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Sodium Dodecyl Sulfate-Freeze-Fracture Immunolabeling of Gap Junctions

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Connexin Methods and Protocols

Part of the book series: Methods In Molecular Biology™ ((MIMB,volume 154))

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Abstract

By the mid 1960s, pioneering work using high-resolution electron microscopy, new fixation methods, and negative staining of isolated liver plasma membranes allowed the identification of a geometric subunit pattern likely associated with junctional domains (1,2). Furthermore, the application of tissue impregnation with electron-dense tracers revealed that the minute “gap” (2 nm wide) between the closely adjoining junctional membranes comprised an hexagonal subunit pattern. This type of membrane-membrane interaction, distinct from tight junctions, adhesion plaques, and desmosomes, was originally called “gap junction” (3).

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References

  1. Robertson J. D. (1963) The occurrence of a subunit pattern in the unit membrane of club endings in Mauthner cell synapses in goldfish brain. J. Cell Biol. 19, 201–221.

    Article  PubMed  CAS  Google Scholar 

  2. Benedetti E. L. and Emmelot P. (1965) Electron microscopic observations on negatively stained plasma membranes isolated from rat liver. J. Cell Biol. 26, 166–174.

    Article  Google Scholar 

  3. Revel J. P. and Karnovsky M. J. (1967) Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J. Cell Biol. 33, C7.

    Article  PubMed  CAS  Google Scholar 

  4. Kumar N. M. and Gilula N. B. (1996) The gap junction communication channel. Cell 84. 381–388.

    Article  PubMed  CAS  Google Scholar 

  5. Bruzzone R., White T. W., and Goodenough D. A. (1996) The cellular internet: on-line with connexins. BioEssays 18, 709–718.

    Article  PubMed  CAS  Google Scholar 

  6. Nicholson S. and Bruzzone R. (1997) Gap junctions: getting the message through. Curr. Biol. 7, 340–344.

    Article  Google Scholar 

  7. Jiang J. X. and Goodenough D. A. (1996) Heteromeric connexons in lens gap junction channels. Proc. Natl. Acad. Sci. USA 93, 1287–1291.

    Article  PubMed  CAS  Google Scholar 

  8. Bennett M. V. L. and Spray D. C. (1985) Gap Junctions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  9. Staehelin L. A. (1979) A simple guide to the evaluation of the quality of afreeze-fracture replica, in Freeze-Fracture Methods, Artifacts, and Interpretations (Rash J. E. and Hudson C. S., eds.), Raven Press, New York, pp. 11–17.

    Google Scholar 

  10. Peracchia C. and Peracchia L. L. (1988) Gap junction dynamics: reversible effect of hydrogen ions. J. Cell Biol. 87, 719–727.

    Article  Google Scholar 

  11. Pinto Da Silva P., Parkison C., and Dwyer N. (1981) Fracture label: cytochemistry of freeze-fracture faces in the erythrocyte membrane. Proc. Natl. Acad. Sci. USA 78, 343–347.

    Article  PubMed  CAS  Google Scholar 

  12. Pinto Da Silva P. and Kan F. W. K. (1984) Label fracture: a method for high resolution labeling of cell surfaces. J. Cell Biol., 99, 1156–1161.

    Article  PubMed  CAS  Google Scholar 

  13. Fujimoto K. and Ogawa K. (1991) Fracture-flip and fracture-flip cytochemistry to study macromolecular architecture of membrane surfaces: practical procedures, interpretation and application. Acta Histochem. Cytochem. 24, 111–117.

    CAS  Google Scholar 

  14. Fujimoto K. and Pinto Da Silva P. (1992) Fracture-flip/Triton X-100 reveals the cytoplasmic surface of human erythocyte membranes. Acta Histochem. Cytochem. 25, 255–263.

    Google Scholar 

  15. Fujimoto K. (1997) SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application. Histochem. Cell Biol. 107, 87–96.

    Article  PubMed  CAS  Google Scholar 

  16. Rash J. E. and Yasumura T. (1999) Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis. Tissue Cell Res. 296, 307–321.

