Skip to main content

Analysis of Centrosome Function and Microtubule Dynamics by Time-Lapse Microscopy in Xenopus Egg Extracts

  • Protocol
  • First Online:
Book cover Cytoskeleton Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 586))

Summary

Centrosomes are essential organelles that organize the microtubule cytoskeleton during interphase and mitosis. Centrosomes are assembled from tens to hundreds of proteins, but how these proteins are organized into functional microtubule nucleating and organizing centers is not yet clear. An important step in understanding the role of individual proteins in centrosome function is to understand whether they are involved in forming, stabilizing, or anchoring microtubules. It is becoming increasingly clear that the analysis of fixed samples is inadequate for a true understanding of the dynamics that drive cell biological processes. In this chapter we focus on methods to analyze microtubule nucleation, organization, and dynamics using assays based on mitotic Xenopus egg extracts and in vitro reactions. These methods can easily be adapted to the study of interphase processes, or to the study of other cytoskeletal proteins and their dynamics.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wiese, C., and Zheng, Y. (2006) Microtubule nucleation: γ-tubulin and beyond. J. Cell Sci 119, 4143–4153

    Article  CAS  PubMed  Google Scholar 

  2. Andersen, J. S., Wilkinson, C. J., Mayor, T., Mortensen, P., Nigg, E. A., and Mann, M. (2003) Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426, 570–574

    Article  CAS  PubMed  Google Scholar 

  3. Azimzadeh, J., and Bornens, M. (2007) Structure and duplication of the centrosome. J Cell Sci 120, 2139–2142

    Article  CAS  PubMed  Google Scholar 

  4. Bettencourt-Dias, M., and Glover, D. M. (2007) Centrosome biogenesis and function: centrosomics brings new understanding. Nat Rev Mol Cell Biol 8, 451–463

    Article  CAS  PubMed  Google Scholar 

  5. Gadde, S., and Heald, R. (2004) Mechanisms and molecules of the mitotic spindle. Curr Biol 14, R797–R805

    Article  CAS  PubMed  Google Scholar 

  6. Belmont, L. D., Hyman, A. A., Sawin, K. E., and Mitchison, T. J. (1990) Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62, 579–589

    Article  CAS  PubMed  Google Scholar 

  7. Lohka, M. J. and Masui, Y. (1983) Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220, 719–721

    Article  CAS  PubMed  Google Scholar 

  8. Felix, M.A., Antony, C., Wright, M. and Maro B. (1994) Centrosome assembly in vitro: role of γ-tubulin recruitment in Xenopus sperm aster formation. J Cell Biol 124, 19–31

    Article  CAS  PubMed  Google Scholar 

  9. Murray, A. W. (1991). Cell cycle extracts. Methods Cell Biol 36, 581–605

    Article  CAS  PubMed  Google Scholar 

  10. Newmeyer, D.D., and Wilson, K. L. (1991) Egg extracts for nuclear import and nuclear assembly reactions. Methods Cell Biol 36, 607–634

    Article  CAS  PubMed  Google Scholar 

  11. Mitchison, T. J., and Kirschner, M. W. (1986) Isolation of mammalian centrosomes. Methods Enzymol 134, 261–268

    Article  CAS  PubMed  Google Scholar 

  12. Mitchison, T. J., and Kirschner, M. W. (1984). Dynamic instability of microtubule growth. Nature 312, 237–242

    Article  CAS  PubMed  Google Scholar 

  13. Moritz, M., Zheng, Y., Alberts, B. M., and Oegema, K. (1998) Recruitment of the g-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. J Cell Biol 142, 775–786

    Article  CAS  PubMed  Google Scholar 

  14. Schnackenberg, B. J., Khodjakov, A., Rieder, C. L., and Palazzo, R. E. (1998) The disassembly and reassembly of functional centrosomes in vitro. Proc Natl Acad Sci USA 95, 9295–9300

    Article  CAS  PubMed  Google Scholar 

  15. Gard, D. L., and Kirschner, M. W. (1987) A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end. J Cell Biol 105, 2203–2215

    Article  CAS  PubMed  Google Scholar 

  16. Popov, A. V., Severin, F., and Karsenti, E. (2002) XMAP215 regulates microtubule-nucleating activity of centrosomes. Curr Biol 12, 1326–1330

    Article  CAS  PubMed  Google Scholar 

  17. Albee, A. J., and Wiese, C. (2008) Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome. Mol Biol Cell 19, 3347–3356

    Article  CAS  PubMed  Google Scholar 

  18. Hyman, A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordeman, L., and Mitchison, T. J. (1991) Preparation of modified tubulins. Methods Enzymol 196, 478–485

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Work in the Wiese Lab is supported by NSF CAREER award MCB-0643878 (to CW).

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

Wiese, C., Mayers, J.R., Albee, A.J. (2009). Analysis of Centrosome Function and Microtubule Dynamics by Time-Lapse Microscopy in Xenopus Egg Extracts. In: Gavin, R. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology, vol 586. Humana Press. https://doi.org/10.1007/978-1-60761-376-3_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-376-3_5

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-375-6

  • Online ISBN: 978-1-60761-376-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics