Protocol

Xenopus Protocols

Volume 322 of the series Methods in Molecular Biology™ pp 459-474

Methods for Studying Spindle Assembly and Chromosome Condensation in Xenopus Egg Extracts

  • Thomas J. MarescaAffiliated withDepartment of Molecular and Cell Biology, University of California Berkeley
  • , Rebecca HealdAffiliated withDepartment of Molecular and Cell Biology, University of California Berkeley

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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.

Key Words

Anaphase chromosome condensation cytoplasmic extract microtubule spindle assembly time-lapse fluorescence microscopy Xenopus laevis egg