    Article  CAS  Google Scholar 

  17. Hertzberg E. L., Anderson D. J., Friedlander M., and Gilula N. B. (1982) Comparative analysis of the major polypeptides from liver gap junctions and lens fiber junctions. J. Cell Biol. 92, 53–59.

    Article  PubMed  CAS  Google Scholar 

  18. Dunia I., Manenti S., Rousselet A., and Benedetti E. L. (1987) Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers. J. Cell Biol. 105, 1679–1689.

    Article  PubMed  CAS  Google Scholar 

  19. Gong X., Klier G., Huang Q., Wu Y., Lei H., Kumar N. M., Horwitz J., and Gilula N. B. (1997) Disruption of α3 connexin gene leads to proteolysis and cataractogenesis in mice. Cell 91, 833–843.

    Article  PubMed  CAS  Google Scholar 

  20. White T. W., Bruzzone R., Goodenough D. A., and Paul D. L (1992) Mouse C×50, a potential member of the connexin family of gap junction proteins, is the lens fiber protein MP70. Mol. Biol. Cell. 3, 711–720.

    PubMed  CAS  Google Scholar 

  21. Kistler J., B. Kirkland, and S. Bullivant. (1985) Identification of a 70,000kD protein in lens membrane junctional domains. J. Cell Biol. 101, 28–35.

    Article  PubMed  CAS  Google Scholar 

  22. Friedlander M. (1980) Immunological approaches to the study of myogenesis and lens fiber junction formation. Curr. Top. Dev. Biol. 14, 321–358.

    Article  PubMed  CAS  Google Scholar 

  23. Broekhuyse R. M., Kuhlmann E. D., and Stols A. L. H. (1976) Lens membrane II. Isolation and characterization of the main intrinsic polypeptide (MIP) of bovine lens fiber membranes. Exp. Eye Res. 23, 365–371.

    Article  PubMed  CAS  Google Scholar 

  24. Vallon O., Dunia I., Favard-Sereno C., Hoebeke J., and Benedetti E. L. (1985) MP26 in the bovine lens: a post-embedding immunocytochemical study. Biol. Cell 53, 85–88.

    PubMed  CAS  Google Scholar 

  25. Milks L. C., Kumar N. M., Houghten N., Unwin N., and Gilula N. B. (1988) Topology of the 32-kD liver gap junction protein determined by site-directed antibody localizations. EMBO J. 7, 2967–2975.

    PubMed  CAS  Google Scholar 

  26. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., and Lerner R. A. (1982) Immunogenic structure of the influenza virus hemagglutinin. Cell 28, 477–487.

    Article  PubMed  CAS  Google Scholar 

  27. Paul D. L., Ebihara L., Takemoto L. J., Swenson K. I., and Goodenough D. A. (1991) Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J. Cell Biol. 115, 1077–1089.

    Article  PubMed  CAS  Google Scholar 

  28. Yeager M. and Gilula N. B. (1992) Membrane topology and quaternary structure of cardiac gap junction ion channels. J. Mol. Biol. 223, 929–948.

    Article  PubMed  CAS  Google Scholar 

  29. Risek B., Guthrie S., Kumar N., and Gilula N. B. (1990) Modulation of gap junction transcript and protein expression during pregnancy in the rat. J. Cell Biol. 110, 269–282.

    Article  PubMed  CAS  Google Scholar 

  30. Jarvis L. J., Kumar N. M., and Louis C. F. (1993) The developmental expression of three mammalian lens fiber cell membrane proteins. Invest. Ophthalmol. Vis. Sci. 34, 613–620.

    PubMed  CAS  Google Scholar 

  31. Gruijters W. T., Kistler J., Bullivant S., and Goodenough D. A. (1987) Immunolocalization of MP70 in lens fiber 16–17-nm intercellular junctions. J. Cell Biol., 104, 565–572.

    Article  PubMed  CAS  Google Scholar 

  32. Kistler J. and Bullivant S. (1987) Protein processing in lens intercellular junctions: cleavage of MP70 to MP38. Invest. Ophthalmol. Vis. Sci. 28, 1687–1692.

