Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Jan 1;108(1):13–22. doi: 10.1083/jcb.108.1.13

Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2)

PMCID: PMC2115365  PMID: 2642910

Abstract

The polarized delivery of membrane proteins to the cell surface and the initial secretion of lysosomal proteins into the culture medium were studied in the polarized human intestinal adenocarcinoma cell line Caco- 2 in the presence or absence of the microtubule-active drug nocodazole. The appearance of newly synthesized proteins at the plasma membrane was measured by their sensitivity to proteases added either to the apical or the basolateral surface of cells grown on nitrocellulose filters. Nocodazole was found to reduce the delivery to the cell surface of an apical membrane protein, aminopeptidase N, and to lead to its partial missorting to the basolateral surface, whereas the drug had no influence on the delivery of a basolateral 120-kD membrane protein defined by a monoclonal antibody. Furthermore, nocodazole selectively blocked the apical secretion of two lysosomal proteins, cathepsin D and acid alpha-glucosidase, whereas the drug had no influence on their basolateral secretion. These results suggest that in Caco-2 cells an intact microtubular network is important for the transport of newly synthesized proteins to the apical cell surface.

Full Text

The Full Text of this article is available as a PDF (3.0 MB).

Selected References

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

  1. Ahnen D. J., Brown W. R., Kloppel T. M. Secretory component: the polymeric immunoglobulin receptor. What's in it for the gastroenterologist and hepatologist? Gastroenterology. 1985 Sep;89(3):667–682. doi: 10.1016/0016-5085(85)90467-6. [DOI] [PubMed] [Google Scholar]
  2. Artvinli S. Biochemical aspects of aldehyde fixation and a new formaldehyde fixative. Histochem J. 1975 Sep;7(5):435–450. doi: 10.1007/BF01003881. [DOI] [PubMed] [Google Scholar]
  3. Bartles J. R., Feracci H. M., Stieger B., Hubbard A. L. Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. J Cell Biol. 1987 Sep;105(3):1241–1251. doi: 10.1083/jcb.105.3.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartles J. R., Hubbard A. L. Plasma membrane protein sorting in epithelial cells: do secretory pathways hold the key? Trends Biochem Sci. 1988 May;13(5):181–184. doi: 10.1016/0968-0004(88)90147-8. [DOI] [PubMed] [Google Scholar]
  5. Bennett G., Carlet E., Wild G., Parsons S. Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucose injection. Am J Anat. 1984 Aug;170(4):545–566. doi: 10.1002/aja.1001700404. [DOI] [PubMed] [Google Scholar]
  6. Blok J., Ginsel L. A., Mulder-Stapel A. A., Onderwater J. J., Daems W. T. The effect of colchicine on the intracellular transport of 3H-fucose-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue. An autoradiographical and biochemical study. Cell Tissue Res. 1981;215(1):1–12. doi: 10.1007/BF00236244. [DOI] [PubMed] [Google Scholar]
  7. Danielsen E. M., Cowell G. M., Poulsen S. S. Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules. Biochem J. 1983 Oct 15;216(1):37–42. doi: 10.1042/bj2160037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ellinger A., Pavelka M., Gangl A. Effect of colchicine on rat small intestinal absorptive cells. II. Distribution of label after incorporation of [3H]fucose into plasma membrane glycoproteins. J Ultrastruct Res. 1983 Dec;85(3):260–271. doi: 10.1016/s0022-5320(83)90038-2. [DOI] [PubMed] [Google Scholar]
  9. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fuller S., von Bonsdorff C. H., Simons K. Vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line, MDCK. Cell. 1984 Aug;38(1):65–77. doi: 10.1016/0092-8674(84)90527-0. [DOI] [PubMed] [Google Scholar]
  11. Galfre G., Howe S. C., Milstein C., Butcher G. W., Howard J. C. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature. 1977 Apr 7;266(5602):550–552. doi: 10.1038/266550a0. [DOI] [PubMed] [Google Scholar]
  12. Gorbsky G., Borisy G. G. Microtubule distribution in cultured cells and intact tissues: improved immunolabeling resolution through the use of reversible embedment cytochemistry. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6889–6893. doi: 10.1073/pnas.82.20.6889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  14. Hagen S. J., Allan C. H., Trier J. S. Demonstration of microtubules in the terminal web of mature absorptive cells from the small intestine of the rat. Cell Tissue Res. 1987 Jun;248(3):709–711. doi: 10.1007/BF00216503. [DOI] [PubMed] [Google Scholar]
  15. Hauri H. P., Bucher K. Immunoblotting with monoclonal antibodies: importance of the blocking solution. Anal Biochem. 1986 Dec;159(2):386–389. doi: 10.1016/0003-2697(86)90357-x. [DOI] [PubMed] [Google Scholar]
  16. Hauri H. P., Sterchi E. E., Bienz D., Fransen J. A., Marxer A. Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells. J Cell Biol. 1985 Sep;101(3):838–851. doi: 10.1083/jcb.101.3.838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  18. Herbst J. J., Hurwitz R., Sunshine P., Kretchmer N. Effect of colchicine on intestinal disaccharidases: correlation with biochemical aspects of cellular renewal. J Clin Invest. 1970 Mar;49(3):530–536. doi: 10.1172/JCI106263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hilkens J., Tager J. M., Buijs F., Brouwer-Kelder B., Van Thienen G. M., Tegelaers F. P., Hilgers J. Monoclonal antibodies against human acid alpha-glucosidase. Biochim Biophys Acta. 1981 Nov 18;678(1):7–11. doi: 10.1016/0304-4165(81)90041-6. [DOI] [PubMed] [Google Scholar]
