Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Jan 1;108(1):31–41. doi: 10.1083/jcb.108.1.31

Isolation of cell-to-cell adherens junctions from rat liver

PMCID: PMC2115366  PMID: 2463257

Abstract

A new isolation procedure for cell-to-cell adherens junctions has been developed using rat liver. From the bile canaliculi-enriched fraction obtained by homogenization of the liver and sucrose gradient centrifugation, the fraction rich in adherens junction was recovered by detergent treatment followed by sucrose gradient centrifugation. Light and electron microscopy revealed that this final fraction was mainly composed of the belt-like adherens junctions with their associated short actin filaments. Biochemical and immunological analyses have shown that vinculin is highly enriched in this fraction. Considering that vinculin is known to be localized in the cell-to-cell adherens junctions, we can conclude that we have succeeded in isolating the cell- to-cell adherens junctions. Furthermore, the constituents of the undercoat (dense layer underlying the membrane) of adherens junctions were selectively extracted from the fraction rich in junctions. Upon SDS electrophoresis of this extract, 10 polypeptides including vinculin, alpha-actinin, and actin were dominant. The results obtained are discussed with special reference to the molecular organization of the undercoats of cell-to-cell adherens junctions.

Full Text

The Full Text of this article is available as a PDF (5.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Burridge K., Connell L. A new protein of adhesion plaques and ruffling membranes. J Cell Biol. 1983 Aug;97(2):359–367. doi: 10.1083/jcb.97.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burridge K., Mangeat P. An interaction between vinculin and talin. Nature. 1984 Apr 19;308(5961):744–746. doi: 10.1038/308744a0. [DOI] [PubMed] [Google Scholar]
  3. Cowin P., Kapprell H. P., Franke W. W., Tamkun J., Hynes R. O. Plakoglobin: a protein common to different kinds of intercellular adhering junctions. Cell. 1986 Sep 26;46(7):1063–1073. doi: 10.1016/0092-8674(86)90706-3. [DOI] [PubMed] [Google Scholar]
  4. De Blas A. L., Cherwinski H. M. Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies. Anal Biochem. 1983 Aug;133(1):214–219. doi: 10.1016/0003-2697(83)90245-2. [DOI] [PubMed] [Google Scholar]
  5. Drochmans P., Freudenstein C., Wanson J. C., Laurent L., Keenan T. W., Stadler J., Leloup R., Franke W. W. Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis. J Cell Biol. 1978 Nov;79(2 Pt 1):427–443. doi: 10.1083/jcb.79.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Franke W. W., Mueller H., Mittnacht S., Kapprell H. P., Jorcano J. L. Significance of two desmosome plaque-associated polypeptides of molecular weights 75 000 and 83 000. EMBO J. 1983;2(12):2211–2215. doi: 10.1002/j.1460-2075.1983.tb01725.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
  9. Geiger B. Membrane-cytoskeleton interaction. Biochim Biophys Acta. 1983 Aug 11;737(3-4):305–341. doi: 10.1016/0304-4157(83)90005-9. [DOI] [PubMed] [Google Scholar]
  10. Geiger B., Tokuyasu K. T., Dutton A. H., Singer S. J. Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4127–4131. doi: 10.1073/pnas.77.7.4127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geiger B., Volk T., Volberg T. Molecular heterogeneity of adherens junctions. J Cell Biol. 1985 Oct;101(4):1523–1531. doi: 10.1083/jcb.101.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goodenough D. A., Stoeckenius W. The isolation of mouse hepatocyte gap junctions. Preliminary chemical characterization and x-ray diffraction. J Cell Biol. 1972 Sep;54(3):646–656. doi: 10.1083/jcb.54.3.646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorbsky G., Cohen S. M., Shida H., Giudice G. J., Steinberg M. S. Isolation of the non-glycosylated proteins of desmosomes and immunolocalization of a third plaque protein: desmoplakin III. Proc Natl Acad Sci U S A. 1985 Feb;82(3):810–814. doi: 10.1073/pnas.82.3.810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gorbsky G., Steinberg M. S. Isolation of the intercellular glycoproteins of desmosomes. J Cell Biol. 1981 Jul;90(1):243–248. doi: 10.1083/jcb.90.1.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Green K. J., Geiger B., Jones J. C., Talian J. C., Goldman R. D. The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes. J Cell Biol. 1987 May;104(5):1389–1402. doi: 10.1083/jcb.104.5.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ingalls H. M., Goodloe-Holland C. M., Luna E. J. Junctional plasma membrane domains isolated from aggregating Dictyostelium discoideum amebae. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4779–4783. doi: 10.1073/pnas.83.13.4779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones J. C., Goldman R. D. Intermediate filaments and the initiation of desmosome assembly. J Cell Biol. 1985 Aug;101(2):506–517. doi: 10.1083/jcb.101.2.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kensler R. W., Goodenough D. A. Isolation of mouse myocardial gap junctions. J Cell Biol. 1980 Sep;86(3):755–764. doi: 10.1083/jcb.86.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Lazarides E., Burridge K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell. 1975 Nov;6(3):289–298. doi: 10.1016/0092-8674(75)90180-4. [DOI] [PubMed] [Google Scholar]
  21. Mueller H., Franke W. W. Biochemical and immunological characterization of desmoplakins I and II, the major polypeptides of the desmosomal plaque. J Mol Biol. 1983 Feb 5;163(4):647–671. doi: 10.1016/0022-2836(83)90116-x. [DOI] [PubMed] [Google Scholar]
  22. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  23. Owaribe K., Kodama R., Eguchi G. Demonstration of contractility of circumferential actin bundles and its morphogenetic significance in pigmented epithelium in vitro and in vivo. J Cell Biol. 1981 Aug;90(2):507–514. doi: 10.1083/jcb.90.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Owaribe K., Masuda H. Isolation and characterization of circumferential microfilament bundles from retinal pigmented epithelial cells. J Cell Biol. 1982 Oct;95(1):310–315. doi: 10.1083/jcb.95.1.310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pardo J. V., Siliciano J. D., Craig S. W. A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements ("costameres") mark sites of attachment between myofibrils and sarcolemma. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1008–1012. doi: 10.1073/pnas.80.4.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Phillips D. R., Jennings L. K., Edwards H. H. Identification of membrane proteins mediating the interaction of human platelets. J Cell Biol. 1980 Jul;86(1):77–86. doi: 10.1083/jcb.86.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rohrschneider L. R. Adhesion plaques of Rous sarcoma virus-transformed cells contain the src gene product. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3514–3518. doi: 10.1073/pnas.77.6.3514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sheetz M. P., Sawyer D. Triton shells of intact erythrocytes. J Supramol Struct. 1978;8(4):399–412. doi: 10.1002/jss.400080403. [DOI] [PubMed] [Google Scholar]
  29. Shriver K., Rohrschneider L. Organization of pp60src and selected cytoskeletal proteins within adhesion plaques and junctions of Rous sarcoma virus-transformed rat cells. J Cell Biol. 1981 Jun;89(3):525–535. doi: 10.1083/jcb.89.3.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Skerrow C. J., Matoltsy A. G. Chemical characterization of isolated epidermal desmosomes. J Cell Biol. 1974 Nov;63(2 Pt 1):524–530. doi: 10.1083/jcb.63.2.524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Skerrow C. J., Matoltsy A. G. Isolation of epidermal desmosomes. J Cell Biol. 1974 Nov;63(2 Pt 1):515–523. doi: 10.1083/jcb.63.2.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Song C. S., Rubin W., Rifkind A. B., Kappas A. Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi. J Cell Biol. 1969 Apr;41(1):124–132. doi: 10.1083/jcb.41.1.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Staehelin L. A. Structure and function of intercellular junctions. Int Rev Cytol. 1974;39:191–283. doi: 10.1016/s0074-7696(08)60940-7. [DOI] [PubMed] [Google Scholar]
  34. Stevenson B. R., Goodenough D. A. Zonulae occludentes in junctional complex-enriched fractions from mouse liver: preliminary morphological and biochemical characterization. J Cell Biol. 1984 Apr;98(4):1209–1221. doi: 10.1083/jcb.98.4.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tsukita S., Tsukita S. Desmocalmin: a calmodulin-binding high molecular weight protein isolated from desmosomes. J Cell Biol. 1985 Dec;101(6):2070–2080. doi: 10.1083/jcb.101.6.2070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vaessen R. T., Kreike J., Groot G. S. Protein transfer to nitrocellulose filters. A simple method for quantitation of single proteins in complex mixtures. FEBS Lett. 1981 Feb 23;124(2):193–196. doi: 10.1016/0014-5793(81)80134-2. [DOI] [PubMed] [Google Scholar]
  37. Volk T., Geiger B. A 135-kd membrane protein of intercellular adherens junctions. EMBO J. 1984 Oct;3(10):2249–2260. doi: 10.1002/j.1460-2075.1984.tb02123.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Watt F. M., Mattey D. L., Garrod D. R. Calcium-induced reorganization of desmosomal components in cultured human keratinocytes. J Cell Biol. 1984 Dec;99(6):2211–2215. doi: 10.1083/jcb.99.6.2211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yu J., Fischman D. A., Steck T. L. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J Supramol Struct. 1973;1(3):233–248. doi: 10.1002/jss.400010308. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES