Skip to main content
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1994 Feb;5(2):217–226. doi: 10.1091/mbc.5.2.217

Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.

K E Sawin 1, T J Mitchison 1
PMCID: PMC301027  PMID: 8019007

Abstract

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.

Full text

PDF
217

Images in this article

Selected References

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

  1. Belmont L. D., Hyman A. A., Sawin K. E., Mitchison T. J. Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell. 1990 Aug 10;62(3):579–589. doi: 10.1016/0092-8674(90)90022-7. [DOI] [PubMed] [Google Scholar]
  2. Coue M., Lombillo V. A., McIntosh J. R. Microtubule depolymerization promotes particle and chromosome movement in vitro. J Cell Biol. 1991 Mar;112(6):1165–1175. doi: 10.1083/jcb.112.6.1165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gorbsky G. J. Chromosome motion in mitosis. Bioessays. 1992 Feb;14(2):73–80. doi: 10.1002/bies.950140202. [DOI] [PubMed] [Google Scholar]
  4. Hill T. L. Theoretical problems related to the attachment of microtubules to kinetochores. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4404–4408. doi: 10.1073/pnas.82.13.4404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hotani H., Horio T. Dynamics of microtubules visualized by darkfield microscopy: treadmilling and dynamic instability. Cell Motil Cytoskeleton. 1988;10(1-2):229–236. doi: 10.1002/cm.970100127. [DOI] [PubMed] [Google Scholar]
  6. Hyman A., Drechsel D., Kellogg D., Salser S., Sawin K., Steffen P., Wordeman L., Mitchison T. Preparation of modified tubulins. Methods Enzymol. 1991;196:478–485. doi: 10.1016/0076-6879(91)96041-o. [DOI] [PubMed] [Google Scholar]
  7. Inoué S. Cell division and the mitotic spindle. J Cell Biol. 1981 Dec;91(3 Pt 2):131s–147s. doi: 10.1083/jcb.91.3.131s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Inoué S., Ritter H., Jr Dynamics of mitotic spindle organization and function. Soc Gen Physiol Ser. 1975;30:3–30. [PubMed] [Google Scholar]
  9. Inoué S., Sato H. Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement. J Gen Physiol. 1967 Jul;50(6 Suppl):259–292. [PMC free article] [PubMed] [Google Scholar]
  10. Karsenti E. Mitotic spindle morphogenesis in animal cells. Semin Cell Biol. 1991 Aug;2(4):251–260. [PubMed] [Google Scholar]
  11. Koshland D. E., Mitchison T. J., Kirschner M. W. Polewards chromosome movement driven by microtubule depolymerization in vitro. Nature. 1988 Feb 11;331(6156):499–504. doi: 10.1038/331499a0. [DOI] [PubMed] [Google Scholar]
  12. Le Guellec R., Paris J., Couturier A., Roghi C., Philippe M. Cloning by differential screening of a Xenopus cDNA that encodes a kinesin-related protein. Mol Cell Biol. 1991 Jun;11(6):3395–3398. doi: 10.1128/mcb.11.6.3395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Leslie R. J., Pickett-Heaps J. D. Spindle microtubule dynamics following ultraviolet-microbeam irradiations of mitotic diatoms. Cell. 1984 Mar;36(3):717–727. doi: 10.1016/0092-8674(84)90352-0. [DOI] [PubMed] [Google Scholar]
  14. Margolis R. L., Wilson L. Microtubule treadmills--possible molecular machinery. Nature. 1981 Oct 29;293(5835):705–711. doi: 10.1038/293705a0. [DOI] [PubMed] [Google Scholar]
  15. Margolis R. L., Wilson L. Opposite end assembly and disassembly of microtubules at steady state in vitro. Cell. 1978 Jan;13(1):1–8. doi: 10.1016/0092-8674(78)90132-0. [DOI] [PubMed] [Google Scholar]
  16. McIntosh J. R., Koonce M. P. Mitosis. Science. 1989 Nov 3;246(4930):622–628. doi: 10.1126/science.2683078. [DOI] [PubMed] [Google Scholar]
  17. McNally F. J., Vale R. D. Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell. 1993 Nov 5;75(3):419–429. doi: 10.1016/0092-8674(93)90377-3. [DOI] [PubMed] [Google Scholar]
