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Indirect Immunofluorescence for Monitoring Spindle Assembly and Disassembly in Yeast

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Cell Cycle Checkpoints

Part of the book series: Methods in Molecular Biology ((MIMB,volume 782))

Abstract

In yeast like all eukaryotes, microtubules are a crucial element of the mitotic spindle that separates the genetic material during cell division. The assembly status and position of the mitotic spindle, as well as cytoplasmic microtubules, can be monitored easily using indirect immunofluorescence with antibodies against tubulin. A detailed protocol is described for Saccharomyces cerevisiae that involves the fixation of actively growing cells, removal of the cell wall by enzymatic digestion, post-fixation, and the application of tubulin antibodies. The use of secondary antibodies conjugated to a fluorescent moiety permit visualization of the mitotic spindle by fluorescence microscopy. Methods for the reduction of background and pre-absorption of antibodies are discussed.

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References

  1. Sullivan, D. S. and Huffaker, T. C. (1992) Astral Microtubules are not required for anaphase B in Saccharomyces cerevisiae. J. Cell Biol. 119, 379–388.

    Article  PubMed  CAS  Google Scholar 

  2. Carminati, J. L. and Stearns, T. (1997) Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex. J. Cell Biol. 138, 629–641.

    Article  PubMed  CAS  Google Scholar 

  3. Miller, R. K. and Rose, M. D. (1998) Kar9p is a novel cortical protein required for cytoplasmic microtubule orientation in yeast. J. Cell Biol. 140, 377–390.

    Article  PubMed  CAS  Google Scholar 

  4. Miller, R. K., Cheng, S.-C., and Rose, M. D. (2000) Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules. Mol. Biol. Cell 11, 2949–2959.

    PubMed  CAS  Google Scholar 

  5. Beach, D. L., Thibodeaux, J., Maddox, P., Yeh, E., and Bloom, K. (2000) The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast. Curr. Biol. 10, 1497–1506.

    Article  PubMed  CAS  Google Scholar 

  6. Byers, B. (1981) Cytology of the yeast life cycle. In The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. J. Strathern, E. W. Jones, and J. R. Broach, editors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. pp. 59–96.

    Google Scholar 

  7. Robinow, C. F. and Marak, J. (1966) A fiber apparatus in the nucleus of the yeast cell. J. Cell Biol. 29, 129.

    Article  PubMed  CAS  Google Scholar 

  8. Byers, B. and Goetsch, L. (1974) Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harbor Symp. Quant. Biol. 38, 123–131.

    Article  PubMed  CAS  Google Scholar 

  9. Adams, A. E. M. and Pringle, J. (1984) Relationship of actin and tubulin distribution to bud growth in wildtype and morphogenetic-mutant Saccharomyces cerevisiae. J. Cell Biol. 98, 934–945.

    Article  PubMed  CAS  Google Scholar 

  10. Kilmartin, J. and Adams, A. E. M. (1984) Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J. Cell Biol. 98, 922–933.

    Article  PubMed  CAS  Google Scholar 

  11. Botstein, D., Amberg, D., Mulholland, J., Huffaker, T., Adams, A., Drubin, D., and Stearns, T. (1997) The yeast cytoskeleton. In The Molecular and Cellular Biology of the Yeast Saccharomyces. J. R. Pringle, J. R. Broach, and E. W. Jones, editors. Cold Spring Harbor Laboratory, Cold spring Harbor, NY. pp. 1–90.

    Google Scholar 

  12. Byers, B. and Goetsch, L. (1975) Behavior of spindles and spindle plagues in the cell cycle and conjugation of Saccharomyces cerevisiae. J. Bacteriol. 124, 511–523.

    PubMed  CAS  Google Scholar 

  13. Lew, D. J., Weinert, T., and Pringle, J. R. (1997) Cell cycle control in Saccharomyces cerevisiae. In The Molecular and Cellular Biology of the Yeast Saccharomyces. J. R. Pringle, J. R. Broach, and E. W. Jones, editors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. pp. 607–695.

    Google Scholar 

  14. Winey, M., Mamay, C. L., O’Toole, E. T., Mastronarde, D. N., Giddings, T. H., McDonald, K. L., and McIntosh, J. R. (1995) Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J. Cell Biol. 129, 1601–1615.

    Article  PubMed  CAS  Google Scholar 

  15. Segal, M. and Bloom, K. (2001) Control of spindle polarity and orientation in Saccharomyces cerevisiae. Trends Cell Biol. 11, 160–166.

    Article  CAS  Google Scholar 

  16. Kusch, J., Meyer, A., Snyder, M. P., and Barral, Y. 2002. Microtubule capture by the cleavage apparatus is required for proper spindle positioning in yeast. Genes Dev. 16, 1627–1639.

    Article  PubMed  CAS  Google Scholar 

  17. Kahana, J. A., Schnapp, B. J., and Silver, P. A. (1995) Kinetics of spindle pole body separation in budding yeast. Proc. Nat’l. Acad. Sci. USA 92, 9707–9711.

    Article  CAS  Google Scholar 

  18. Yeh, E., Skibbens, R. V., Cheng, J. W., Salmon, E. D., and Bloom, K. (1995) Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae. J. Cell Biol. 130, 687–700.

    Article  PubMed  CAS  Google Scholar 

  19. Tirnauer, J. S., O’Toole, E., Berrueta, L., Bierer, B. E., and Pellman, D. (1999) Yeast Bim1p promotes the G1-specific dynamics of microtubules. J. Cell Biol. 145, 993–1007.

    Article  PubMed  CAS  Google Scholar 

  20. Adames, N. R. and Cooper, J. A. (2000) Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J. Cell Biol. 149, 863–874.

    Article  PubMed  CAS  Google Scholar 

  21. Rose, M. D., Winston, F., and Hieter, P. (1990) Methods of Yeast Genetics, a laboratory course manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. pp. 97–102.

    Google Scholar 

  22. Miller, R. K. (2004) Monitoring spindle assembly and disassembly in yeast by indirect immunofluorescence. In Cell Cycle Checkpoint Control Protocols. H. B. Lieberman, editors. Humana Press, Totowa, NJ. Methods in Molecular Biology series. Vol. 241. pp. 341–352.

    Google Scholar 

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Acknowledgments

We thank Mark Rose for his continued support and helpful comments. We thank Nelda Driggs for administrative assistance. This work was supported in part by a Basil O’Connor Starter Scholar Research Award from the March of Dimes (#5-FY01-523), the Oklahoma Health Research Program of the Oklahoma Center for the Advancement of Science and Technology (OCAST #HR09-150S), and start-up funds to R.K.M from O.S.U. and the Oklahoma Agricultural Experiment Station. J.W.P.K. was supported by a Niblack Research Scholarship.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, USA

    Jacob W. P. Keeling & Rita K. Miller

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  1. Jacob W. P. Keeling
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  2. Rita K. Miller
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Correspondence to Rita K. Miller .

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Editors and Affiliations

  1. , Department of Medicine, University of California, Gilman Drive #0063 9500, La Jolla, 92093-0063, California, USA

    Willis X. Li

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Keeling, J.W.P., Miller, R.K. (2011). Indirect Immunofluorescence for Monitoring Spindle Assembly and Disassembly in Yeast. In: Li, W. (eds) Cell Cycle Checkpoints. Methods in Molecular Biology, vol 782. Humana Press. https://doi.org/10.1007/978-1-61779-273-1_17

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  • DOI: https://doi.org/10.1007/978-1-61779-273-1_17

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-272-4

  • Online ISBN: 978-1-61779-273-1

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