CaMKII induces microtubule instability in anaphase. CSF extracts containing Cy3-tubulin and in vitro translated securin were preincubated for 10 min in the presence of RanQ69L to preassemble microtubules. (A) Reactions were treated with constitutively active CaMKII (CaMKII*), the catalytically inactive mutant (CaMKII*K42A), or CaMKII* together with cyclinBΔ90 (CaMKII* + Δ90) or XErpND (CamKII* + XErpND) as indicated. Microtubule assemblies were visualized by direct fluorescence. (B) Quantification of microtubule assemblies counted in A after 40 min. Error bars represent SD from three independent experiments; metaphase was set to 100%. (C) Reactions were performed as in A; the amounts of endogenous cyclin B (X.l. cyclin B) were determined by immunoblotting, and the amounts of exogenously added in vitro translated (IVT) securin were determined by autoradiography; Cdk1 activities were measured by histone H1 phosphorylation (pHistone H1). (D) Inhibition of endogenous CaMKII activity; CSF extracts containing Cy3-tubulin and RanQ69L were left untreated (Meta) or incubated with calcium in the absence (−) or presence (+) of the CaMKII inhibitor IINtide. (left) Microtubule structures were visualized by direct fluorescence of Cy3-tubulin. (right) Cyclin B (X.l. cyclin B) and Cdk1 activity (pHistone H1) were determined. (D, bottom) Reactions were chased using CaMKII* for the indicated time points and were visualized. Bars, 5 μm.
