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. 1974 Apr 1;61(1):70–82. doi: 10.1083/jcb.61.1.70

EFFECTS OF COLCHICINE, CYTOCHALASIN B, AND 2-DEOXYGLUCOSE ON THE TOPOGRAPHICAL ORGANIZATION OF SURFACE-BOUND CONCANAVALIN A IN NORMAL AND TRANSFORMED FIBROBLASTS

Thomas E Ukena 1, Joan Z Borysenko 1, Morris J Karnovsky 1, Richard D Berlin 1
PMCID: PMC2109267  PMID: 4132067

Abstract

The distribution of surface-bound concanavalin A on the membranes of 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts was examined using a shadow-cast replica technique with a hemocyanin marker. When cells were prefixed in paraformaldehyde, the binding site distribution was always random on both cell types. On the other hand, labeling of transformed cells with concanavalin A (Con A) and hemocyanin at 37°C resulted in the organization of Con A binding sites (CABS) into clusters (primary organization) which were not present on the pseudopodia and other peripheral areas of the membrane (secondary organization). Treatment of transformed cells with colchicine, cytochalasin B, or 2-deoxyglucose did not alter the inherent random distribution of binding sites as determined by fixation before labeling. However, these drugs produced marked changes in the secondary (but not the primary) organization of CABS on transformed cells labeled at 37°C. Colchicine treatment resulted in the formation of a caplike aggregation of binding site clusters near the center of the cell, whereas cytochalasin B and 2-deoxyglucose led to the formation of patches of CABS over the entire membrane, eliminating the inward displacement of patches observed on untreated cells. The distribution of bound Con A on normal cells (3T3) at 37°C was always random, in both control and drug-treated preparations. Pretreatment of cells with Con A enhanced the effect of colchicine on cell morphology, but inhibited the morphological effects of cytochalasin B. The mechanisms that determine receptor movement and disposition are discussed.

