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. 1983 Mar 1;96(3):613–624. doi: 10.1083/jcb.96.3.613

Effects of extracellular calcium depletion on membrane topography and occluding junctions of mammary epithelial cells in culture

PMCID: PMC2112402  PMID: 6403552

Abstract

Ca2+ dependence of occluding junction structure and permeability, well documented in explanted or cultured epithelial sheets, presumably reflects inherent control mechanisms. As an approach to identification of these mechanisms, we induced disassembly of zonulae occludentes in confluent monolayers of mouse mammary epithelial cells by exposure to low concentrations of the chelators, EGTA or sodium citrate. Stages in disassembly were monitored during treatment by phase-contrast microscopy and prepared for transmission and scanning electron microscopy. Cellular response included several events affecting occluding junctions: (a) Centripetal cytoplasmic contraction created tension on junction membranes and displaced intramembrane strands along lines determined by the axis of tension. (b) Destabilization of junction position, probably through increased membrane fluidity, augmented tension-induced movement of strands, resulting in fragmentation of the junction belt. (c) Active ruffling and retraction of freed peripheral membranes remodeled cell borders to produce many filopodia, distally attached by occluding-junction fragments to neighboring cell membranes. Filopodia generally persisted until mechanically ruptured, when endocytosis of the junction and adhering cytoplasmic bleb ensued. Junction disassembly thus resulted from mechanical tensions generated by initial centripetal contraction and subsequent peripheral cytoskeletal activity, combined with destabilization of the junction's intramembrane strand pattern.

