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. 1993 Jan 1;120(1):141–151. doi: 10.1083/jcb.120.1.141

Three-dimensional reconstruction and analysis of mitotic spindles from the yeast, Schizosaccharomyces pombe

PMCID: PMC2119489  PMID: 8416984

Abstract

Mitotic spindles of Schizosaccharomyces pombe have been studied by EM, using serial cross sections to reconstruct 12 spindles from cells that were ultrarapidly frozen and fixed by freeze substitution. The resulting distributions of microtubules (MTs) have been analyzed by computer. Short spindles contain two kinds of MTs: continuous ones that run from pole to pole and MTs that originate at one pole and end in the body of the spindle. Among the latter there are three pairs of MT bundles that end on fibrous, darkly staining structures that we interpret as kinetochores. The number of MTs ending at each putative kinetochore ranges from two to four; all kinetochore-associated MTs disappear as the spindle elongates from 3-6 microns. At this and greater spindle lengths, there are no continuous MTs, only polar MTs that interdigitate at the spindle midzone, but the spindle continues to elongate. An analysis of the density of neighboring MTs at the midzone of long spindles shows that their most common spacing is approximately 40 nm, center to center, and that there is a preferred angular separation of 90 degrees. Only hints of such square-packing are found at the midzone of short spindles, and near the poles there is no apparent order at any mitotic stage. Our data suggest that the kinetochore MTs (KMTs) do not interact directly with nonkinetochore MTs, but that interdigitating MTs from the two spindle poles do interact to form a mechanically stable bundle that connects the poles. As the spindle elongates, the number of MTs decreases while the mean length of the MTs that remain increases. We conclude that the chromosomes of S. pombe become attached to the spindle by kinetochore MTs, that these MTs disappear as the chromosomes segregate, that increased separation of daughter nuclei is accompanied by a sliding apart of anti-parallel MTs, and that the mitotic processes of S. pombe are much like those in other eukaryotic cells.

