Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Dec 1;105(6):2847–2854. doi: 10.1083/jcb.105.6.2847

Temperature-dependent reversible assembly of taxol-treated microtubules

PMCID: PMC2114708  PMID: 2891714

Abstract

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Berkowitz S. A., Katagiri J., Binder H. K., Williams R. C., Jr Separation and characterization of microtubule proteins from calf brain. Biochemistry. 1977 Dec 13;16(25):5610–5617. doi: 10.1021/bi00644a035. [DOI] [PubMed] [Google Scholar]
  2. Bloom G. S., Luca F. C., Collins C. A., Vallee R. B. Use of multiple monoclonal antibodies to characterize the major microtubule-associated protein in sea urchin eggs. Cell Motil. 1985;5(6):431–446. doi: 10.1002/cm.970050602. [DOI] [PubMed] [Google Scholar]
  3. Bloom G. S., Luca F. C., Vallee R. B. Microtubule-associated protein 1B: identification of a major component of the neuronal cytoskeleton. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5404–5408. doi: 10.1073/pnas.82.16.5404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bloom G. S., Luca F. C., Vallee R. B. Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules. J Cell Biol. 1984 Jan;98(1):331–340. doi: 10.1083/jcb.98.1.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bloom G. S., Schoenfeld T. A., Vallee R. B. Widespread distribution of the major polypeptide component of MAP 1 (microtubule-associated protein 1) in the nervous system. J Cell Biol. 1984 Jan;98(1):320–330. doi: 10.1083/jcb.98.1.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borisy G. G., Marcum J. M., Olmsted J. B., Murphy D. B., Johnson K. A. Purification of tubulin and associated high molecular weight proteins from porcine brain and characterization of microtubule assembly in vitro. Ann N Y Acad Sci. 1975 Jun 30;253:107–132. doi: 10.1111/j.1749-6632.1975.tb19196.x. [DOI] [PubMed] [Google Scholar]
  7. Bulinski J. C., Borisy G. G. Self-assembly of microtubules in extracts of cultured HeLa cells and the identification of HeLa microtubule-associated proteins. Proc Natl Acad Sci U S A. 1979 Jan;76(1):293–297. doi: 10.1073/pnas.76.1.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cabral F., Wible L., Brenner S., Brinkley B. R. Taxol-requiring mutant of Chinese hamster ovary cells with impaired mitotic spindle assembly. J Cell Biol. 1983 Jul;97(1):30–39. doi: 10.1083/jcb.97.1.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cleveland D. W., Hwo S. Y., Kirschner M. W. Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin. J Mol Biol. 1977 Oct 25;116(2):207–225. doi: 10.1016/0022-2836(77)90213-3. [DOI] [PubMed] [Google Scholar]
  10. Coluccio L. M., Tilney L. G. Phalloidin enhances actin assembly by preventing monomer dissociation. J Cell Biol. 1984 Aug;99(2):529–535. doi: 10.1083/jcb.99.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Correia J. J., Williams R. C., Jr Mechanisms of assembly and disassembly of microtubules. Annu Rev Biophys Bioeng. 1983;12:211–235. doi: 10.1146/annurev.bb.12.060183.001235. [DOI] [PubMed] [Google Scholar]
  12. De Brabander M., Geuens G., Nuydens R., Willebrords R., De Mey J. Taxol induces the assembly of free microtubules in living cells and blocks the organizing capacity of the centrosomes and kinetochores. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5608–5612. doi: 10.1073/pnas.78.9.5608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Detrich H. W., 3rd, Wilson L. Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. Biochemistry. 1983 May 10;22(10):2453–2462. doi: 10.1021/bi00279a023. [DOI] [PubMed] [Google Scholar]
  14. Gaskin F., Cantor C. R., Shelanski M. L. Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules. J Mol Biol. 1974 Nov 15;89(4):737–755. doi: 10.1016/0022-2836(74)90048-5. [DOI] [PubMed] [Google Scholar]
  15. Horwitz S. B., Lothstein L., Manfredi J. J., Mellado W., Parness J., Roy S. N., Schiff P. B., Sorbara L., Zeheb R. Taxol: mechanisms of action and resistance. Ann N Y Acad Sci. 1986;466:733–744. doi: 10.1111/j.1749-6632.1986.tb38455.x. [DOI] [PubMed] [Google Scholar]
  16. Janson J. C. Adsorption phenomena on Sephadex. J Chromatogr. 1967 May;28(1):12–20. doi: 10.1016/s0021-9673(01)85920-3. [DOI] [PubMed] [Google Scholar]
  17. Karr T. L., Kristofferson D., Purich D. L. Calcium ion induces endwise depolymerization of bovine brain microtubules. J Biol Chem. 1980 Dec 25;255(24):11853–11856. [PubMed] [Google Scholar]
  18. Kumar N. Taxol-induced polymerization of purified tubulin. Mechanism of action. J Biol Chem. 1981 Oct 25;256(20):10435–10441. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Marcum J. M., Borisy G. G. Characterization of microtubule protein oligomers by analytical ultracentrifugation. J Biol Chem. 1978 Apr 25;253(8):2825–2833. [PubMed] [Google Scholar]