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Selected References

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  1. Amsterdam A., Jamieson J. D. Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells. J Cell Biol. 1974 Dec;63(3):1037–1056. doi: 10.1083/jcb.63.3.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Batten B. E., Anderson E. Effects of Ca+2 and Mg+2 deprivation of cell shape in cultured ovarian granulosa cells. Am J Anat. 1981 May;161(1):101–114. doi: 10.1002/aja.1001610108. [DOI] [PubMed] [Google Scholar]
  3. Ben-Ze'ev A., Duerr A., Solomon F., Penman S. The outer boundary of the cytoskeleton: a lamina derived from plasma membrane proteins. Cell. 1979 Aug;17(4):859–865. doi: 10.1016/0092-8674(79)90326-x. [DOI] [PubMed] [Google Scholar]
  4. Bentzel C. J., Hainau B., Edelman A., Anagnostopoulos T., Benedetti E. L. Effect of plant cytokinins on microfilaments and tight junction permeability. Nature. 1976 Dec 16;264(5587):666–668. doi: 10.1038/264666a0. [DOI] [PubMed] [Google Scholar]
  5. Bentzel C. J., Hainau B., Ho S., Hui S. W., Edelman A., Anagnostopoulos T., Benedetti E. L. Cytoplasmic regulation of tight-junction permeability: effect of plant cytokinins. Am J Physiol. 1980 Sep;239(3):C75–C89. doi: 10.1152/ajpcell.1980.239.3.C75. [DOI] [PubMed] [Google Scholar]
  6. Berry M. N., Friend D. S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec;43(3):506–520. doi: 10.1083/jcb.43.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bisbee C. A., Machen T. E., Bern H. A. Mouse mammary epithelial cells on floating collagen gels: transepithelial ion transport and effects of prolactin. Proc Natl Acad Sci U S A. 1979 Jan;76(1):536–540. doi: 10.1073/pnas.76.1.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bouchaud C., Bouvier D. Fine structure of tight junctions between rat choroidal cells after osmotic opening induced by urea and sucrose. Tissue Cell. 1978;10(2):331–342. doi: 10.1016/0040-8166(78)90027-7. [DOI] [PubMed] [Google Scholar]
  9. Brightman M. W., Hori M., Rapoport S. I., Reese T. S., Westergaard E. Osmotic opening of tight junctions in cerebral endothelium. J Comp Neurol. 1973 Dec 15;152(4):317–325. doi: 10.1002/cne.901520402. [DOI] [PubMed] [Google Scholar]
  10. Britch M., Allen T. D. The modulation of cellular contractility and adhesion by trypsin and EGTA. Exp Cell Res. 1980 Jan;125(1):221–231. doi: 10.1016/0014-4827(80)90206-2. [DOI] [PubMed] [Google Scholar]
  11. Campisi J., Scandella C. J. Calcium-induced decrease in membrane fluidity of sea urchin egg cortex after fertilization. Nature. 1980 Jul 10;286(5769):185–186. doi: 10.1038/286185a0. [DOI] [PubMed] [Google Scholar]
  12. Carson J. L., Collier A. M., Hu S. S. Ultrastructural studies of hamster tracheal epithelium in vivo and in vitro. J Ultrastruct Res. 1980 Jan;70(1):70–81. doi: 10.1016/s0022-5320(80)90023-4. [DOI] [PubMed] [Google Scholar]
  13. Cereijido M., Robbins E. S., Dolan W. J., Rotunno C. A., Sabatini D. D. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J Cell Biol. 1978 Jun;77(3):853–880. doi: 10.1083/jcb.77.3.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Claude P. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J Membr Biol. 1978 Mar 10;39(2-3):219–232. doi: 10.1007/BF01870332. [DOI] [PubMed] [Google Scholar]
  15. Cohen-Solal L., Cohen-Solal M., Glimcher M. J. Identification of gamma-glutamyl phosphate in the alpha 2 chains of chicken bone collagen. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4327–4330. doi: 10.1073/pnas.76.9.4327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cooper M., Teichberg S., Lifshitz F. Alterations in rat jejunal permeability to a macromolecular tracer during a hyperosmotic load. Lab Invest. 1978 Apr;38(4):447–454. [PubMed] [Google Scholar]
  17. Decker R. S. Disassembly of the zonula occludens during amphibian neurulation. Dev Biol. 1981 Jan 15;81(1):12–22. doi: 10.1016/0012-1606(81)90343-2. [DOI] [PubMed] [Google Scholar]
  18. Dermietzel R., Meller K., Tetzlaff W., Waelsch M. In vivo and in vitro formation of the junctional complex in choroid epithelium. A freeze-etching study. Cell Tissue Res. 1977 Jul 19;181(4):427–441. doi: 10.1007/BF00221766. [DOI] [PubMed] [Google Scholar]
  19. Duffey M. E., Hainau B., Ho S., Bentzel C. J. Regulation of epithelial tight junction permeability by cyclic AMP. Nature. 1981 Dec 3;294(5840):451–453. doi: 10.1038/294451a0. [DOI] [PubMed] [Google Scholar]
  20. Elson E. L., Reidler J. A. Analysis of cell surface interactions by measurements of lateral mobility. J Supramol Struct. 1979;12(4):481–489. doi: 10.1002/jss.400120408. [DOI] [PubMed] [Google Scholar]
  21. Emerman J. T., Burwen S. J., Pitelka D. R. Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture. Tissue Cell. 1979;11(1):109–119. doi: 10.1016/0040-8166(79)90011-9. [DOI] [PubMed] [Google Scholar]
  22. Erlij D., Martínez-Palomo A. Opening of tight junctions in frog skin by hypertonic urea solutions. J Membr Biol. 1972;9(3):229–240. [PubMed] [Google Scholar]
