Changes in microtubule stability from metaphase to anaphase in Xenopus egg extracts. (A) Calcium was added to CSF-arrested Xenopus egg extracts (0 min) in the presence of cyclinBΔ90, and samples were withdrawn at the indicated times to determine the activity of Cdk1 toward histone H1 (pHistone H1, top) and the amounts of endogenous cyclin B (X.I. cyclin B, bottom). (B) Microtubules or spindles were assembled in metaphase-arrested extracts (left), calcium was added in the presence of cyclinBΔ90 (right), and microtubules were visualized by direct fluorescence of added Cy3-tubulin. Sperm nuclei, RanQ69L, or chromatin beads were used as a nucleating source. (C) Immunoprecipitation using anti-TPX2 antibodies from metaphase or anaphase extracts. Immunocomplexes were eluted with high salt, and aurora A/Eg2 was determined by immunoblotting. (D) The microtubule assembly assay is as described in B; purified human centrosomes were used as a nucleating source. Cycled, microtubule asters were preassembled in metaphase before calcium addition; Meta, metaphase; Ana, anaphase. Bars, 10 μm.