Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Jul 2;122(2):361–372. doi: 10.1083/jcb.122.2.361

The force-producing mechanism for centrosome separation during spindle formation in vertebrates is intrinsic to each aster

PMCID: PMC2119639  PMID: 8320259

Abstract

A popular hypothesis for centrosome separation during spindle formation and anaphase is that pushing forces are generated between interacting microtubules (MTs) of opposite polarity, derived from opposing centrosomes. However, this mechanism is not consistent with the observation that centrosomes in vertebrate cells continue to separate during prometaphase when their MT arrays no longer overlap (i.e., during anaphase-like prometaphase). To evaluate whether centrosome separation during prophase/prometaphase, anaphase-like prometaphase and anaphase is mediated by a common mechanism we compared their behavior in vivo at a high spatial and temporal resolution. We found that the two centrosomes possess a considerable degree of independence throughout all stages of separation, i.e., the direction and migration rate of one centrosome does not impart a predictable behavior to the other, and both exhibit frequent and rapid (4-6 microns/min) displacements toward random points within the cell including the other centrosome. The kinetic behavior of individual centrosomes as they separate to form the spindle is the same whether or not their MT arrays overlap. The characteristics examined include, e.g., total displacement per minute, the vectorial rate of motion toward and away from the other centrosome, the frequency of toward and away motion as well as motion not contributing to separation, and the rate contributed by each centrosome to the separation process. By contrast, when compared with prometaphase, anaphase centrosomes separated at significantly faster rates even though the average vectorial rate of motion away from the other centrosome was the same as in prophase/prometaphase. The difference in separation rates arises because anaphase centrosomes spend less time moving toward one another than in prophase/prometaphase, and at a significantly slower rate. From our data we conclude that the force for centrosome separation during vertebrate spindle formation is not produced by MT-MT interactions between opposing asters, i.e., that the mechanism is intrinsic to each aster. Our results also strongly support the contention that forces generated independently by each aster also contribute substantially to centrosome separation during anaphase, but that the process is modified by interactions between opposing astral MTs in the interzone.

Full Text

The Full Text of this article is available as a PDF (4.8 MB).

Selected References

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

  1. Aist J. R., Bayles C. J., Tao W., Berns M. W. Direct experimental evidence for the existence, structural basis and function of astral forces during anaphase B in vivo. J Cell Sci. 1991 Oct;100(Pt 2):279–288. doi: 10.1242/jcs.100.2.279. [DOI] [PubMed] [Google Scholar]
  2. Aist J. R., Berns M. W. Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfecti): new evidence from ultrastructural and laser microbeam experiments. J Cell Biol. 1981 Nov;91(2 Pt 1):446–458. doi: 10.1083/jcb.91.2.446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aist J. R., Liang H., Berns M. W. Astral and spindle forces in PtK2 cells during anaphase B: a laser microbeam study. J Cell Sci. 1993 Apr;104(Pt 4):1207–1216. doi: 10.1242/jcs.104.4.1207. [DOI] [PubMed] [Google Scholar]
  4. Alexander S. P., Rieder C. L. Chromosome motion during attachment to the vertebrate spindle: initial saltatory-like behavior of chromosomes and quantitative analysis of force production by nascent kinetochore fibers. J Cell Biol. 1991 May;113(4):805–815. doi: 10.1083/jcb.113.4.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Amin-Hanjani S., Wadsworth P. Inhibition of spindle elongation by taxol. Cell Motil Cytoskeleton. 1991;20(2):136–144. doi: 10.1002/cm.970200206. [DOI] [PubMed] [Google Scholar]
  6. Aubin J. E., Osborn M., Weber K. Variations in the distribution and migration of centriole duplexes in mitotic PtK2 cells studied by immunofluorescence microscopy. J Cell Sci. 1980 Jun;43:177–194. doi: 10.1242/jcs.43.1.177. [DOI] [PubMed] [Google Scholar]
  7. Bajer A. S. Functional autonomy of monopolar spindle and evidence for oscillatory movement in mitosis. J Cell Biol. 1982 Apr;93(1):33–48. doi: 10.1083/jcb.93.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bajer A. S., Molè-Bajer J. Asters, poles, and transport properties within spindlelike microtubule arrays. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):263–283. doi: 10.1101/sqb.1982.046.01.029. [DOI] [PubMed] [Google Scholar]
  9. Brenner S., Branch A., Meredith S., Berns M. W. The absence of centrioles from spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division in vitro. J Cell Biol. 1977 Feb;72(2):368–379. doi: 10.1083/jcb.72.2.368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brinkley B. R., Cartwright J., Jr Ultrastructural analysis of mitotic spindle elongation in mammalian cells in vitro. Direct microtubule counts. J Cell Biol. 1971 Aug;50(2):416–431. doi: 10.1083/jcb.50.2.416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brinkley B. R., Stubblefield E., Hsu T. C. The effects of colcemid inhibition and reversal on the fine structure of the mitotic apparatus of Chinese hamster cells in vitro. J Ultrastruct Res. 1967 Jul;19(1):1–18. doi: 10.1016/s0022-5320(67)80057-1. [DOI] [PubMed] [Google Scholar]
  12. Bré M. H., Kreis T. E., Karsenti E. Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules. J Cell Biol. 1987 Sep;105(3):1283–1296. doi: 10.1083/jcb.105.3.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cande W. Z., Hogan C. J. The mechanism of anaphase spindle elongation. Bioessays. 1989 Jul;11(1):5–9. doi: 10.1002/bies.950110103. [DOI] [PubMed] [Google Scholar]
  14. Goldstein L. S. Functional redundancy in mitotic force generation. J Cell Biol. 1993 Jan;120(1):1–3. doi: 10.1083/jcb.120.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hayden J. H., Bowser S. S., Rieder C. L. Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells. J Cell Biol. 1990 Sep;111(3):1039–1045. doi: 10.1083/jcb.111.3.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heneen W. K. In situ analysis of normal and abnormal patterns of the mitotic apparatus in cultured rat-kangaroo cells. Chromosoma. 1970;29(1):88–117. doi: 10.1007/BF01183663. [DOI] [PubMed] [Google Scholar]
  17. Hiramoto Y., Nakano Y. Micromanipulation studies of the mitotic apparatus in sand dollar eggs. Cell Motil Cytoskeleton. 1988;10(1-2):172–184. doi: 10.1002/cm.970100122. [DOI] [PubMed] [Google Scholar]
  18. Hogan C. J., Cande W. Z. Antiparallel microtubule interactions: spindle formation and anaphase B. Cell Motil Cytoskeleton. 1990;16(2):99–103. doi: 10.1002/cm.970160203. [DOI] [PubMed] [Google Scholar]
  19. Hyman A. A., White J. G. Determination of cell division axes in the early embryogenesis of Caenorhabditis elegans. J Cell Biol. 1987 Nov;105(5):2123–2135. doi: 10.1083/jcb.105.5.2123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Joshi H. C., Palacios M. J., McNamara L., Cleveland D. W. Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature. 1992 Mar 5;356(6364):80–83. doi: 10.1038/356080a0. [DOI] [PubMed] [Google Scholar]
