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. 1991 Mar 2;112(6):1177–1187. doi: 10.1083/jcb.112.6.1177

Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein

PMCID: PMC2288889  PMID: 1671864

Abstract

Taxol, a microtubule stabilizing drug, induces the formation of numerous microtubule asters in the cytoplasm of mitotic cells (De Brabander, M., G. Geuens, R. Nuydens, R. Willebrords, J. DeMey. 1981. Proc. Natl. Acad. Sci. USA. 78:5608-5612). The center of these asters share with spindle poles some characteristics such as the presence of centrosomal material and calmodulin. We have recently reproduced the assembly of taxol asters in a cell-free system (Buendia, B., C. Antony, F. Verde, M. Bornens, and E. Karsenti. 1990. J. Cell Sci. 97:259-271) using extracts of Xenopus eggs. In this paper, we show that taxol aster assembly requires phosphorylation, and that they do not grow from preformed centers, but rather by a reorganization of microtubules first crosslinked into bundles. This process seems to involve sliding of microtubules along each other and we show that cytoplasmic dynein is required for taxol aster assembly. This result provides a possible functional basis to the recent findings, that dynein is present in the spindle and enriched near spindle poles (Pfarr, C. M., M. Cove, P. M. Grissom, T. S. Hays, M. E. Porter, and J. R. McIntosh. 1990. Nature (Lond.). 345:263-265; Steuer, E. R., L. Wordeman, T. A. Schroer, and M. P. Sheetz. 1990. Nature (Lond.). 345:266-268).

