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. 1988 Mar 1;106(3):677–685. doi: 10.1083/jcb.106.3.677

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis

PMCID: PMC2115081  PMID: 3346322

Abstract

The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.

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Selected References

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  1. Arnn J., Staehelin L. A. The structure and function of spot desmosomes. Int J Dermatol. 1981 Jun;20(5):330–339. doi: 10.1111/j.1365-4362.1981.tb00815.x. [DOI] [PubMed] [Google Scholar]
  2. Cohen S. M., Gorbsky G., Steinberg M. S. Immunochemical characterization of related families of glycoproteins in desmosomes. J Biol Chem. 1983 Feb 25;258(4):2621–2627. [PubMed] [Google Scholar]
  3. Cowin P., Garrod D. R. Antibodies to epithelial desmosomes show wide tissue and species cross-reactivity. Nature. 1983 Mar 10;302(5904):148–150. doi: 10.1038/302148a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Cowin P., Kapprell H. P., Franke W. W. The complement of desmosomal plaque proteins in different cell types. J Cell Biol. 1985 Oct;101(4):1442–1454. doi: 10.1083/jcb.101.4.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cowin P., Mattey D., Garrod D. Distribution of desmosomal components in the tissues of vertebrates, studied by fluorescent antibody staining. J Cell Sci. 1984 Mar;66:119–132. doi: 10.1242/jcs.66.1.119. [DOI] [PubMed] [Google Scholar]
  7. Cowin P., Mattey D., Garrod D. Identification of desmosomal surface components (desmocollins) and inhibition of desmosome formation by specific Fab'. J Cell Sci. 1984 Aug;70:41–60. doi: 10.1242/jcs.70.1.41. [DOI] [PubMed] [Google Scholar]
  8. Docherty R. J., Edwards J. G., Garrod D. R., Mattey D. L. Chick embryonic pigmented retina is one of the group of epithelioid tissues that lack cytokeratins and desmosomes and have intermediate filaments composed of vimentin. J Cell Sci. 1984 Oct;71:61–74. doi: 10.1242/jcs.71.1.61. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Edelman G. M. Cell adhesion and the molecular processes of morphogenesis. Annu Rev Biochem. 1985;54:135–169. doi: 10.1146/annurev.bi.54.070185.001031. [DOI] [PubMed] [Google Scholar]
  11. Engvall E., Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971 Sep;8(9):871–874. doi: 10.1016/0019-2791(71)90454-x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Fey E. G., Wan K. M., Penman S. Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition. J Cell Biol. 1984 Jun;98(6):1973–1984. doi: 10.1083/jcb.98.6.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Franke W. W., Schmid E., Grund C., Müller H., Engelbrecht I., Moll R., Stadler J., Jarasch E. D. Antibodies to high molecular weight polypeptides of desmosomes: specific localization of a class of junctional proteins in cells and tissue. Differentiation. 1981;20(3):217–241. doi: 10.1111/j.1432-0436.1981.tb01178.x. [DOI] [PubMed] [Google Scholar]
  16. Garrod D. R. Desmosomes, cell adhesion molecules and the adhesive properties of cells in tissues. J Cell Sci Suppl. 1986;4:221–237. doi: 10.1242/jcs.1986.supplement_4.14. [DOI] [PubMed] [Google Scholar]
  17. Garrod D. R. Formation of desmosomes in polarized and non-polarized epithelial cells: implications for epithelial morphogenesis. Biochem Soc Trans. 1986 Feb;14(1):172–175. doi: 10.1042/bst0140172. [DOI] [PubMed] [Google Scholar]
  18. Geiger B., Schmid E., Franke W. W. Spatial distribution of proteins specific for desmosomes and adhaerens junctions in epithelial cells demonstrated by double immunofluorescence microscopy. Differentiation. 1983;23(3):189–205. doi: 10.1111/j.1432-0436.1982.tb01283.x. [DOI] [PubMed] [Google Scholar]