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Selected References

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  1. Aist J. R., Williams P. H. Ultrastructure and time course of mitosis in the fungus Fusarium oxysporum. J Cell Biol. 1972 Nov;55(2):368–389. doi: 10.1083/jcb.55.2.368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carbon J., Clarke L. Centromere structure and function in budding and fission yeasts. New Biol. 1990 Jan;2(1):10–19. [PubMed] [Google Scholar]
  3. Clarke L., Baum M. P. Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences. Mol Cell Biol. 1990 May;10(5):1863–1872. doi: 10.1128/mcb.10.5.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dahl R., Staehelin L. A. High-pressure freezing for the preservation of biological structure: theory and practice. J Electron Microsc Tech. 1989 Nov;13(3):165–174. doi: 10.1002/jemt.1060130305. [DOI] [PubMed] [Google Scholar]
  5. Fischer P., Binder M., Wintersberger U. A study of the chromosomes of the yeast Schizosaccharomyces pombe by light and electron microscopy. Exp Cell Res. 1975 Nov;96(1):15–22. doi: 10.1016/s0014-4827(75)80031-0. [DOI] [PubMed] [Google Scholar]
  6. Hagan I. M., Hyams J. S. The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe. J Cell Sci. 1988 Mar;89(Pt 3):343–357. doi: 10.1242/jcs.89.3.343. [DOI] [PubMed] [Google Scholar]
  7. Hagan I., Yanagida M. Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene. Nature. 1990 Oct 11;347(6293):563–566. doi: 10.1038/347563a0. [DOI] [PubMed] [Google Scholar]
  8. Hayles J., Nurse P. A review of mitosis in the fission yeast Schizosaccharomyces pombe. Exp Cell Res. 1989 Oct;184(2):273–286. doi: 10.1016/0014-4827(89)90327-3. [DOI] [PubMed] [Google Scholar]
  9. Heath I. B. Mitosis in the fungus Thraustotheca clavata. J Cell Biol. 1974 Jan;60(1):204–220. doi: 10.1083/jcb.60.1.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Howard R. J., Aist J. R. Hyphal tip cell ultrastructure of the fungus Fusarium: improved preservation by freeze-substitution. J Ultrastruct Res. 1979 Mar;66(3):224–234. doi: 10.1016/s0022-5320(79)90120-5. [DOI] [PubMed] [Google Scholar]
  11. Hoyt M. A., Stearns T., Botstein D. Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes. Mol Cell Biol. 1990 Jan;10(1):223–234. doi: 10.1128/mcb.10.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kanbe T., Hiraoka Y., Tanaka K., Yanagida M. The transition of cells of the fission yeast beta-tubulin mutant nda3-311 as seen by freeze-substitution electron microscopy. Requirement of functional tubulin for spindle pole body duplication. J Cell Sci. 1990 Jun;96(Pt 2):275–282. doi: 10.1242/jcs.96.2.275. [DOI] [PubMed] [Google Scholar]
  13. King S. M., Hyams J. S., Luba A. Absence of microtubule sliding and an analysis of spindle formation and elongation in isolated mitotic spindles from the yeast Saccharomyces cerevisiae. J Cell Biol. 1982 Aug;94(2):341–349. doi: 10.1083/jcb.94.2.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. King S. M., Hyams J. S., Luba A. Ultrastructure of mitotic spindles isolated from a cell division cycle mutant of the yeast, Saccharomyces cerevisiae. Eur J Cell Biol. 1982 Aug;28(1):98–102. [PubMed] [Google Scholar]
  15. 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]
  16. Masuda H., Hirano T., Yanagida M., Cande W. Z. In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells. J Cell Biol. 1990 Feb;110(2):417–425. doi: 10.1083/jcb.110.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Masuda H., Sevik M., Cande W. Z. In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts. J Cell Biol. 1992 Jun;117(5):1055–1066. doi: 10.1083/jcb.117.5.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mc2ntosh J. R., Cande Z., Snyder J., Vanderslice K. Studies on the mechanism of mitosis. Ann N Y Acad Sci. 1975 Jun 30;253:407–427. doi: 10.1111/j.1749-6632.1975.tb19217.x. [DOI] [PubMed] [Google Scholar]
  19. McCully E. K., Robinow C. F. Mitosis in the fission yeast Schizosaccharomyces pombe: a comparative study with light and electron microscopy. J Cell Sci. 1971 Sep;9(2):475–507. doi: 10.1242/jcs.9.2.475. [DOI] [PubMed] [Google Scholar]
  20. McDonald K. L., Edwards M. K., McIntosh J. R. Cross-sectional structure of the central mitotic spindle of Diatoma vulgare. Evidence for specific interactions between antiparallel microtubules. J Cell Biol. 1979 Nov;83(2 Pt 1):443–461. doi: 10.1083/jcb.83.2.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McDonald K. L., O'Toole E. T., Mastronarde D. N., McIntosh J. R. Kinetochore microtubules in PTK cells. J Cell Biol. 1992 Jul;118(2):369–383. doi: 10.1083/jcb.118.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McDonald K., Pickett-Heaps J. D., McIntosh J. R., Tippit D. H. On the mechanism of anaphase spindle elongation in Diatoma vulgare. J Cell Biol. 1977 Aug;74(2):377–388. doi: 10.1083/jcb.74.2.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McIntosh J. R., McDonald K. L., Edwards M. K., Ross B. M. Three-dimensional structure of the central mitotic spindle of Diatoma vulgare. J Cell Biol. 1979 Nov;83(2 Pt 1):428–442. doi: 10.1083/jcb.83.2.428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McIntosh J. R., Roos U. P., Neighbors B., McDonald K. L. Architecture of the microtubule component of mitotic spindles from Dictyostelium discoideum. J Cell Sci. 1985 Apr;75:93–129. doi: 10.1242/jcs.75.1.93. [DOI] [PubMed] [Google Scholar]
  25. Mitchison J. M. The fission yeast, Schizosaccharomyces pombe. Bioessays. 1990 Apr;12(4):189–191. doi: 10.1002/bies.950120409. [DOI] [PubMed] [Google Scholar]
  26. Moens P. B. Spindle and kinetochore morphology of Dictyostelium discoideum. J Cell Biol. 1976 Jan;68(1):113–122. doi: 10.1083/jcb.68.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nicolas G. Advantages of fast-freeze fixation followed by freeze-substitution for the preservation of cell integrity. J Electron Microsc Tech. 1991 Aug;18(4):395–405. doi: 10.1002/jemt.1060180408. [DOI] [PubMed] [Google Scholar]
  28. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. doi: 10.1038/344503a0. [DOI] [PubMed] [Google Scholar]
  29. Peterson J. B., Ris H. Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae. J Cell Sci. 1976 Nov;22(2):219–242. doi: 10.1242/jcs.22.2.219. [DOI] [PubMed] [Google Scholar]
  30. Robinow C. F. The Number of Chromosomes in SCHIZOSACCHAROMYCES POMBE: Light Microscopy of Stained Preparations. Genetics. 1977 Nov;87(3):491–497. doi: 10.1093/genetics/87.3.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Roos U. P. Mitosis in the cellular slime mold Polysphondylium violaceum. J Cell Biol. 1975 Feb;64(2):480–491. doi: 10.1083/jcb.64.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Tanaka K., Kanbe T. Mitosis in the fission yeast Schizosaccharomyces pombe as revealed by freeze-substitution electron microscopy. J Cell Sci. 1986 Feb;80:253–268. doi: 10.1242/jcs.80.1.253. [DOI] [PubMed] [Google Scholar]
  34. Tippit D. H., Schulz D., Pickett-Heaps J. D. Analysis of the distribution of spindle microtubules in the diatom Fragilaria. J Cell Biol. 1978 Dec;79(3):737–763. doi: 10.1083/jcb.79.3.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Uzawa S., Yanagida M. Visualization of centromeric and nucleolar DNA in fission yeast by fluorescence in situ hybridization. J Cell Sci. 1992 Feb;101(Pt 2):267–275. doi: 10.1242/jcs.101.2.267. [DOI] [PubMed] [Google Scholar]
  36. Winey M., Goetsch L., Baum P., Byers B. MPS1 and MPS2: novel yeast genes defining distinct steps of spindle pole body duplication. J Cell Biol. 1991 Aug;114(4):745–754. doi: 10.1083/jcb.114.4.745. [DOI] [PMC free article] [PubMed] [Google Scholar]

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