMed
    1. Kaoutzani, P.,
    2. Parkos, C. A.,
    3. Delp-Archer, C. and
    4. Madara, J. L.
    (1993). Isolation of plasma membrane fractions from the intestinal epithelial model T84. Am. J. Physiol 264, 1327–35
    OpenUrl
    1. Li, S.,
    2. Okamoto, T.,
    3. Chun, M.,
    4. Sargiacomo, M.,
    5. Casanova, J. E.,
    6. Hansen, S. H.,
    7. Nishimoto, I. and
    8. Lisanti, M. P.
    (1995). Evidence for a regulated interaction between heterotrimeric G proteins and caveolin. J. Biol. Chem 270, 15693–701
    OpenUrlAbstract/FREE Full Text
    1. Lisanti, M. P.,
    2. Scherer, P. E.,
    3. Vidugiriene, J.,
    4. Tang, Z.,
    5. Hermanowski-Vosatka, A.,
    6. Tu, Y. H.,
    7. Cook, R. F. and
    8. Sargiacomo, M.
    (1994). Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J. Cell Biol 126, 111–26
    OpenUrlAbstract/FREE Full Text
    1. Lisanti, M. P.,
    2. Tang, Z.,
    3. Scherer, P. E.,
    4. Kubler, E.,
    5. Koleske, A. J. and
    6. Sargiacomo, M.
    (1995). Caveolae, transmembrane signalling and cellular transformation. Mol. Membr. Biol 12, 121–4
    OpenUrlCrossRefPubMedWeb of Science
    1. Lisanti, M. P.,
    2. Tang, Z. L. and
    3. Sargiacomo, M.
    (1993). Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae. J. Cell Biol 123, 595–604
    OpenUrlAbstract/FREE Full Text
    1. Madara, J. L.
    (1988). Tight junction dynamics: is paracellular transport regulated?. Cell 53, 497–8
    OpenUrlCrossRefPubMedWeb of Science
    1. Madara, J. L. and
    2. Dharmsathaphorn, K.
    (1985). Occluding junction structure-function relationships in a cultured epithelial monolayer. J. Cell Biol 101, 2124–2133
    OpenUrlAbstract/FREE Full Text
    1. Madara, J. L.,
    2. Moore, R. and
    3. Carlson, S.
    (1987). Alteration of intestinal tight junction structure and permeability by cytoskeletal contraction. Am. J. Physiol 253, 854–61
    OpenUrl
    1. Madara, J. L.,
    2. Parkos, C.,
    3. Colgan, S.,
    4. Nusrat, A.,
    5. Atisook, K. and
    6. Kaoutzani, P.
    (1992). The movement of solutes and cells across tight junctions. Ann. NY Acad. Sci 664, 47–60
    OpenUrlCrossRefPubMedWeb of Science
    1. Madara, J. L.,
    2. Stafford, J.,
    3. Barenberg, D. and
    4. Carlson, S.
    (1988). Functional coupling of tight junctions and microfilaments in T84 monolayers. Am. J. Physiol 254, 416–23
    OpenUrl
    1. Madara, J. L.,
    2. Stafford, J.,
    3. Dharmsathaphorn, K. and
    4. Carlson, S.
    (1987). Structural analysis of a human intestinal epithelial cell line. Gastroenterology 92, 1133–45
    OpenUrlPubMedWeb of Science
    1. Nusrat, A.,
    2. Parkos, C. A.,
    3. Bacarra, A. E.,
    4. Godowski, P. J.,
    5. Delp-Archer, C.,
    6. Rosen, E. M. and
    7. Madara, J. L.
    (1994). Hepatocyte growth factor/scatter factor effects on epithelia. Regulation of intercellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium. J. Clin. Invest 93, 2056–65
    OpenUrlCrossRefPubMedWeb of Science
    1. Parkos, C. A.,
    2. Delp, C.,
    3. Arnaout, M. A. and
    4. Madara, J. L.
    (1991). Neutrophil Migration Across a Cultured Intestinal Epithelium: Dependence on a CD1lb/CD18—Mediated Event and Enhanced Efficiency in the Physiologic Direction. J. Clin. Invest 88, 1605–12