  22. Margolis R. L., Rauch C. T., Job D. Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior. Proc Natl Acad Sci U S A. 1986 Feb;83(3):639–643. doi: 10.1073/pnas.83.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Olmsted J. B., Borisy G. G. Characterization of microtubule assembly in porcine brain extracts by viscometry. Biochemistry. 1973 Oct 9;12(21):4282–4289. doi: 10.1021/bi00745a037. [DOI] [PubMed] [Google Scholar]
  24. Olmsted J. B., Borisy G. G. Ionic and nucleotide requirements for microtubule polymerization in vitro. Biochemistry. 1975 Jul;14(13):2996–3005. doi: 10.1021/bi00684a032. [DOI] [PubMed] [Google Scholar]
  25. Parness J., Horwitz S. B. Taxol binds to polymerized tubulin in vitro. J Cell Biol. 1981 Nov;91(2 Pt 1):479–487. doi: 10.1083/jcb.91.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Paschal B. M., Shpetner H. S., Vallee R. B. MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. J Cell Biol. 1987 Sep;105(3):1273–1282. doi: 10.1083/jcb.105.3.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schiff P. B., Fant J., Horwitz S. B. Promotion of microtubule assembly in vitro by taxol. Nature. 1979 Feb 22;277(5698):665–667. doi: 10.1038/277665a0. [DOI] [PubMed] [Google Scholar]
  28. Schiff P. B., Horwitz S. B. Taxol assembles tubulin in the absence of exogenous guanosine 5'-triphosphate or microtubule-associated proteins. Biochemistry. 1981 May 26;20(11):3247–3252. doi: 10.1021/bi00514a041. [DOI] [PubMed] [Google Scholar]
  29. Schiff P. B., Horwitz S. B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1561–1565. doi: 10.1073/pnas.77.3.1561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Suprenant K. A., Marsh J. C. Temperature and pH govern the self-assembly of microtubules from unfertilized sea-urchin egg extracts. J Cell Sci. 1987 Feb;87(Pt 1):71–84. doi: 10.1242/jcs.87.1.71. [DOI] [PubMed] [Google Scholar]
  32. Suprenant K. A., Rebhun L. I. Assembly of unfertilized sea urchin egg tubulin at physiological temperatures. J Biol Chem. 1983 Apr 10;258(7):4518–4525. [PubMed] [Google Scholar]
  33. Theurkauf W. E., Vallee R. B. Extensive cAMP-dependent and cAMP-independent phosphorylation of microtubule-associated protein 2. J Biol Chem. 1983 Jun 25;258(12):7883–7886. [PubMed] [Google Scholar]
  34. Vale R. D., Reese T. S., Sheetz M. P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell. 1985 Aug;42(1):39–50. doi: 10.1016/s0092-8674(85)80099-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vallee R. B. A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs). J Cell Biol. 1982 Feb;92(2):435–442. doi: 10.1083/jcb.92.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vallee R. B., Bloom G. S. Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule-associated proteins with monoclonal antibodies. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6259–6263. doi: 10.1073/pnas.80.20.6259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vallee R. B., Borisy G. G. The non-tubulin component of microtubule protein oligomers. Effect on self-association and hydrodynamic properties. J Biol Chem. 1978 Apr 25;253(8):2834–2845. [PubMed] [Google Scholar]
  38. Vallee R. B., Collins C. A. Purification of microtubules and microtubule-associated proteins from sea urchin eggs and cultured mammalian cells using taxol, and use of exogenous taxol-stabilized brain microtubules for purifying microtubule-associated proteins. Methods Enzymol. 1986;134:116–127. doi: 10.1016/0076-6879(86)34080-1. [DOI] [PubMed] [Google Scholar]
  39. Vallee R. B., Davis S. E. Low molecular weight microtubule-associated proteins are light chains of microtubule-associated protein 1 (MAP 1). Proc Natl Acad Sci U S A. 1983 Mar;80(5):1342–1346. doi: 10.1073/pnas.80.5.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vallee R. B. Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments. Methods Enzymol. 1986;134:89–104. doi: 10.1016/0076-6879(86)34078-3. [DOI] [PubMed] [Google Scholar]
  41. Wani M. C., Taylor H. L., Wall M. E., Coggon P., McPhail A. T. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc. 1971 May 5;93(9):2325–2327. doi: 10.1021/ja00738a045. [DOI] [PubMed] [Google Scholar]
  42. Weatherbee J. A., Luftig R. B., Weihing R. R. In vitro polymerization of microtubules from HeLa cells. J Cell Biol. 1978 Jul;78(1):47–57. doi: 10.1083/jcb.78.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Webb B. C., Wilson L. Cold-stable microtubules from brain. Biochemistry. 1980 Apr 29;19(9):1993–2001. doi: 10.1021/bi00550a041. [DOI] [PubMed] [Google Scholar]
  44. Wehland J., Henkart M., Klausner R., Sandoval I. V. Role of microtubules in the distribution of the Golgi apparatus: effect of taxol and microinjected anti-alpha-tubulin antibodies. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4286–4290. doi: 10.1073/pnas.80.14.4286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Weisenberg R. C., Deery W. J. The mechanism of calcium-induced microtubule disassembly. Biochem Biophys Res Commun. 1981 Oct 15;102(3):924–931. doi: 10.1016/0006-291x(81)91626-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES