Fig. 2. Defects in metaphase chromosome alignment and anaphase segregation in the absence of cohesin.(A) Fluorescence images showing spindles assembled around Xenopus sperm nuclei in mock- and cohesin-depleted extracts, in the presence of Rhodamine-labeled tubulin (red). After 60 minutes in metaphase, reactions were spun down onto coverslips and processed for immunofluorescence with an antibody against the kinetochore protein Ndc80 (green). DNA was stained with Hoechst-33258 dye (blue), and microtubules are red. Bar, 10 µm. (B) Quantification of chromosome misalignment. The number of chromosomes that had strayed completely away from the metaphase plate in each spindle were counted. Bars represent averages for three independent experiments and a total of n=328 and n=317 spindles in mock- and cohesin-depleted extracts, respectively. Error bars are standard deviations (± s.d.). (C) Quantification of metaphase plate compaction relative to spindle size in mock- and cohesin-depleted spindles. Bars represent the area of the metaphase plate divided by the area of the spindle, to account for differences in spindle size. At least ten spindles were measured in each of three independent experiments, using Metamorph software (Molecular Devices). Error bars are standard deviations. (D) Fluorescence images showing microtubules (red), kinetochore component Ndc80 (green) and DNA (blue) in spindles undergoing anaphase in mock- and cohesin-depleted extracts. Reactions were spun onto coverslips 20 minutes post anaphase induction and processed for immunofluorescence. Bar, 10 µm.