  19. Giudice G. J., Cohen S. M., Patel N. H., Steinberg M. S. Immunological comparison of desmosomal components from several bovine tissues. J Cell Biochem. 1984;26(1):35–45. doi: 10.1002/jcb.240260104. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Jones J. C., Goldman A. E., Steinert P. M., Yuspa S., Goldman R. D. Dynamic aspects of the supramolecular organization of intermediate filament networks in cultured epidermal cells. Cell Motil. 1982;2(3):197–213. doi: 10.1002/cm.970020302. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Kapprell H. P., Cowin P., Franke W. W., Ponstingl H., Opferkuch H. J. Biochemical characterization of desmosomal proteins isolated from bovine muzzle epidermis: amino acid and carbohydrate composition. Eur J Cell Biol. 1985 Mar;36(2):217–229. [PubMed] [Google Scholar]
  24. Kelly D. E. Fine structure of desmosomes. , hemidesmosomes, and an adepidermal globular layer in developing newt epidermis. J Cell Biol. 1966 Jan;28(1):51–72. doi: 10.1083/jcb.28.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kelly D. E., Shienvold F. L. The desmosome: fine structural studies with freeze-fracture replication and tannic acid staining of sectioned epidermis. Cell Tissue Res. 1976 Sep 20;172(3):309–323. doi: 10.1007/BF00399514. [DOI] [PubMed] [Google Scholar]
  26. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  27. Mattey D. L., Garrod D. R. Calcium-induced desmosome formation in cultured kidney epithelial cells. J Cell Sci. 1986 Sep;85:95–111. doi: 10.1242/jcs.85.1.95. [DOI] [PubMed] [Google Scholar]
  28. Mattey D. L., Garrod D. R. Splitting and internalization of the desmosomes of cultured kidney epithelial cells by reduction in calcium concentration. J Cell Sci. 1986 Sep;85:113–124. doi: 10.1242/jcs.85.1.113. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. OVERTON J. Desmosome development in normal and reassociating cells in the early chick blastoderm. Dev Biol. 1962 Jun;4:532–548. doi: 10.1016/0012-1606(62)90056-8. [DOI] [PubMed] [Google Scholar]
  33. Overton J., DeSalle R. Control of desmosome formation in aggregating embryonic chick cells. Dev Biol. 1980 Mar;75(1):168–176. doi: 10.1016/0012-1606(80)90152-9. [DOI] [PubMed] [Google Scholar]
  34. Overton J. Experimental manipulation of desmosome formation. J Cell Biol. 1973 Mar;56(3):636–646. doi: 10.1083/jcb.56.3.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Overton J. Experiments with junctions of the adhaerens type. Curr Top Dev Biol. 1975;10:1–34. doi: 10.1016/s0070-2153(08)60037-x. [DOI] [PubMed] [Google Scholar]
  36. Overton J. Selective formation of desmosomes in chick cell reaggregates. Dev Biol. 1974 Aug;39(2):210–225. doi: 10.1016/0012-1606(74)90236-x. [DOI] [PubMed] [Google Scholar]
  37. Overton J. The fat of desmosomes in trypsinized tissue. J Exp Zool. 1968 Jun;168(2):203–214. doi: 10.1002/jez.1401680208. [DOI] [PubMed] [Google Scholar]
  38. Penn E. J., Hobson C., Rees D. A., Magee A. I. Structure and assembly of desmosome junctions: biosynthesis, processing, and transport of the major protein and glycoprotein components in cultured epithelial cells. J Cell Biol. 1987 Jul;105(1):57–68. doi: 10.1083/jcb.105.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pirbazari M., Kelly D. E. Analysis of desmosomal intramembrane particle populations and cytoskeletal elements: detergent extraction and freeze-fracture. Cell Tissue Res. 1985;241(2):341–351. doi: 10.1007/BF00217179. [DOI] [PubMed] [Google Scholar]
  40. Skerrow C. J., Hunter I., Skerrow D. Dissection of the bovine epidermal desmosome into cytoplasmic protein and membrane glycoprotein domains. J Cell Sci. 1987 Apr;87(Pt 3):411–421. doi: 10.1242/jcs.87.3.411. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. 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]
  44. Suhrbier A., Garrod D. An investigation of the molecular components of desmosomes in epithelial cells of five vertebrates. J Cell Sci. 1986 Mar;81:223–242. doi: 10.1242/jcs.81.1.223. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Warren S. L., Nelson W. J. Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures. Mol Cell Biol. 1987 Apr;7(4):1326–1337. doi: 10.1128/mcb.7.4.1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]

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