  18. Mitchison T. J., Kirschner M. W. Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J Cell Biol. 1985 Sep;101(3):766–777. doi: 10.1083/jcb.101.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mitchison T. J. Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. J Cell Biol. 1989 Aug;109(2):637–652. doi: 10.1083/jcb.109.2.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mitchison T. J., Salmon E. D. Poleward kinetochore fiber movement occurs during both metaphase and anaphase-A in newt lung cell mitosis. J Cell Biol. 1992 Nov;119(3):569–582. doi: 10.1083/jcb.119.3.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mitchison T. J., Sawin K. E. Tubulin flux in the mitotic spindle: where does it come from, where is it going? Cell Motil Cytoskeleton. 1990;16(2):93–98. doi: 10.1002/cm.970160202. [DOI] [PubMed] [Google Scholar]
  22. Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. doi: 10.1038/312237a0. [DOI] [PubMed] [Google Scholar]
  23. Nicklas R. B. Mitosis. Adv Cell Biol. 1971;2:225–297. doi: 10.1007/978-1-4615-9588-5_5. [DOI] [PubMed] [Google Scholar]
  24. Rieder C. L., Davison E. A., Jensen L. C., Cassimeris L., Salmon E. D. Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle. J Cell Biol. 1986 Aug;103(2):581–591. doi: 10.1083/jcb.103.2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Salmon E. D., Leslie R. J., Saxton W. M., Karow M. L., McIntosh J. R. Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. J Cell Biol. 1984 Dec;99(6):2165–2174. doi: 10.1083/jcb.99.6.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sawin K. E., LeGuellec K., Philippe M., Mitchison T. J. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature. 1992 Oct 8;359(6395):540–543. doi: 10.1038/359540a0. [DOI] [PubMed] [Google Scholar]
  27. Sawin K. E., Mitchison T. J. Mitotic spindle assembly by two different pathways in vitro. J Cell Biol. 1991 Mar;112(5):925–940. doi: 10.1083/jcb.112.5.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sawin K. E., Mitchison T. J. Poleward microtubule flux mitotic spindles assembled in vitro. J Cell Biol. 1991 Mar;112(5):941–954. doi: 10.1083/jcb.112.5.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sawin K. E., Mitchison T. J., Wordeman L. G. Evidence for kinesin-related proteins in the mitotic apparatus using peptide antibodies. J Cell Sci. 1992 Feb;101(Pt 2):303–313. doi: 10.1242/jcs.101.2.303. [DOI] [PubMed] [Google Scholar]
  30. Saxton W. M., Stemple D. L., Leslie R. J., Salmon E. D., Zavortink M., McIntosh J. R. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shiina N., Gotoh Y., Nishida E. A novel homo-oligomeric protein responsible for an MPF-dependent microtubule-severing activity. EMBO J. 1992 Dec;11(13):4723–4731. doi: 10.1002/j.1460-2075.1992.tb05577.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Simon S. M., Peskin C. S., Oster G. F. What drives the translocation of proteins? Proc Natl Acad Sci U S A. 1992 May 1;89(9):3770–3774. doi: 10.1073/pnas.89.9.3770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tao W., Walter R. J., Berns M. W. Laser-transected microtubules exhibit individuality of regrowth, however most free new ends of the microtubules are stable. J Cell Biol. 1988 Sep;107(3):1025–1035. doi: 10.1083/jcb.107.3.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vale R. D. Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts. Cell. 1991 Feb 22;64(4):827–839. doi: 10.1016/0092-8674(91)90511-v. [DOI] [PubMed] [Google Scholar]
  35. Verde F., Berrez J. M., Antony C., Karsenti E. Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein. J Cell Biol. 1991 Mar;112(6):1177–1187. doi: 10.1083/jcb.112.6.1177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Walker R. A., Inoué S., Salmon E. D. Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro. J Cell Biol. 1989 Mar;108(3):931–937. doi: 10.1083/jcb.108.3.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Walker R. A., O'Brien E. T., Pryer N. K., Soboeiro M. F., Voter W. A., Erickson H. P., Salmon E. D. Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. J Cell Biol. 1988 Oct;107(4):1437–1448. doi: 10.1083/jcb.107.4.1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wilson P. J., Forer A. Ultraviolet microbeam irradiation of chromosomal spindle fibres shears microtubules and permits study of the new free ends in vivo. J Cell Sci. 1988 Dec;91(Pt 4):455–468. doi: 10.1242/jcs.91.4.455. [DOI] [PubMed] [Google Scholar]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES