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. 1996 May 1;133(3):595–604. doi: 10.1083/jcb.133.3.595

DSK1, a novel kinesin-related protein from the diatom Cylindrotheca fusiformis that is involved in anaphase spindle elongation

PMCID: PMC2120814  PMID: 8636234

Abstract

We have identified an 80-kD protein that is involved in mitotic spindle elongation in the diatom Cylindrotheca fusiformis. DSK1 (Diatom Spindle Kinesin 1) was isolated using a peptide antibody raised against a conserved region in the motor domain of the kinesin superfamily. By sequence homology, DSK1 belongs to the central motor family of kinesin- related proteins. Immunoblots using an antibody raised against a non- conserved region of DSK1 show that DSK1 is greatly enriched in mitotic spindle preparations. Anti-DSK1 stains in diatom central spindle with a bias toward the midzone, and staining is retained in the spindle midzone during spindle elongation in vitro. Furthermore, preincubation with anti-DSK1 blocks function in an in vitro spindle elongation assay. This inhibition of spindle elongation can be rescued by preincubating concurrently with the fusion protein against which anti-DSK1 was raised. We conclude that DSK1 is involved in spindle elongation and is likely to be responsible for pushing hal-spindles apart in the spindle midzone.

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Selected References

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  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. Aizawa H., Sekine Y., Takemura R., Zhang Z., Nangaku M., Hirokawa N. Kinesin family in murine central nervous system. J Cell Biol. 1992 Dec;119(5):1287–1296. doi: 10.1083/jcb.119.5.1287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Cande W. Z., McDonald K. L. In vitro reactivation of anaphase spindle elongation using isolated diatom spindles. Nature. 1985 Jul 11;316(6024):168–170. doi: 10.1038/316168a0. [DOI] [PubMed] [Google Scholar]
  7. Cande W. Z., McDonald K. Physiological and ultrastructural analysis of elongating mitotic spindles reactivated in vitro. J Cell Biol. 1986 Aug;103(2):593–604. doi: 10.1083/jcb.103.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Enos A. P., Morris N. R. Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans. Cell. 1990 Mar 23;60(6):1019–1027. doi: 10.1016/0092-8674(90)90350-n. [DOI] [PubMed] [Google Scholar]
  10. Goldsmith M., Leyland S., Connolly J. A., van der Kooy D. A unique tubulin antiserum attenuates the rate of poleward chromosome movement in anaphase. Eur J Cell Biol. 1992 Aug;58(2):346–355. [PubMed] [Google Scholar]
  11. Gorbsky G. J., Sammak P. J., Borisy G. G. Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends. J Cell Biol. 1987 Jan;104(1):9–18. doi: 10.1083/jcb.104.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorbsky G. J., Sammak P. J., Borisy G. G. Microtubule dynamics and chromosome motion visualized in living anaphase cells. J Cell Biol. 1988 Apr;106(4):1185–1192. doi: 10.1083/jcb.106.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hagan I., Yanagida M. Kinesin-related cut7 protein associates with mitotic and meiotic spindles in fission yeast. Nature. 1992 Mar 5;356(6364):74–76. doi: 10.1038/356074a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Hamaguchi Y., Toriyama M., Sakai H., Hiramoto Y. Redistribution of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs and the effects of taxol. Cell Struct Funct. 1987 Feb;12(1):43–52. doi: 10.1247/csf.12.43. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Hogan C. J., Neale P. J., Lee M., Cande W. Z. The diatom central spindle as a model system for studying antiparallel microtubule interactions during spindle elongation in vitro. Methods Cell Biol. 1993;39:277–292. doi: 10.1016/s0091-679x(08)60177-6. [DOI] [PubMed] [Google Scholar]