    PubMed  CAS  Google Scholar 

  33. Li J. S., Fitzgerald S., Dong Y., Knight C., Donaldson P., and Kistler J. (1997) Processing of the gap junction connexin 50 in the ocular lens is accomplished by calpain. Eur. J. Cell Biol. 73, 141–149.

    Article  Google Scholar 

  34. Branton D. (1966) Fracture faces of frozen membranes. Proc. Natl. Acad. Sci. USA 55, 1048–1056.

    Article  PubMed  CAS  Google Scholar 

  35. Deamer D. W. and Branton D. (1967) Fracture planes in a ice-bilayer model membrane system. Science 158, 655–657.

    Article  PubMed  CAS  Google Scholar 

  36. Pinto Da Silva P. and Branton D. (1970) Membrane splitting in freeze-teching: covalently bound ferritin as a membrane marker. J. Cell Biol. 45, 598–604.

    Article  PubMed  CAS  Google Scholar 

  37. Moor H. (1973) Cryotechnology for the structural analysis of biological material, in Freeze-Etching, Techniques and Applications (Benedetti E. L. and Favard P., eds.), French Society of Electron Microscopy, Paris, pp. 11–30.

    Google Scholar 

  38. Benedetti E. L. and Favard P. (1973) Freeze-etching, techniques and applications. French Society of Electron Microscopy, Paris, France.

    Google Scholar 

  39. Satir B. H. and Satir P. (1979) Partitioning of intramembrane particles during the freeze-fracture procedure, in Freeze-Fracture Methods, Artifact, and Interpretations (Rash J. E. and Hudson C. S., eds.), Raven Press, New York, pp. 43–49.

    Google Scholar 

  40. Torrisi M. R. and Mancini P. (1996) Freeze-fracture immunogold labeling. Histochem. CellBiol., 106, 19–30.

    Article  CAS  Google Scholar 

  41. Verkleij J. (1984) Lipidic intramembranous particles. Biochim. Biophys. Acta, 779, 43–63.

    PubMed  CAS  Google Scholar 

  42. Hanna R. B., Ornberg R. L., and Reese T. S. (1985) Structural Details of Rapid Frozen Gap Junctions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 23–32.

    Google Scholar 

  43. Hirokawa N. and Heuser J. (1982) The inside and outside of gap-junction membranes visualized by deep etching. Cell 30, 395–406.

    Article  PubMed  CAS  Google Scholar 

  44. Hoh J. H., Sosinsky G., Revel J. P., and Hansena P. K. (1993) Structure of the extracellular surface of the gap junction by atomic force microscopy. Biophys. J. 65, 149–163.

    Article  PubMed  CAS  Google Scholar 

  45. Perkins G., Goodenough D., and Sosinsky G. (1997) Tree-dimensional structure of the gap junction connexon. Biophys. J. 72, 533–544.

    Article  PubMed  CAS  Google Scholar 

  46. Unger V. M., Kumar N. M., Gilula N. B., and Yaeger M. (1997) Proyection structure of a gap junction membrane channel at 7 Å resolution. Nat. Struct. Biol. 4, 39–43.

    Article  PubMed  CAS  Google Scholar 

  47. Unger V. M., Kumar N. M., Gilula N. B., and Yaeger M. (1999) Three-dimensional structure of a recombinant gap junction membrane channel. Science 283, 1176–1179.

    Article  PubMed  CAS  Google Scholar 

  48. Foote C. I., Zan Zhou, and Nicholson B. J. (1998) The pattern of disulfide linkages in the extracellular loop regions of Connexin 32 suggests a model for the docking interface of gap junctions. J. Cell Biol. 140, 1187–1197.

    Article  PubMed  CAS  Google Scholar 

  49. White T. W., Bruzzone R., Wolfram S., Paul D. L., and Goodenough D. A. (1994) Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins. J. Cell Biol. 125, 879–892.

    Article  PubMed  CAS  Google Scholar 

  50. Sosinsky G. (1996) Molecular organization of gap junction membrane channels. J. Bioenerg. Biomembr. 28, 297–309.

    Article  PubMed  CAS  Google Scholar 

  51. Ghroshroy R., Goodenough D. A., and Sosisnky E. (1995) Preparation, characterization, and structure of half gap junctional layers split with urea and EGTA. J. Membr. Biol. 146, 15–28.

