Abstract
Methods are presented for preparing cytoplasmic extracts from Xenopus laevis eggs and their utilization to reconstitute and monitor events of the cell cycle in vitro. Addition of sperm nuclei to crude extracts and “cycling” of the reaction through interphase and back into metaphase promotes formation of bipolar spindles capable of segregating their duplicated chromosomes. Reactions can be “spun down” onto cover slips for immunofluorescence analysis. High-speed extracts support mitotic chromosome condensation, which can be observed live by fluorescence time-lapse video microscopy. Because of the biochemically accessible nature of the egg extract system, a wide array of biochemical techniques can be combined with spindle and chromosome assembly reactions to evaluate the roles of specific proteins in these processes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Tunquist, B. J. and Maller, J. L. (2003) Under arrest: cytostatic factor (CSF)-mediated metaphase arrest in vertebrate eggs. Genes Dev. 17, 683–710.
Murray, A. W. (1991) Cell cycle extracts. Methods Cell Biol. 36, 581–605.
Hyman, A. A., Dreschel, D., Kellogg, D., et al. (1991) Preparation of modified tubulins, in Methods in Enzymology (Vallee, R. B., ed.), Academic Press, San Diego, CA, pp. 478–485.
Sawin, K. E. and Mitchison, T. J. (1991) Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol. 112, 925–940.
Shamu, C. E. and Murray, A. W. (1992) Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase. J. Cell Biol. 117, 921–934.
Murray, A. W., Desai, A. B., and Salmon, E. D. (1996) Real time observation of anaphase in vitro. Proc. Nat. Acad. Sci. USA 93, 12,327–12,332.
Wignall, S. M. and Heald, R. (2001) Methods for the study of centrosome-independent spindle assembly in Xenopus extracts. Methods Cell Biol. 67, 241–256.
Heald, R., Tournebize, R., Habermann, A., Karsenti, E., and Hyman, A. (1997) Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization. J. Cell Biol. 138, 615–628.
Mitchison, T. J., Maddox, P., Groen, A., et al. (2004) Bipolarization and poleward flux correlate during Xenopus extract spindle assembly. Mol. Biol. Cell. 15, 5603–5615.
Murray, A. W. and Kirschner, M. W. (1989) Cyclin synthesis drives the early embryonic cell cycle. Nature 339, 275–280.
Hirano, T. and Mitchison, T. J. (1993) Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts. J. Cell Biol. 120, 601–612.
Losada, A., Yokochi, T., Kobayashi, R., and Hirano, T. (2000) Identification and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes. J. Cell Biol. 150, 405–16.
Wignall, S. M., Deehan, R., Maresca, T. J., and Heald, R. (2003) The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts. J. Cell Biol. 161, 1041–1051.
Hirano, T., Kobayashi, R., and Hirano M. (1997) Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89, 511–521.
Funabiki, H. and Murray, A. W. (2000) The chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement in Xenopus egg extracts. Cell 102, 411–424.
Wignall, S. M., Gray, N. S., Chang, Y. T., et al. (2004) Identification of a novel protein regulating microtubule stability through a chemical approach. Chem. Biol. 11, 135–146.
Peterson, J. R., Lokey, R. S., Mitchison, T. J., and Kirschner, M. W. (2001) A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions. Proc. Natl. Acad. Sci. USA 98, 10,624–10,629.
Ono, T., Losada, A., Hirano, M., Myers, M. P., Neuwald, A. F., and Hirano, T. (2003) Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115, 109–121.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Maresca, T.J., Heald, R. (2006). Methods for Studying Spindle Assembly and Chromosome Condensation in Xenopus Egg Extracts. In: Liu, X.J. (eds) Xenopus Protocols. Methods in Molecular Biology™, vol 322. Humana Press. https://doi.org/10.1007/978-1-59745-000-3_33
Download citation
DOI: https://doi.org/10.1007/978-1-59745-000-3_33
Publisher Name: Humana Press
Print ISBN: 978-1-58829-362-6
Online ISBN: 978-1-59745-000-3
eBook Packages: Springer Protocols