  23. Ernst S. A., Dodson W. C., Karnaky K. J., Jr Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus). J Cell Biol. 1980 Nov;87(2 Pt 1):488–497. doi: 10.1083/jcb.87.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Galli P., Brenna A., Camilli de P., Meldolesi J. Extracellular calcium and the organization of tight junctions in pancreatic acinar cells. Exp Cell Res. 1976 Apr;99(1):178–183. doi: 10.1016/0014-4827(76)90694-7. [DOI] [PubMed] [Google Scholar]
  26. Geiger B., Dutton A. H., Tokuyasu K. T., Singer S. J. Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J Cell Biol. 1981 Dec;91(3 Pt 1):614–628. doi: 10.1083/jcb.91.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Glässer D., Rattke W., Iwig M. Bovine lens epithelium: a suitable model for studying growth control mechanisms. C6-substituted purines inhibit cell flattening and growth stimulation of G0 cells. Exp Cell Res. 1979 Sep;122(2):281–292. doi: 10.1016/0014-4827(79)90305-7. [DOI] [PubMed] [Google Scholar]
  28. Goodenough D. A., Gilula N. B. The splitting of hepatocyte gap junctions and zonulae occludentes with hypertonic disaccharides. J Cell Biol. 1974 Jun;61(3):575–590. doi: 10.1083/jcb.61.3.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Gordon L. M., Sauerheber R. D., Esgate J. A. Spin label studies on rat liver and heart plasma membranes: effects of temperature, calcium, and lanthanum on membrane fluidity. J Supramol Struct. 1978;9(3):299–326. doi: 10.1002/jss.400090303. [DOI] [PubMed] [Google Scholar]
  30. Handler J. S., Steele R. E., Sahib M. K., Wade J. B., Preston A. S., Lawson N. L., Johnson J. P. Toad urinary bladder epithelial cells in culture: maintenance of epithelial structure, sodium transport, and response to hormones. Proc Natl Acad Sci U S A. 1979 Aug;76(8):4151–4155. doi: 10.1073/pnas.76.8.4151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hull B. E., Staehelin L. A. Functional significance of the variations in the geometrical organization of tight junction networks. J Cell Biol. 1976 Mar;68(3):688–704. doi: 10.1083/jcb.68.3.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hull B. E., Staehelin L. A. The terminal web. A reevaluation of its structure and function. J Cell Biol. 1979 Apr;81(1):67–82. doi: 10.1083/jcb.81.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kachar B., Pinto da Silva P. Rapid massive assembly of tight junction strands. Science. 1981 Jul 31;213(4507):541–544. doi: 10.1126/science.7244652. [DOI] [PubMed] [Google Scholar]
  34. Koppel D. E., Sheetz M. P., Schindler M. Matrix control of protein diffusion in biological membranes. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3576–3580. doi: 10.1073/pnas.78.6.3576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lane N. J., Swales L. S. Dispersal of junctional particles, not internalization, during the in vivo disappearance of gap junctions. Cell. 1980 Mar;19(3):579–586. doi: 10.1016/s0092-8674(80)80034-1. [DOI] [PubMed] [Google Scholar]
  36. Lee H. C., Auersperg N. Calcium in epithelial cell contraction. J Cell Biol. 1980 May;85(2):325–336. doi: 10.1083/jcb.85.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Leighton J., Brada Z., Estes L. W., Justh G. Secretory activity and oncogenicity of a cell line (MDCK) derived from canine kidney. Science. 1969 Jan 31;163(3866):472–473. doi: 10.1126/science.163.3866.472. [DOI] [PubMed] [Google Scholar]
  38. Martinez-Palomo A., Meza I., Beaty G., Cereijido M. Experimental modulation of occluding junctions in a cultured transporting epithelium. J Cell Biol. 1980 Dec;87(3 Pt 1):736–745. doi: 10.1083/jcb.87.3.736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Meldolesi J., Castiglioni G., Parma R., Nassivera N., De Camilli P. Ca++-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs. J Cell Biol. 1978 Oct;79(1):156–172. doi: 10.1083/jcb.79.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Meza I., Ibarra G., Sabanero M., Martínez-Palomo A., Cereijido M. Occluding junctions and cytoskeletal components in a cultured transporting epithelium. J Cell Biol. 1980 Dec;87(3 Pt 1):746–754. doi: 10.1083/jcb.87.3.746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Misfeldt D. S., Hamamoto S. T., Pitelka D. R. Transepithelial transport in cell culture. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1212–1216. doi: 10.1073/pnas.73.4.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Montesano R., Gabbiani G., Perrelet A., Orci L. In vivo induction of tight junction proliferation in rat liver. J Cell Biol. 1976 Mar;68(3):793–798. doi: 10.1083/jcb.68.3.793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nicolson G. L. Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over surface components. Biochim Biophys Acta. 1976 Apr 13;457(1):57–108. doi: 10.1016/0304-4157(76)90014-9. [DOI] [PubMed] [Google Scholar]
  44. Orci L., Amherdt M., Henquin J. C., Lambert A. E., Unger R. H., Renold A. E. Pronase effect on pancreatic beta cell secretion and morphology. Science. 1973 May 11;180(4086):647–649. doi: 10.1126/science.180.4086.647. [DOI] [PubMed] [Google Scholar]