    OpenUrlCrossRefPubMedWeb of Science
    1. Parton, R. G. and
    2. Simons, K.
    (1995). Digging into caveolae [comment]. Science 269, 1398–9
    OpenUrlFREE Full Text
    1. Pinto da Silva, P. and
    2. Kachar, B.
    (1982). On tight-junction structure. Cell 28, 441–50
    OpenUrlCrossRefPubMedWeb of Science
    1. Saitou, M.,
    2. Fujimoto, K.,
    3. Doi, Y.,
    4. Itoh, M.,
    5. Fujimoto, T.,
    6. Furuse, M.,
    7. Takano, H.,
    8. Noda, T. and
    9. Tsukita, S.
    (1998). Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J. Cell Biol 141, 397–408
    OpenUrlAbstract/FREE Full Text
    1. Sakakibara, A.,
    2. Furuse, M.,
    3. Saitou, M.,
    4. Ando-Akatsuka, Y. and
    5. Tsukita, S.
    (1997). Possible involvement of phosphorylation of occludin in tight junction formation. J. Cell Biol 137, 1393–401
    OpenUrlAbstract/FREE Full Text
    1. Sargiacomo, M.,
    2. Sudol, M.,
    3. Tang, Z. and
    4. Lisanti, M. P.
    (1993). Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J. Cell Biol 122, 789–807
    OpenUrlAbstract/FREE Full Text
    1. Scheiffele, P.,
    2. Verkade, P.,
    3. Fra, A. M.,
    4. Virta, H.,
    5. Simons, K. and
    6. Ikonen, E.
    (1998). Caveolin-1 and-2 in the exocytic pathway of MDCK cells. J. Cell Biol 140, 795–806
    OpenUrlAbstract/FREE Full Text
    1. Scherer, P. E.,
    2. Okamoto, T.,
    3. Chun, M.,
    4. Nishimoto, I.,
    5. Lodish, H. F. and
    6. Lisanti, M. P.
    (1996). Identification, sequence, and expression of caveolin-2 defines a caveolin gene family. Proc. Natl. Acad. Sci. USA 93, 131–5
    OpenUrlAbstract/FREE Full Text
    1. Simons, K. and
    2. Ikonen, E.
    (1997). Functional rafts in cell membranes. Nature 387, 569–72
    OpenUrlCrossRefPubMedWeb of Science
    1. Song, S. K.,
    2. Li, S.,
    3. Okamoto, T.,
    4. Quilliam, L. A.,
    5. Sargiacomo, M. and
    6. Lisanti, M. P.
    (1996). Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. J. Biol. Chem 271, 9690–7
    OpenUrlAbstract/FREE Full Text
    1. Stankewich, M. C.,
    2. Francis, S. A.,
    3. Vu, Q. U.,
    4. Schneeberger, E. E. and
    5. Lynch, R. D.
    (1996). Alterations in cell cholesterol content modulate Ca(2+)-induced tight junction assembly by MDCK cells. Lipids 31, 817–28
    OpenUrlCrossRefPubMedWeb of Science
    1. Stevenson, B. R.,
    2. Anderson, J. M.,
    3. Goodenough, D. A. and
    4. Mooseker, M. S.
    (1988). Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance. J. Cell Biol 107, 2401–8
    OpenUrlAbstract/FREE Full Text
    1. Stevenson, B. R. and
    2. Goodenough, D. A.
    (1984). Zonulae occludentes injunctional complex-enriched fractions from mouse liver: preliminary morphological and biochemical characterization. J. Cell Biol 98, 1209–21
    OpenUrlAbstract/FREE Full Text
    1. Stevenson, B. R.,
    2. Siliciano, J. D.,
    3. Mooseker, M. S. and
    4. Goodenough, D. A.
    (1986). Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J. Cell Biol 103, 755–66
    OpenUrlAbstract/FREE Full Text
    1. Tsukita, S.,
    2. Furuse, M. and
    3. Itoh, M.
    (1996). Molecular dissection of tight junctions. Cell Struct. Funct 21, 381–5
    OpenUrlCrossRefPubMedWeb of Science
    1. Varma, R. and
    2. Mayor, S.
    (1998). GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394, 798–801
    OpenUrlCrossRefPubMedWeb of Science
    1. Weber, E.,
    2. Berta, G.,
    3. Tousson, A.,
    4. St John, P.,
    5. Green, M. W.,
    6. Gopalokrishnan, U.,
    7. Jilling, T.,
    8. Sorscher, E. J.,
    9. Elton, T. S.,
    10. Abrahamson, D. R.,
    11. et al.
    (1994). Expression and polarized targeting of a rab3 isoform in epithelial cells. J. Cell Biol 125, 583–94
    OpenUrlAbstract/FREE Full Text
    1. Wolf, A. A.,
    2. Jobling, M. G.,
    3. Wimer-Mackin, S.,
    4. Ferguson-Maltzman, M.,
    5. Madara, J. L.,
    6. Holmes, R. K. and
    7. Lencer, W. I.
    (1998). Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia. J. Cell Biol 141, 917–27
    OpenUrlAbstract/FREE Full Text
    1. Wong, V.
    (1997). Phosphorylation of occludin correlates with occludin localization and function at the tight junction. Am. J. Physiol 273, 1859–67
    OpenUrl
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Journal Article
Tight junctions are membrane microdomains
A. Nusrat, C.A. Parkos, P. Verkade, C.S. Foley, T.W. Liang, W. Innis-Whitehouse, K.K. Eastburn, J.L. Madara
Journal of Cell Science 2000 113: 1771-1781;
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