Mitosis pp 243-258

Part of the Methods in Molecular Biology book series (MIMB, volume 545)

The Spindle Checkpoint: Assays for the Analysis of Spindle Checkpoint Arrest and Recovery

  • Josefin Fernius
  • Kevin G. Hardwick


The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation by inhibiting anaphase onset until all chromosomes have established stable bipolar attachments. Here we describe a number of protocols that can be used to assay the ability of budding and fission yeast cells to (1) establish and maintain a spindle checkpoint arrest, and (2) segregate chromosomes efficiently upon recovery from mitotic arrest. We focus on experimental detail of the budding yeast protocols, but also point out important differences between budding and fission yeast assays.

Key words

Checkpoint segregation recovery biorientation 


  1. 1.
    Hoyt, M. A., Totis, L., and Roberts, B.T. (1991) S.cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66, 507–17.PubMedCrossRefGoogle Scholar
  2. 2.
    Li, R., and Murray, A. W. (1991) Feedback control of mitosis in budding yeast. Cell 66, 519–31.PubMedCrossRefGoogle Scholar
  3. 3.
    Straight, A. F., and Murray, A. W. (1997) The spindle assembly checkpoint in budding yeast. Methods Enzymol 283, 425–40.PubMedCrossRefGoogle Scholar
  4. 4.
    Hwang, L. H., Lau, L. F., Smith, D. L., Mistrot, C. A., Hardwick, K. G., Hwang, E. S., Amon, A., and Murray, A. W. (1998) Budding yeast Cdc20: a target of the spindle checkpoint. Science 279, 1041–4.PubMedCrossRefGoogle Scholar
  5. 5.
    Kim, S. H., Lin, D. P., Matsumoto, S., Kitazono, A., and Matsumoto, T. (1998) Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint. Science 279, 1045–7.PubMedCrossRefGoogle Scholar
  6. 6.
    Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–61.PubMedCrossRefGoogle Scholar
  7. 7.
    Straight, A. F., Belmont, A. S., Robinett, C. C., and Murray, A. W. (1996) GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol 6, 1599–608.PubMedCrossRefGoogle Scholar
  8. 8.
    Biggins, S., and Murray, A. W. (2001) The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint. Genes Dev 15, 3118–29.PubMedCrossRefGoogle Scholar
  9. 9.
    Indjeian, V. B., Stern, B. M., and Murray, A. W. (2005) The centromeric protein Sgo1 is required to sense lack of tension on mitotic chromosomes. Science 307, 130–3.PubMedCrossRefGoogle Scholar
  10. 10.
    Fernius, J., and Hardwick, K. G. (2007) Bub1 kinase targets Sgo1 to ensure efficient chromosome bi-orientation in budding yeast mitosis. PLoS Genetics 3, e213.PubMedCrossRefGoogle Scholar
  11. 11.
    Pinsky, B. A., Kung, C., Shokat, K. M., and Biggins, S. (2006) The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores. Nat Cell Biol 8, 78–83.PubMedCrossRefGoogle Scholar
  12. 12.
    Pinsky, B. A., Tatsutani, S. Y., Collins, K. A., and Biggins, S. (2003) An Mtw1 complex promotes kinetochore biorientation that is monitored by the Ipl1/Aurora protein kinase. Dev Cell 5, 735–45.PubMedCrossRefGoogle Scholar
  13. 13.
    He, X., Asthana, S., and Sorger, P. K. (2000) Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast. Cell 101, 763–75.PubMedCrossRefGoogle Scholar
  14. 14.
    Indjeian, V. B., and Murray, A. W. (2007) Budding yeast mitotic chromosomes have an intrinsic bias to biorient on the spindle. Curr Biol. 17, 1837–46.PubMedCrossRefGoogle Scholar
  15. 15.
    Funabiki, H., Yamano, H., Kumada, K., Nagao, K., Hunt, T., and Yanagida, M. (1996) Cut2 proteolysis required for sister-chromatid seperation in fission yeast. Nature 381, 438–41.PubMedCrossRefGoogle Scholar
  16. 16.
    Sawin, K. E., and Snaith, H. A. (2004) Role of microtubules and tea1p in establishment and maintenance of fission yeast cell polarity. J Cell Sci 117, 689–700.PubMedCrossRefGoogle Scholar
  17. 17.
    Hiraoka, Y., Toda, T., and Yanagida, M. (1984) The nda3+ gene of fission yeast encodes beta-tubulin: a cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell 39, 349–58.PubMedCrossRefGoogle Scholar
  18. 18.
    Millband, D. N., and Hardwick, K. G. (2002) Fission yeast Mad3p is required for Mad2p to inhibit the anaphase-promoting complex and localises to kinetochores in a Bub1p, Bub3p and Mph1p dependent manner. Mol Cell Biol 22, 2728–42.PubMedCrossRefGoogle Scholar
  19. 19.
    Hagan, I., and Yanagida, M. (1990) Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene. Nature 347, 563–66.PubMedCrossRefGoogle Scholar
  20. 20.
    Toyoda, Y., Furuya, K., Goshima, G., Nagao, K., Takahashi, K., and Yanagida, M. (2002) Requirement of chromatid cohesion proteins Rad21/Scc1 and Mis4/Scc2 for normal spindle-kinetochore interaction in fission yeast. Curr Biol 12, 347–58.PubMedCrossRefGoogle Scholar
  21. 21.
    Vanoosthuyse, V., Valsdottir, R., Javerzat, J. P., and Hardwick, K. G. (2004) Kinetochore targeting of fission yeast Mad and Bub proteins is essential for spindle checkpoint function but not for all chromosome segregation roles of Bub1p. Mol Cell Biol 24, 9786–801.PubMedCrossRefGoogle Scholar
  22. 22.
    Kawashima, S. A., Tsukahara, T., Langegger, M., Hauf, S., Kitajima, T. S., and Watanabe, Y. (2007) Shugoshin enables tension-generating attachment of kinetochores by loading Aurora to centromeres. Genes Dev 21, 420–35.PubMedCrossRefGoogle Scholar
  23. 23.
    Nabeshima, K., Saitoh, S., and Yanagida, M. (1997) Use of green fluorescent protein for intracellular protein localization in living fission yeast cells. Methods Enzymol 283, 459–71.PubMedCrossRefGoogle Scholar
  24. 24.
    Nabeshima, K., Nakagawa, T., Straight, A. F., Murray, A., Chikashige, Y., Yamashita, Y. M., Hiraoka, Y., and Yanagida, M. (1998) Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle. Mol Biol Cell 9, 3211–25.PubMedGoogle Scholar
  25. 25.
    Ding, R., West, R. R., Morphew, D. M., Oakley, B. R., and McIntosh, J. R. (1997) The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds. Mol Biol Cell 8, 1461–79.PubMedGoogle Scholar
  26. 26.
    Bernard, P., Hardwick, K., and Javerzat, J. P. (1998) Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis. J Cell Biol 143, 1775–87.PubMedCrossRefGoogle Scholar
  27. 27.
    Pidoux, A. L., Uzawa, S., Perry, P. E., Cande, W. Z., and Allshire, R. C. (2000) Live analysis of lagging chromosomes during anaphase and their effect on spindle elongation rate in fission yeast. J Cell Sci 113 Pt 23, 4177–91.Google Scholar
  28. 28.
    Gregan, J., Riedel, C. G., Pidoux, A. L., Katou, Y., Rumpf, C., Schleiffer, A., Kearsey, S. E., Shirahige, K., Allshire, R. C., and Nasmyth, K. (2007) The kinetochore proteins Pcs1 and Mde4 and heterochromatin are required to prevent merotelic orientation. Curr Biol 17, 1190–200.PubMedCrossRefGoogle Scholar
  29. 29.
    Ding, D. Q., Yamamoto, A., Haraguchi, T., and Hiraoka, Y. (2004) Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast. Dev Cell 6, 329–41.PubMedCrossRefGoogle Scholar
  30. 30.
    Matsumoto, T., Murakami, S., Niwa, O., and Yanagida, M. (1990) Construction and characterization of centric circular and acentric linear chromosomes in fission yeast. Curr Genet 18, 323–30.CrossRefGoogle Scholar
  31. 31.
    Niwa, O., Matsumoto, T., Chikashige, Y., and Yanagida, M. (1989) Characterization of Schizosaccharomyces pombe minichromosome deletion derivatives and a functional allocation of their centromere. Embo J 8, 3045–52.PubMedGoogle Scholar
  32. 32.
    Vanoosthuyse, V., Prykhozhij, S. and Hardwick, K.G. (2007) Shugoshin 2 regulates localisation of the chromosomal passenger proteins in fission yeast mitosis. Mol Biol Cell 18, 1657–69.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Josefin Fernius
    • 1
  • Kevin G. Hardwick
    • 1
  1. 1.Wellcome Trust Centre for Cell BiologyUniversity of EdinburghEdinburghUK

Personalised recommendations