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Selected References

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  1. Amos L. A. Brain dynein crossbridges microtubules into bundles. J Cell Sci. 1989 May;93(Pt 1):19–28. doi: 10.1242/jcs.93.1.19. [DOI] [PubMed] [Google Scholar]
  2. Buendia B., Antony C., Verde F., Bornens M., Karsenti E. A centrosomal antigen localized on intermediate filaments and mitotic spindle poles. J Cell Sci. 1990 Oct;97(Pt 2):259–271. doi: 10.1242/jcs.97.2.259. [DOI] [PubMed] [Google Scholar]
  3. Centonze V. E., Borisy G. G. Nucleation of microtubules from mitotic centrosomes is modulated by a phosphorylated epitope. J Cell Sci. 1990 Mar;95(Pt 3):405–411. doi: 10.1242/jcs.95.3.405. [DOI] [PubMed] [Google Scholar]
  4. Dabora S. L., Sheetz M. P. Cultured cell extracts support organelle movement on microtubules in vitro. Cell Motil Cytoskeleton. 1988;10(4):482–495. doi: 10.1002/cm.970100405. [DOI] [PubMed] [Google Scholar]
  5. De Brabander M., Geuens G., Nuydens R., Willebrords R., Aerts F., De Mey J. Microtubule dynamics during the cell cycle: the effects of taxol and nocodazole on the microtubule system of Pt K2 cells at different stages of the mitotic cycle. Int Rev Cytol. 1986;101:215–274. doi: 10.1016/s0074-7696(08)60250-8. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Evans L., Mitchison T., Kirschner M. Influence of the centrosome on the structure of nucleated microtubules. J Cell Biol. 1985 Apr;100(4):1185–1191. doi: 10.1083/jcb.100.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Felix M. A., Pines J., Hunt T., Karsenti E. A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity. EMBO J. 1989 Oct;8(10):3059–3069. doi: 10.1002/j.1460-2075.1989.tb08457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gard D. L., Kirschner M. W. A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end. J Cell Biol. 1987 Nov;105(5):2203–2215. doi: 10.1083/jcb.105.5.2203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gibbons I. R., Lee-Eiford A., Mocz G., Phillipson C. A., Tang W. J., Gibbons B. H. Photosensitized cleavage of dynein heavy chains. Cleavage at the "V1 site" by irradiation at 365 nm in the presence of ATP and vanadate. J Biol Chem. 1987 Feb 25;262(6):2780–2786. [PubMed] [Google Scholar]
  11. Heidemann S. R., Gallas P. T. The effect of taxol on living eggs of Xenopus laevis. Dev Biol. 1980 Dec;80(2):489–494. doi: 10.1016/0012-1606(80)90421-2. [DOI] [PubMed] [Google Scholar]
  12. Hiller G., Weber K. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues. Cell. 1978 Aug;14(4):795–804. doi: 10.1016/0092-8674(78)90335-5. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Johnson K. A. Pathway of the microtubule-dynein ATPase and the structure of dynein: a comparison with actomyosin. Annu Rev Biophys Biophys Chem. 1985;14:161–188. doi: 10.1146/annurev.bb.14.060185.001113. [DOI] [PubMed] [Google Scholar]
  15. Kallenbach R. J. Ultrastructural analysis of the initiation and development of cytasters in sea-urchin eggs. J Cell Sci. 1985 Feb;73:261–278. doi: 10.1242/jcs.73.1.261. [DOI] [PubMed] [Google Scholar]
  16. Karsenti E., Newport J., Hubble R., Kirschner M. Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs. J Cell Biol. 1984 May;98(5):1730–1745. doi: 10.1083/jcb.98.5.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keryer G., Ris H., Borisy G. G. Centriole distribution during tripolar mitosis in Chinese hamster ovary cells. J Cell Biol. 1984 Jun;98(6):2222–2229. doi: 10.1083/jcb.98.6.2222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Labbé J. C., Capony J. P., Caput D., Cavadore J. C., Derancourt J., Kaghad M., Lelias J. M., Picard A., Dorée M. MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B. EMBO J. 1989 Oct;8(10):3053–3058. doi: 10.1002/j.1460-2075.1989.tb08456.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lye R. J., Porter M. E., Scholey J. M., McIntosh J. R. Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans. Cell. 1987 Oct 23;51(2):309–318. doi: 10.1016/0092-8674(87)90157-7. [DOI] [PubMed] [Google Scholar]
  20. Maro B., Howlett S. K., Webb M. Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes. J Cell Biol. 1985 Nov;101(5 Pt 1):1665–1672. doi: 10.1083/jcb.101.5.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moreno S., Nurse P. Substrates for p34cdc2: in vivo veritas? Cell. 1990 May 18;61(4):549–551. doi: 10.1016/0092-8674(90)90463-o. [DOI] [PubMed] [Google Scholar]
  22. Neant I., Guerrier P. 6-Dimethylaminopurine blocks starfish oocyte maturation by inhibiting a relevant protein kinase activity. Exp Cell Res. 1988 May;176(1):68–79. doi: 10.1016/0014-4827(88)90121-8. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Paschal B. M., Vallee R. B. Retrograde transport by the microtubule-associated protein MAP 1C. Nature. 1987 Nov 12;330(6144):181–183. doi: 10.1038/330181a0. [DOI] [PubMed] [Google Scholar]
  25. Pepperkok R., Bré M. H., Davoust J., Kreis T. E. Microtubules are stabilized in confluent epithelial cells but not in fibroblasts. J Cell Biol. 1990 Dec;111(6 Pt 2):3003–3012. doi: 10.1083/jcb.111.6.3003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pfarr C. M., Coue M., Grissom P. M., Hays T. S., Porter M. E., McIntosh J. R. Cytoplasmic dynein is localized to kinetochores during mitosis. Nature. 1990 May 17;345(6272):263–265. doi: 10.1038/345263a0. [DOI] [PubMed] [Google Scholar]
  27. Porter M. E., Scholey J. M., Stemple D. L., Vigers G. P., Vale R. D., Sheetz M. P., McIntosh J. R. Characterization of the microtubule movement produced by sea urchin egg kinesin. J Biol Chem. 1987 Feb 25;262(6):2794–2802. [PubMed] [Google Scholar]
  28. Rebhun L. I., Sawada N. Augmentation and dispersion of the in vivo mitotic apparatus of living marine eggs. Protoplasma. 1969;68(1):1–22. doi: 10.1007/BF01247894. [DOI] [PubMed] [Google Scholar]
  29. Sager P. R., Rothfield N. L., Oliver J. M., Berlin R. D. A novel mitotic spindle pole component that originates from the cytoplasm during prophase. J Cell Biol. 1986 Nov;103(5):1863–1872. doi: 10.1083/jcb.103.5.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Schroer T. A., Steuer E. R., Sheetz M. P. Cytoplasmic dynein is a minus end-directed motor for membranous organelles. Cell. 1989 Mar 24;56(6):937–946. doi: 10.1016/0092-8674(89)90627-2. [DOI] [PubMed] [Google Scholar]
  33. Seals J. R., McDonald J. M., Bruns D., Jarett L. A sensitive and precise isotopic assay of ATPase activity. Anal Biochem. 1978 Oct 15;90(2):785–795. doi: 10.1016/0003-2697(78)90169-0. [DOI] [PubMed] [Google Scholar]
  34. Steuer E. R., Wordeman L., Schroer T. A., Sheetz M. P. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature. 1990 May 17;345(6272):266–268. doi: 10.1038/345266a0. [DOI] [PubMed] [Google Scholar]
  35. Vale R. D. Microtubule-based motor proteins. Curr Opin Cell Biol. 1990 Feb;2(1):15–22. doi: 10.1016/s0955-0674(05)80025-0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Vandre D. D., Davis F. M., Rao P. N., Borisy G. G. Phosphoproteins are components of mitotic microtubule organizing centers. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4439–4443. doi: 10.1073/pnas.81.14.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Verde F., Labbé J. C., Dorée M., Karsenti E. Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature. 1990 Jan 18;343(6255):233–238. doi: 10.1038/343233a0. [DOI] [PubMed] [Google Scholar]
  39. Wadsworth P., McGrail M. Interphase microtubule dynamics are cell type-specific. J Cell Sci. 1990 Jan;95(Pt 1):23–32. doi: 10.1242/jcs.95.1.23. [DOI] [PubMed] [Google Scholar]
  40. Wadsworth P., Salmon E. D. Microtubule dynamics in mitotic spindles of living cells. Ann N Y Acad Sci. 1986;466:580–592. doi: 10.1111/j.1749-6632.1986.tb38434.x. [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]

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