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.
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References
Wiese, C., and Zheng, Y. (2006) Microtubule nucleation: γ-tubulin and beyond. J. Cell Sci 119, 4143–4153
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
Azimzadeh, J., and Bornens, M. (2007) Structure and duplication of the centrosome. J Cell Sci 120, 2139–2142
Bettencourt-Dias, M., and Glover, D. M. (2007) Centrosome biogenesis and function: centrosomics brings new understanding. Nat Rev Mol Cell Biol 8, 451–463
Gadde, S., and Heald, R. (2004) Mechanisms and molecules of the mitotic spindle. Curr Biol 14, R797–R805
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
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
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
Murray, A. W. (1991). Cell cycle extracts. Methods Cell Biol 36, 581–605
Newmeyer, D.D., and Wilson, K. L. (1991) Egg extracts for nuclear import and nuclear assembly reactions. Methods Cell Biol 36, 607–634
Mitchison, T. J., and Kirschner, M. W. (1986) Isolation of mammalian centrosomes. Methods Enzymol 134, 261–268
Mitchison, T. J., and Kirschner, M. W. (1984). Dynamic instability of microtubule growth. Nature 312, 237–242
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
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
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
Popov, A. V., Severin, F., and Karsenti, E. (2002) XMAP215 regulates microtubule-nucleating activity of centrosomes. Curr Biol 12, 1326–1330
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
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
Acknowledgments
Work in the Wiese Lab is supported by NSF CAREER award MCB-0643878 (to CW).
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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
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