  18. Hogan C. J., Stephens L., Shimizu T., Cande W. Z. Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles. J Cell Biol. 1992 Dec;119(5):1277–1286. doi: 10.1083/jcb.119.5.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hogan C. J., Wein H., Wordeman L., Scholey J. M., Sawin K. E., Cande W. Z. Inhibition of anaphase spindle elongation in vitro by a peptide antibody that recognizes kinesin motor domain. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6611–6615. doi: 10.1073/pnas.90.14.6611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hoyt M. A., He L., Loo K. K., Saunders W. S. Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly. J Cell Biol. 1992 Jul;118(1):109–120. doi: 10.1083/jcb.118.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hyman A. A., Middleton K., Centola M., Mitchison T. J., Carbon J. Microtubule-motor activity of a yeast centromere-binding protein complex. Nature. 1992 Oct 8;359(6395):533–536. doi: 10.1038/359533a0. [DOI] [PubMed] [Google Scholar]
  22. Hyman A. A., Mitchison T. J. Two different microtubule-based motor activities with opposite polarities in kinetochores. Nature. 1991 May 16;351(6323):206–211. doi: 10.1038/351206a0. [DOI] [PubMed] [Google Scholar]
  23. Ingold A. L., Cohn S. A., Scholey J. M. Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains. J Cell Biol. 1988 Dec;107(6 Pt 2):2657–2667. doi: 10.1083/jcb.107.6.2657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Leslie R. J., Pickett-Heaps J. D. Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation. J Cell Biol. 1983 Feb;96(2):548–561. doi: 10.1083/jcb.96.2.548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lombillo V. A., Nislow C., Yen T. J., Gelfand V. I., McIntosh J. R. Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro. J Cell Biol. 1995 Jan;128(1-2):107–115. doi: 10.1083/jcb.128.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Masuda H., McDonald K. L., Cande W. Z. The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation. J Cell Biol. 1988 Aug;107(2):623–633. doi: 10.1083/jcb.107.2.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. McDonald K. L., Pfister K., Masuda H., Wordeman L., Staiger C., Cande W. Z. Comparison of spindle elongation in vivo and in vitro in Stephanopyxis turris. J Cell Sci Suppl. 1986;5:205–227. doi: 10.1242/jcs.1986.supplement_5.14. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. Mitchison T., Evans L., Schulze E., Kirschner M. Sites of microtubule assembly and disassembly in the mitotic spindle. Cell. 1986 May 23;45(4):515–527. doi: 10.1016/0092-8674(86)90283-7. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Noda Y., Sato-Yoshitake R., Kondo S., Nangaku M., Hirokawa N. KIF2 is a new microtubule-based anterograde motor that transports membranous organelles distinct from those carried by kinesin heavy chain or KIF3A/B. J Cell Biol. 1995 Apr;129(1):157–167. doi: 10.1083/jcb.129.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Pickett-Heaps J. D., Tippit D. H. The diatom spindle in perspective. Cell. 1978 Jul;14(3):455–467. doi: 10.1016/0092-8674(78)90232-5. [DOI] [PubMed] [Google Scholar]
  42. Rebhun L. I., Palazzo R. E. In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg. Cell Motil Cytoskeleton. 1988;10(1-2):197–209. doi: 10.1002/cm.970100124. [DOI] [PubMed] [Google Scholar]
  43. Roof D. M., Meluh P. B., Rose M. D. Kinesin-related proteins required for assembly of the mitotic spindle. J Cell Biol. 1992 Jul;118(1):95–108. doi: 10.1083/jcb.118.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Saunders W. S., Koshland D., Eshel D., Gibbons I. R., Hoyt M. A. Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation. J Cell Biol. 1995 Feb;128(4):617–624. doi: 10.1083/jcb.128.4.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Shelden E., Wadsworth P. Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to-pole motion. J Cell Biol. 1992 Mar;116(6):1409–1420. doi: 10.1083/jcb.116.6.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  49. Sperry A. O., Zhao L. P. Kinesin-related proteins in the mammalian testes: candidate motors for meiosis and morphogenesis. Mol Biol Cell. 1996 Feb;7(2):289–305. doi: 10.1091/mbc.7.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Wein H., Brady B., Cande W. Z. Isolating the plant mitotic apparatus: a procedure for isolating spindles from the diatom Cylindrotheca fusiformis. Methods Cell Biol. 1995;50:177–187. doi: 10.1016/s0091-679x(08)61030-4. [DOI] [PubMed] [Google Scholar]
  52. Wordeman L., Mitchison T. J. Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis. J Cell Biol. 1995 Jan;128(1-2):95–104. doi: 10.1083/jcb.128.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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