  21. Kirschner M., Mitchison T. Beyond self-assembly: from microtubules to morphogenesis. Cell. 1986 May 9;45(3):329–342. doi: 10.1016/0092-8674(86)90318-1. [DOI] [PubMed] [Google Scholar]
  22. Mazia D. The chromosome cycle and the centrosome cycle in the mitotic cycle. Int Rev Cytol. 1987;100:49–92. doi: 10.1016/s0074-7696(08)61698-8. [DOI] [PubMed] [Google Scholar]
  23. McIntosh J. R., Hering G. E. Spindle fiber action and chromosome movement. Annu Rev Cell Biol. 1991;7:403–426. doi: 10.1146/annurev.cb.07.110191.002155. [DOI] [PubMed] [Google Scholar]
  24. McIntosh J. R., Pfarr C. M. Mitotic motors. J Cell Biol. 1991 Nov;115(3):577–585. doi: 10.1083/jcb.115.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mitchison T. J. Mitosis: basic concepts. Curr Opin Cell Biol. 1989 Feb;1(1):67–74. doi: 10.1016/s0955-0674(89)80039-0. [DOI] [PubMed] [Google Scholar]
  26. Nislow C., Lombillo V. A., Kuriyama R., McIntosh J. R. A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature. 1992 Oct 8;359(6395):543–547. doi: 10.1038/359543a0. [DOI] [PubMed] [Google Scholar]
  27. Nislow C., Sellitto C., Kuriyama R., McIntosh J. R. A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression. J Cell Biol. 1990 Aug;111(2):511–522. doi: 10.1083/jcb.111.2.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Oppenheim D. S., Hauschka B. T., McIntosh J. R. Anaphase motions in dilute colchicine. Evidence of two phases in chromosome segregation. Exp Eye Res. 1973 Apr;79(1):95–105. [PubMed] [Google Scholar]
  29. Paintrand M., Moudjou M., Delacroix H., Bornens M. Centrosome organization and centriole architecture: their sensitivity to divalent cations. J Struct Biol. 1992 Mar-Apr;108(2):107–128. doi: 10.1016/1047-8477(92)90011-x. [DOI] [PubMed] [Google Scholar]
  30. Palazzo R. E., Lutz D. A., Rebhun L. I. Reactivation of isolated mitotic apparatus: metaphase versus anaphase spindles. Cell Motil Cytoskeleton. 1991;18(4):304–318. doi: 10.1002/cm.970180407. [DOI] [PubMed] [Google Scholar]
  31. Rattner J. B., Berns M. W. Centriole behavior in early mitosis of rat kangaroo cells (PTK2). Chromosoma. 1976 Mar 10;54(4):387–395. doi: 10.1007/BF00292817. [DOI] [PubMed] [Google Scholar]
  32. Rieder C. L., Alexander S. P. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J Cell Biol. 1990 Jan;110(1):81–95. doi: 10.1083/jcb.110.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rieder C. L. Formation of the astral mitotic spindle: ultrastructural basis for the centrosome-kinetochore interaction. Electron Microsc Rev. 1990;3(2):269–300. doi: 10.1016/0892-0354(90)90005-d. [DOI] [PubMed] [Google Scholar]
  34. Rieder C. L., Hard R. Newt lung epithelial cells: cultivation, use, and advantages for biomedical research. Int Rev Cytol. 1990;122:153–220. doi: 10.1016/s0074-7696(08)61208-5. [DOI] [PubMed] [Google Scholar]
  35. Roos U. P. Light and electron microscopy of rat kangaroo cells in mitosis. I. Formation and breakdown of the mitotic apparatus. Chromosoma. 1973;40(1):43–82. doi: 10.1007/BF00319836. [DOI] [PubMed] [Google Scholar]
  36. Saxton W. M., McIntosh J. R. Interzone microtubule behavior in late anaphase and telophase spindles. J Cell Biol. 1987 Aug;105(2):875–886. doi: 10.1083/jcb.105.2.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shelden E., Wadsworth P. Interzonal microtubules are dynamic during spindle elongation. J Cell Sci. 1990 Oct;97(Pt 2):273–281. doi: 10.1242/jcs.97.2.273. [DOI] [PubMed] [Google Scholar]
  38. Sluder G., Rieder C. L. Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes. J Cell Biol. 1985 Mar;100(3):897–903. doi: 10.1083/jcb.100.3.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Snyder J. A., Golub R. J., Berg S. P. Sucrose-induced spindle elongation in mitotic PtK-1 cells. Eur J Cell Biol. 1984 Sep;35(1):62–69. [PubMed] [Google Scholar]
  40. Sullivan D. S., Huffaker T. C. Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae. J Cell Biol. 1992 Oct;119(2):379–388. doi: 10.1083/jcb.119.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. TAYLOR E. W. Dynamics of spindle formation and its inhibition by chemicals. J Biophys Biochem Cytol. 1959 Oct;6:193–196. doi: 10.1083/jcb.6.2.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]

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

RESOURCES