Figure 2.

ATP-dependent weakening of native kinetochores requires Mps1 kinase activity. (A) The Mps1 inhibitor reversine reduces phosphorylation of Spc105, Dsn1, and Ndc80 by copurifying kinase activity. Kinetochores purified by immunoprecipitation of Dsn1-His-Flag from WT cells (SBY8253) were incubated with γ-³²P-labeled ATP, either alone or with 5 µM of reversine, and then visualized by silver stain or autoradiography after SDS-PAGE. Asterisk marks an additional unidentified phosphoprotein. (B) Median rupture strengths (left) and corresponding survival probability distributions (right) for WT kinetochores (from SBY8253) measured under indicated conditions. The ATP-dependent weakening (red) was blocked by addition of 5 µM reversine (purple). Values inside bars indicate numbers of events for each condition. Error bars represent ± 95% confidence intervals calculated by bootstrapping. P values for all pairwise strength comparisons (from log-rank tests) are provided in Table S5.