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. 1976 Apr;73(4):1227–1231. doi: 10.1073/pnas.73.4.1227

Reversible in vitro polymerization of tubulin from a cultured cell line (rat glial cell clone C6).

G Wiche, R D Cole
PMCID: PMC430235  PMID: 1063405

Abstract

Tubulin from cultures of the rat glial cell clone C6 could be polymerized in vitro into intact microtubules. The polymerization was reversible and spontaneous, i.e., no addition of heterologous nucleation centers was necessary. Two cycles of polymerization/depolymerization yielded tubulin preparations of 95% purity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy was used to show that the microtubules assembled in vitro by two cycles of polymerization/depolymerization were morphologically intact and temperature sensitive. In contrast, tubulin from neuroblastoma cells, clone Neuro-2A, could not be polymerized in a reversible fashion. The discovery of a cell line from which tubulin can be reversibly polymerized in vitro establishes a model system for studies of cell-cycle- and cell-type-dependent regulatory mechanisms controlling the assembly of microtubules.

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Selected References

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

  1. Ames G. F. Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. Membrane, soluble, and periplasmic fractions. J Biol Chem. 1974 Jan 25;249(2):634–644. [PubMed] [Google Scholar]
  2. Borisy G. G., Olmsted J. B., Klugman R. A. In vitro aggregation of cytoplasmic microtubule subunits. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2890–2894. doi: 10.1073/pnas.69.10.2890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borisy G. G., Olmsted J. B., Marcum J. M., Allen C. Microtubule assembly in vitro. Fed Proc. 1974 Feb;33(2):167–174. [PubMed] [Google Scholar]
  4. Castle A. G., Crawford N. Isolation of tubulin from pig platelets. FEBS Lett. 1975 Mar 1;51(1):195–200. doi: 10.1016/0014-5793(75)80886-6. [DOI] [PubMed] [Google Scholar]
  5. Eipper B. A. Properties of rat brain tubulin. J Biol Chem. 1974 Mar 10;249(5):1407–1416. [PubMed] [Google Scholar]
  6. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  7. Kirschner M. W., Honig L. S., Williams R. C. Quantitative electron microscopy of microtubule assembly in vitro. J Mol Biol. 1975 Dec 5;99(2):263–276. doi: 10.1016/s0022-2836(75)80144-6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Olmsted J. B., Borisy G. G. Microtubules. Annu Rev Biochem. 1973;42:507–540. doi: 10.1146/annurev.bi.42.070173.002451. [DOI] [PubMed] [Google Scholar]
  11. Rebhun L. I., Jemiolo D., Ivy N., Mellon M., Nath J. Regulation of the in vivo mitotic apparatus by glycols and metabolic inhibitors. Ann N Y Acad Sci. 1975 Jun 30;253:362–377. doi: 10.1111/j.1749-6632.1975.tb19214.x. [DOI] [PubMed] [Google Scholar]
  12. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sheterline P., Schofield J. G. Endogenous phosphorylation and dephosphorylation of microtubule-associated proteins isolated from bovine anterior pituitary. FEBS Lett. 1975 Aug 15;56(2):297–302. doi: 10.1016/0014-5793(75)81113-6. [DOI] [PubMed] [Google Scholar]
  14. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Weisenberg R. C. Microtubule formation in vitro in solutions containing low calcium concentrations. Science. 1972 Sep 22;177(4054):1104–1105. doi: 10.1126/science.177.4054.1104. [DOI] [PubMed] [Google Scholar]
  16. Yahara I., Edelman G. M. Modulation of lymphocyte receptor mobility by locally bound concanavalin A. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1579–1583. doi: 10.1073/pnas.72.4.1579. [DOI] [PMC free article] [PubMed] [Google Scholar]

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