    Google Scholar 

  52. Fujimoto K., Nagafuchi A., Tsukita S., Kuraoka A., Ohokuma A., and Shibata Y. (1997) Dynamics of connexins, E-cadherin and α-catenin on cell membrane during junction formation. J. Cell Sci. 110, 311–322.

    PubMed  CAS  Google Scholar 

  53. Furuse M., Fujimoto K., Sato N., Hirase T., Tsukita S., and Tsukita S. (1996) Overexpression of occludin, a tight junction-associated integral membrane protein, induces formation of intracellular multilamellar bodies bearing tight junction-structures. J. Cell Sci. 109, 429–435.

    PubMed  CAS  Google Scholar 

  54. Hirase T., Staddon J. M., Saitou M., Ando-Akatsuka Y., Itoh M., Furuse M., Fujimoto K., Tsukita S., and Rubin L. L. (1997) Occludin as a possible determinant of tight junction permeability in endothelial cells. J. Cell Sci. 110, 1603–1613.

    PubMed  CAS  Google Scholar 

  55. Hülser D. F., Rehkopf B., and Traub O. (1997) Dispersed and aggregated gap junction channels identified by immunogold labeling of freeze-fractured membranes. Exp. Cell Res. 233, 240–251.

    Article  PubMed  Google Scholar 

  56. Fujimoto K., Umeda M., and Fujimoto T. (1996) Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling. J. Cell Sci. 109, 2453–2460.

    PubMed  Google Scholar 

  57. Dunia I., Recouvreur M., Nicolas P., Kumar N., Bloemendal H., and Benedetti E. L. (1998) Assembly of connexins and MP26 in lens fiber plasma membranes studied by SDS-fracture immunolabeling. J. Cell Sci. 111, 2109–2120.

    PubMed  Google Scholar 

  58. Benedetti E. L., Dunia I., Dufier J. L., Yit K-S., and Bloemendal H. (1996) Plasma membrane-cytoskeleton complex in the normal and cataractous lens, in The Cytoskeleton, Vol. 3 (Hesketh J. E. and Pryme I. F., eds.), JAI Press, London, pp. 451–518.

    Google Scholar 

  59. Deen P. M. T. and van Os C. H. (1998) Epithelial aquaporins Curr. Opin. Cell Biol. 10, 435–442.

    Article  PubMed  CAS  Google Scholar 

  60. Cullen M. J., Walsh J., Stevenson S. A., Rothery S. and Severs N. J. (1998) Co-localization of dystrophin and p-dystroglycan demonstrated in “en face” view by double-immunogold labeling of freeze-fractured skeletal muscle. J. Histochem. Cytochem. 46, 945–953.

    PubMed  CAS  Google Scholar 

  61. Nomura R. and Fujimoto T. (1999) Tyrosine-phosphorylated caveolin-1: immunolocalization and molecular characterization. Mol. Biol. Cell 10, 975–986.

    PubMed  CAS  Google Scholar 

  62. Reynolds J. A. and Tandford Ch. (1970a) The gross conformation of proteinsodium dodecyl sulfate complexes. J. Biol. Chem. 254, 5161–5165.

    Google Scholar 

  63. Reynolds J. A. and Tandford Ch. (1970b) Binding of dodecyl-sulfate to proteins at high binding ratios. Possible implications for the state of proteins in biological membranes. Proc. Natl. Acad. Sci. USA 66, 1002–1007.

    Article  PubMed  CAS  Google Scholar 

  64. Helenius A. and Simons K. (1975) Solubilization of membranes by detergents. Biochem. Biophys. Acta 415, 29–79.

    PubMed  CAS  Google Scholar 

  65. White F. H. and Wright G. (1984) Effect of structure-forming solutes on chicken eggwhite lysozyme after reductive cleavage of disulfide bonds. Int. J. Pept. Prot. Res. 23, 256–270.