  45. Pickett P. B., Pitelka D. R., Hamamoto S. T., Misfeldt D. S. Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells. J Cell Biol. 1975 Aug;66(2):316–332. doi: 10.1083/jcb.66.2.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pitelka D. R., Hamamoto S. T., Duafala J. G., Nemanic M. K. Cell contacts in the mouse mammary gland. I. Normal gland in postnatal development and the secretory cycle. J Cell Biol. 1973 Mar;56(3):797–818. doi: 10.1083/jcb.56.3.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pitelka D. R., Taggart B. N. Mechanical tension induces lateral movement of intramembrane components of the tight junction: studies on mouse mammary cells in culture. J Cell Biol. 1983 Mar;96(3):606–612. doi: 10.1083/jcb.96.3.606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Polak-Charcon S., Ben-Shaul Y. Degradation of tight junctions in HT29, a human colon adenocarcinoma cell line. J Cell Sci. 1979 Feb;35:393–402. doi: 10.1242/jcs.35.1.393. [DOI] [PubMed] [Google Scholar]
  49. Polak-Charcon S., Shoham J., Ben-Shaul Y. Junction formation in trypsinized cells of human adenocarcinoma cell line. Exp Cell Res. 1978 Oct 1;116(1):1–13. doi: 10.1016/0014-4827(78)90058-7. [DOI] [PubMed] [Google Scholar]
  50. Porvaznik M. Tight junction disruption and recovery after sublethal gamma irradiation. Radiat Res. 1979 May;78(2):233–250. [PubMed] [Google Scholar]
  51. RICHARDSON K. C., JARETT L., FINKE E. H. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 1960 Nov;35:313–323. doi: 10.3109/10520296009114754. [DOI] [PubMed] [Google Scholar]
  52. Rabito C. A., Tchao R., Valentich J., Leighton J. Distribution and characteristics of the occluding junctions in a monolayer of a cell line (MDCK) derived from canine kidney. J Membr Biol. 1978 Nov 8;43(4):351–365. doi: 10.1007/BF01871696. [DOI] [PubMed] [Google Scholar]
  53. Rapoport S. I., Thompson H. K. Osmotic opening of the blood-brain barrier in the monkey without associated neurological deficits. Science. 1973 Jun 1;180(4089):971–971. doi: 10.1126/science.180.4089.971. [DOI] [PubMed] [Google Scholar]
  54. Reeves W., Caulfield J. P., Farquhar M. G. Differentiation of epithelial foot processes and filtration slits: sequential appearance of occluding junctions, epithelial polyanion, and slit membranes in developing glomeruli. Lab Invest. 1978 Aug;39(2):90–100. [PubMed] [Google Scholar]
  55. Revel J. P., Yip P., Chang L. L. Cell junctions in the early chick embryo--a freeze etch study. Dev Biol. 1973 Dec;35(2):302–317. doi: 10.1016/0012-1606(73)90026-2. [DOI] [PubMed] [Google Scholar]
  56. Rhodes R. S., Karnovsky M. J. Loss of macromolecular barrier function associated with surgical trauma to the intestine. Lab Invest. 1971 Sep;25(3):220–229. [PubMed] [Google Scholar]
  57. Rosen J. J., Culp L. A. Morphology and cellular origins of substrate-attached material from mouse fibroblasts. Exp Cell Res. 1977 Jun;107(1):139–149. doi: 10.1016/0014-4827(77)90395-0. [DOI] [PubMed] [Google Scholar]
  58. SEDAR A. W., FORTE J. G. EFFECTS OF CALCIUM DEPLETION ON THE JUNCTIONAL COMPLEX BETWEEN OXYNTIC CELLS OF GASTRIC GLANDS. J Cell Biol. 1964 Jul;22:173–188. doi: 10.1083/jcb.22.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Schneeberger E. E. Heterogeneity of tight junction morphology in extrapulmonary and intrapulmonary airways of the rat. Anat Rec. 1980 Oct;198(2):193–208. doi: 10.1002/ar.1091980207. [DOI] [PubMed] [Google Scholar]
  60. Shimono M., Clementi F. Intercellular junctions of oral epithelium. II. Ultrastructural changes in rat buccal epithelium induced by trypsin digestion. J Ultrastruct Res. 1977 Apr;59(1):101–112. doi: 10.1016/s0022-5320(77)80032-4. [DOI] [PubMed] [Google Scholar]
  61. Singer I. I. The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts. Cell. 1979 Mar;16(3):675–685. doi: 10.1016/0092-8674(79)90040-0. [DOI] [PubMed] [Google Scholar]
  62. Staehelin L. A. Further observations on the fine structure of freeze-cleaved tight junctions. J Cell Sci. 1973 Nov;13(3):763–786. doi: 10.1242/jcs.13.3.763. [DOI] [PubMed] [Google Scholar]
  63. 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]
  64. Talmon A., Ben-Shaul Y. Tight junctions of dissociated and reaggregated embryonic lung cells. Cell Differ. 1979 Dec;8(6):437–448. doi: 10.1016/0045-6039(79)90040-x. [DOI] [PubMed] [Google Scholar]
  65. Teng M. H., Rifkin D. B. Fibronectin from chicken embryo fibroblasts contains covalently bound phosphate. J Cell Biol. 1979 Mar;80(3):784–791. doi: 10.1083/jcb.80.3.784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Tice L. W., Carter R. L., Cahill M. B. Changes in tight junctions of rat intestinal crypt cells associated with changes in their mitotic activity. Tissue Cell. 1979;11(2):293–316. doi: 10.1016/0040-8166(79)90043-0. [DOI] [PubMed] [Google Scholar]
  67. Wade J. B., Karnovsky M. J. Fracture faces of osmotically disrupted zonulae occludentes. J Cell Biol. 1974 Aug;62(2):344–350. doi: 10.1083/jcb.62.2.344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wade J. B., Revel J. P., DiScala V. A. Effect of osmotic gradients on intercellular junctions of the toad bladder. Am J Physiol. 1973 Feb;224(2):407–415. doi: 10.1152/ajplegacy.1973.224.2.407. [DOI] [PubMed] [Google Scholar]
  69. White M. T., Hu A. S., Hamamoto S. T., Nandi S. In vitro analysis of proliferating epithelial cell populations from the mouse mammary gland: fibroblast-free growth and serial passage. In Vitro. 1978 Mar;14(3):271–281. doi: 10.1007/BF02616036. [DOI] [PubMed] [Google Scholar]

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