Cohesin decay leads to abnormal centromere alignment. (A) Representative images of centromere positioning (Cid-EGFP, green) in control (100% Rad21^WT) and D strains. DNA is labeled with His-RFP (red). Times are relative to TEV injection. (B) Intercentromere distances in metaphase upon Rad21^TEV cleavage 10 min after TEV injection and before TEV addition for strain D. 20 centromere pairs were analyzed per embryo (n = 6, 5, 6, 7, and 6 embryos). ***, P < 0.0001, Kruskal-Wallis test relative to 100% Rad21^WT. (C) Chromosome/centromere alignment of His-RFP and Cid-EGFP profiles at 0 and 10 min after TEV injection. His-RFP/Cid-EGFP intensity plot profiles were fit to a Lorentzian function as illustrated, and the width value was used as an alignment readout. Five metaphases were measured per embryo (n = 7 and 6 embryos for control and strain D, respectively). For these analyses, only embryos that did not display chromatid disjunction within the course of the experiment (20 min) were analyzed. Bars, 5 µm.