    Article  CAS  Google Scholar 

  66. Rothe G. M. and Maurer W. D. (1986) One dimensional PAA-gel electrophoretic techniques to separate functional and denaturated proteins, in Gel Electrophoresis of Proteins (Dunn M. J., ed.), Wright Press, Bristol, UK, pp. 37–140.

    Google Scholar 

  67. Benedetti E. L., and Emmelot P. (1968) Hexagonal array of subunits in tight junctions separated from isolated rat liver plasma membranes. J. Cell Biol., 38, 15–24.

    Article  PubMed  CAS  Google Scholar 

  68. Dunia I., Sen-Ghosh K., Benedetti E. L., Zweers A., and Bloemendal H. (1974) Isolation and protein pattern of eye lens fiber junctions. FEBS Lett. 45, 139–144.

    Article  PubMed  CAS  Google Scholar 

  69. Robards A. W. and Sleytr V. B. (1985) Practical Methods of Electron Microscopy, in Low Temperature Methods in Biological Electron Microscopy, Vol. 10 (Glauert A. M., ed.), Elsevier, Amsterdam.

    Google Scholar 

  70. Severs N. J. and Shotton D. M. (1995) Rapid freezing, freeze-fracture and deep etching, in Techniques in Modern Biomedical Microscopy, Wiley-Liss, New York.

    Google Scholar 

  71. Gross H. (1979) Advances in ultrahigh vacuum freeze-fracturing at very low specimen temperature, in Freeze-Fracture Methods, Artifacts and Interpretations (Rash J. E. and Hudson C. S., eds.), Raven Press., New York, pp. 127–139.

    Google Scholar 

  72. Gulik-Krzywicki T. (1997) Freeze-fracture transmission electron microscopy. Curr. Opin. Colloid Interphase Sci. 2, 137–144.

    Article  CAS  Google Scholar 

  73. Raposo G., Kleijmeer M., Posthuma G., Slot J. and Geuze H. (1997) Immunogold labeling of ultrathin cryosections:application in immunology, in Weir’s Handbook of Experimental Immunology, 5th ed., Vol. 4. (Herzenberg L. A., Weir D. and Blackwell C., eds.), Blackwell Science, Cambridge, MA, pp. 1–11.

    Google Scholar 

  74. Slot J. W., Posthuma G., Chang L. Y., Crapo J. D., and Geuze H. (1989) Quantitative aspects of immunogold labeling in embedded and non-embedded sections. Am.J.Anat. 185, 271–281.

    Article  PubMed  CAS  Google Scholar 

  75. Griffiths G. (1993) Fine Structure Immunocytochemistry, Springer-Verlag, New York, New York.

    Google Scholar 

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Author information

Authors and Affiliations

  1. Institut J. Monod, Université Paris VII, Paris, France

    Irene Dunia, Michel Recouvreur, Pierre Nicolas & E. Lucio Benedetti

  2. Department of Cell Biology, The Scripps Research Institute, La Jolla, CA

    Nalin M. Kumar

  3. Department of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands

    Hans Bloemendal

Authors
  1. Irene Dunia
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  2. Michel Recouvreur
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  3. Pierre Nicolas
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  4. Nalin M. Kumar
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  5. Hans Bloemendal
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  6. E. Lucio Benedetti
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Editor information

Editors and Affiliations

  1. Institut Pasteur, Paris, France

    Roberto Bruzzone

  2. Collége de France, Paris, France

    Christian Giaume

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© 2001 Humana Press Inc.

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Dunia, I., Recouvreur, M., Nicolas, P., Kumar, N.M., Bloemendal, H., Benedetti, E.L. (2001). Sodium Dodecyl Sulfate-Freeze-Fracture Immunolabeling of Gap Junctions. In: Bruzzone, R., Giaume, C. (eds) Connexin Methods and Protocols. Methods In Molecular Biology™, vol 154. Humana Press. https://doi.org/10.1385/1-59259-043-8:33

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  • DOI: https://doi.org/10.1385/1-59259-043-8:33

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-658-1

  • Online ISBN: 978-1-59259-043-8

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