  20. Hugon J. S., Bennett G., Pothier P., Ngoma Z. Loss of microtubules and alteration of glycoprotein migration in organ cultures of mouse intestine exposed to nocodazole or colchicine. Cell Tissue Res. 1987 Jun;248(3):653–662. doi: 10.1007/BF00216496. [DOI] [PubMed] [Google Scholar]
  21. Kreis T. E. Microtubules containing detyrosinated tubulin are less dynamic. EMBO J. 1987 Sep;6(9):2597–2606. doi: 10.1002/j.1460-2075.1987.tb02550.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Massey D., Feracci H., Gorvel J. P., Rigal A., Soulié J. M., Maroux S. Evidence for the transit of aminopeptidase N through the basolateral membrane before it reaches the brush border of enterocytes. J Membr Biol. 1987;96(1):19–25. doi: 10.1007/BF01869331. [DOI] [PubMed] [Google Scholar]
  23. Matlin K. S., Simons K. Sorting of an apical plasma membrane glycoprotein occurs before it reaches the cell surface in cultured epithelial cells. J Cell Biol. 1984 Dec;99(6):2131–2139. doi: 10.1083/jcb.99.6.2131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Misek D. E., Bard E., Rodriguez-Boulan E. Biogenesis of epithelial cell polarity: intracellular sorting and vectorial exocytosis of an apical plasma membrane glycoprotein. Cell. 1984 Dec;39(3 Pt 2):537–546. doi: 10.1016/0092-8674(84)90460-4. [DOI] [PubMed] [Google Scholar]
  25. Mostov K. E., Simister N. E. Transcytosis. Cell. 1985 Dec;43(2 Pt 1):389–390. doi: 10.1016/0092-8674(85)90166-7. [DOI] [PubMed] [Google Scholar]
  26. Nagura H., Nakane P. K., Brown W. R. Translocation of dimeric IgA through neoplastic colon cells in vitro. J Immunol. 1979 Nov;123(5):2359–2368. [PubMed] [Google Scholar]
  27. Oude Elferink R. P., Van Doorn-Van Wakeren J., Strijland A., Reuser A. J., Tager J. M. Biosynthesis and intracellular transport of alpha-glucosidase and cathepsin D in normal and mutant human fibroblasts. Eur J Biochem. 1985 Nov 15;153(1):55–63. doi: 10.1111/j.1432-1033.1985.tb09266.x. [DOI] [PubMed] [Google Scholar]
  28. Pavelka M., Ellinger A. Effect of colchicine on the Golgi apparatus and on GERL of rat jejunal absorptive cells. Ultrastructural localization of thiamine pyrophosphatase and acid phosphatase activity. Eur J Cell Biol. 1981 Apr;24(1):53–61. [PubMed] [Google Scholar]
  29. Pavelka M., Ellinger A., Gangl A. Effect of colchicine on rat small intestinal absorptive cells. I Formation of basolateral microvillus borders. J Ultrastruct Res. 1983 Dec;85(3):249–259. doi: 10.1016/s0022-5320(83)90037-0. [DOI] [PubMed] [Google Scholar]
  30. Quaroni A., Kirsch K., Weiser M. M. Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effect of colchicine on the redistribution of L-[1,5,6-3H]fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes. Biochem J. 1979 Jul 15;182(1):213–221. doi: 10.1042/bj1820213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rindler M. J., Ivanov I. E., Plesken H., Sabatini D. D. Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses. J Cell Biol. 1985 Jan;100(1):136–151. doi: 10.1083/jcb.100.1.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rindler M. J., Ivanov I. E., Sabatini D. D. Microtubule-acting drugs lead to the nonpolarized delivery of the influenza hemagglutinin to the cell surface of polarized Madin-Darby canine kidney cells. J Cell Biol. 1987 Feb;104(2):231–241. doi: 10.1083/jcb.104.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Salas P. J., Misek D. E., Vega-Salas D. E., Gundersen D., Cereijido M., Rodriguez-Boulan E. Microtubules and actin filaments are not critically involved in the biogenesis of epithelial cell surface polarity. J Cell Biol. 1986 May;102(5):1853–1867. doi: 10.1083/jcb.102.5.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sandoz D., Lainé M. C., Nicolas G. Distribution of microtubules within the intestinal terminal web as revealed by quick-freezing and cryosubstitution. Eur J Cell Biol. 1986 Jan;39(2):481–484. [PubMed] [Google Scholar]
  35. Schweizer A., Fransen J. A., Bächi T., Ginsel L., Hauri H. P. Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. J Cell Biol. 1988 Nov;107(5):1643–1653. doi: 10.1083/jcb.107.5.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Simons K., Fuller S. D. Cell surface polarity in epithelia. Annu Rev Cell Biol. 1985;1:243–288. doi: 10.1146/annurev.cb.01.110185.001331. [DOI] [PubMed] [Google Scholar]
  37. Slot J. W., Geuze H. J. A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol. 1985 Jul;38(1):87–93. [PubMed] [Google Scholar]
  38. Stieger B., Matter K., Baur B., Bucher K., Höchli M., Hauri H. P. Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface. J Cell Biol. 1988 Jun;106(6):1853–1861. doi: 10.1083/jcb.106.6.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Vale R. D. Intracellular transport using microtubule-based motors. Annu Rev Cell Biol. 1987;3:347–378. doi: 10.1146/annurev.cb.03.110187.002023. [DOI] [PubMed] [Google Scholar]

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

RESOURCES