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Selected References

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  1. ABERCROMBIE M., HEAYSMAN J. E., KARTHAUSER H. M. Social behaviour of cells in tissue culture. III. Mutual influence of sarcoma cells and fibroblasts. Exp Cell Res. 1957 Oct;13(2):276–291. doi: 10.1016/0014-4827(57)90007-1. [DOI] [PubMed] [Google Scholar]
  2. AUB J. C., TIESLAU C., LANKESTER A. REACTIONS OF NORMAL AND TUMOR CELL SURFACES TO ENZYMES. I. WHEAT-GERM LIPASE AND ASSOCIATED MUCOPOLYSACCHARIDES. Proc Natl Acad Sci U S A. 1963 Oct;50:613–619. doi: 10.1073/pnas.50.4.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Abercrombie M., Heaysman J. E., Pegrum S. M. The locomotion of fibroblasts in culture. 3. Movements of particles on the dorsal surface of the leading lamella. Exp Cell Res. 1970 Oct;62(2):389–398. doi: 10.1016/0014-4827(70)90570-7. [DOI] [PubMed] [Google Scholar]
  4. Addanki S., Sotos J. F., Rearick P. D. Rapid determination of picomole quantities of ATP with a liquid scintillation counter. Anal Biochem. 1966 Feb;14(2):261–264. doi: 10.1016/0003-2697(66)90135-7. [DOI] [PubMed] [Google Scholar]
  5. Arndt-Jovin D. J., Berg P. Quantitative binding of 125 I-concanavalin A to normal and transformed cells. J Virol. 1971 Nov;8(5):716–721. doi: 10.1128/jvi.8.5.716-721.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Aub J. C., Sanford B. H., Cote M. N. Studies on reactivity of tumor and normal cells to a wheat germ agglutinin. Proc Natl Acad Sci U S A. 1965 Aug;54(2):396–399. doi: 10.1073/pnas.54.2.396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berlin R. D., Ukena T. E. Effect of colchicine and vinblastine on the agglutination of polymorpho-nuclear leucocytes by concanavalin A. Nat New Biol. 1972 Jul 26;238(82):120–122. doi: 10.1038/newbio238120a0. [DOI] [PubMed] [Google Scholar]
  8. Burger M. M. A difference in the architecture of the surface membrane of normal and virally transformed cells. Proc Natl Acad Sci U S A. 1969 Mar;62(3):994–1001. doi: 10.1073/pnas.62.3.994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burger M. M., Goldberg A. R. Identification of a tumor-specific determinant on neoplastic cell surfaces. Proc Natl Acad Sci U S A. 1967 Feb;57(2):359–366. doi: 10.1073/pnas.57.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cline M. J., Livingston D. C. Binding of 3 H-concanavalin A by normal and transformed cells. Nat New Biol. 1971 Aug 4;232(31):155–156. doi: 10.1038/newbio232155a0. [DOI] [PubMed] [Google Scholar]
  11. Ebstensen R. D., Plagemann P. G. Cytochalasin B: inhibition of glucose and glucosamine transport. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1430–1434. doi: 10.1073/pnas.69.6.1430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Edelman G. M., Yahara I., Wang J. L. Receptor mobility and receptor-cytoplasmic interactions in lymphocytes. Proc Natl Acad Sci U S A. 1973 May;70(5):1442–1446. doi: 10.1073/pnas.70.5.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edidin M., Weiss A. Antigen cap formation in cultured fibroblasts: a reflection of membrane fluidity and of cell motility. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2456–2459. doi: 10.1073/pnas.69.9.2456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. HELMKAMP R. W., GOODLAND R. L., BALE W. F., SPAR I. L., MUTSCHLER L. E. High specific activity iodination of gamma-globulin with iodine-131 monochloride. Cancer Res. 1960 Nov;20:1495–1500. [PubMed] [Google Scholar]
  15. Hatanaka M., Augl C., Gilden R. V. Evidence for a functional change in the plasma membrane of murine sarcoma virus-infected mouse embryo cells. Transport and transport-associated phosphorylation of 14C-2-deoxy-D-glucose. J Biol Chem. 1970 Feb 25;245(4):714–717. [PubMed] [Google Scholar]
  16. Hatanaka M., Hanafusa H. Analysis of a functional change in membrane in the process of cell transformation by Rous sarcoma virus; alteration in the characteristics of sugar transport. Virology. 1970 Aug;41(4):647–652. doi: 10.1016/0042-6822(70)90429-0. [DOI] [PubMed] [Google Scholar]
  17. Inbar M., Sachs L. Interaction of the carbohydrate-binding protein concanavalin A with normal and transformed cells. Proc Natl Acad Sci U S A. 1969 Aug;63(4):1418–1425. doi: 10.1073/pnas.63.4.1418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Karnovsky M. J., Unanue E. R., Leventhal M. Ligand-induced movement of lymphocyte membrane macromolecules. II. Mapping of surface moieties. J Exp Med. 1972 Oct 1;136(4):907–930. doi: 10.1084/jem.136.4.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kletzien R. F., Perdue J. F., Springer A. Cytochalasin A and B. Inhibition of sugar uptake in cultured cells. J Biol Chem. 1972 May 10;247(9):2964–2966. [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Martin G. S., Venuta S., Weber M., Rubin H. Temperature-dependent alterations in sugar transport in cells infected by a temperature-sensitive mutant of Rous sarcoma virus. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2739–2741. doi: 10.1073/pnas.68.11.2739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mizel S. B., Wilson L. Inhibition of the transport of several hexoses in mammalian cells by cytochalasin B. J Biol Chem. 1972 Jun 25;247(12):4102–4105. [PubMed] [Google Scholar]
  23. Nicolson G. L., Blaustein J. The interaction of Ricinus communis agglutinin with normal and tumor cell surfaces. Biochim Biophys Acta. 1972 May 9;266(2):543–547. doi: 10.1016/0005-2736(72)90109-5. [DOI] [PubMed] [Google Scholar]
  24. Nicolson G. L. Temperature-dependent mobility of concanavalin A sites on tumour cell surfaces. Nat New Biol. 1973 Jun 13;243(128):218–220. doi: 10.1038/newbio243218a0. [DOI] [PubMed] [Google Scholar]
  25. Nicolson G. L. Topography of membrane concanavalin A sites modified by proteolysis. Nat New Biol. 1972 Oct 18;239(94):193–197. doi: 10.1038/newbio239193a0. [DOI] [PubMed] [Google Scholar]
  26. Ozanne B., Sambrook J. Binding of radioactively labelled concanavalin A and wheat germ agglutinin to normal and virus-transformed cells. Nat New Biol. 1971 Aug 4;232(31):156–160. doi: 10.1038/newbio232156a0. [DOI] [PubMed] [Google Scholar]
  27. Plagemann P. G., Estensen R. D. Cytochalasin B. VI. Competitive inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells. J Cell Biol. 1972 Oct;55(1):179–185. doi: 10.1083/jcb.55.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rosenblith J. Z., Ukena T. E., Yin H. H., Berlin R. D., Karnovsky M. J. A comparative evaluation of the distribution of concanavalin A-binding sites on the surfaces of normal, virally-transformed, and protease-treated fibroblasts. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1625–1629. doi: 10.1073/pnas.70.6.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sela B. A., Lis H., Sharon N., Sachs L. Quantitation of N-acetyl-D-galactosamine-like sites on the surface membrane of normal and transformed mammalian cells. Biochim Biophys Acta. 1971 Dec 3;249(2):564–568. doi: 10.1016/0005-2736(71)90132-5. [DOI] [PubMed] [Google Scholar]
  30. Smith S. B., Revel J. P. Mapping of concanavalin A binding sites on the surface of several cell types. Dev Biol. 1972 Mar;27(3):434–441. doi: 10.1016/0012-1606(72)90183-2. [DOI] [PubMed] [Google Scholar]
  31. TODARO G. J., GREEN H., GOLDBERG B. D. TRANSFORMATION OF PROPERTIES OF AN ESTABLISHED CELL LINE BY SV40 AND POLYOMA VIRUS. Proc Natl Acad Sci U S A. 1964 Jan;51:66–73. doi: 10.1073/pnas.51.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ukena T. E., Berlin R. D. Effect of colchicine and vinblastine on the topographical separation of membrane functions. J Exp Med. 1972 Jul 1;136(1):1–7. doi: 10.1084/jem.136.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vasiliev J. M., Gelfand I. M., Domnina L. V., Ivanova O. Y., Komm S. G., Olshevskaja L. V. Effect of colcemid on the locomotory behaviour of fibroblasts. J Embryol Exp Morphol. 1970 Nov;24(3):625–640. [PubMed] [Google Scholar]
  34. Veselý P., Entlicher G., Kocourek J. Pea phytohemagglutinin selective agglutination of tumour cells. Experientia. 1972 Sep 15;28(9):1085–1086. doi: 10.1007/BF01918689. [DOI] [PubMed] [Google Scholar]
  35. Warner D. A., Perdue J. F. Cytochalasin B and the adenosine triphosphate content of treated fibroblasts. J Cell Biol. 1972 Oct;55(1):242–244. doi: 10.1083/jcb.55.1.242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Weisenberg R. C., Borisy G. G., Taylor E. W. The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry. 1968 Dec;7(12):4466–4479. doi: 10.1021/bi00852a043. [DOI] [PubMed] [Google Scholar]
  37. Wessells N. K., Spooner B. S., Ash J. F., Bradley M. O., Luduena M. A., Taylor E. L., Wrenn J. T., Yamada K. Microfilaments in cellular and developmental processes. Science. 1971 Jan 15;171(3967):135–143. doi: 10.1126/science.171.3967.135. [DOI] [PubMed] [Google Scholar]
  38. Wilson L., Friedkin M. The biochemical events of mitosis. I. Synthesis and properties of colchicine labeled with tritium in its acetyl moiety. Biochemistry. 1966 Jul;5(7):2463–2468. doi: 10.1021/bi00871a042. [DOI] [PubMed] [Google Scholar]
  39. Wilson L., Friedkin M. The biochemical events of mitosis. II. The in vivo and in vitro binding of colchicine in grasshopper embryos and its possible relation to inhibition of mitosis. Biochemistry. 1967 Oct;6(10):3126–3135. doi: 10.1021/bi00862a021. [DOI] [PubMed] [Google Scholar]
  40. Yahara I., Edelman G. M. Restriction of the mobility of lymphocyte immunoglobulin receptors by concanavalin A. Proc Natl Acad Sci U S A. 1972 Mar;69(3):608–612. doi: 10.1073/pnas.69.3.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yin H. H., Ukena T. E., Berlin R. D. Effect of colchicine, colcemid, and vinblastine on the agglutination, by concanavalin A, of transformed cells. Science. 1972 Nov 24;178(4063):867–868. doi: 10.1126/science.178.4063.867. [DOI] [PubMed] [Google Scholar]

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