Cohesin removal from chromosomes during mitosis. (a) A small increase of soluble cohesin in mitosis. Total cell extracts (T) of an nda3-KM311 strain, either during exponential growth when most cells are in G2, or after arrest in mid-mitosis with condensed chromosomes at 20°C, were separated into soluble (S) and chromosome-bound (C) fractions. The distribution of Rad21-Pk9 was analyzed by western blotting. Cdc2 and histone H3 served as soluble and chromosomal loading controls, respectively. DAPI-stained condensed mitotic chromosomes, and the low septation index, confirmed the arrest. DIC, differential interference contrast. (b) Prophase dissociation and anaphase (ana) cleavage of cohesin during synchronous mitotic progression. The cell fractionation experiment was repeated at time points after release of cells from a cdc25-22-imposed G2 arrest. Mitotic progression was monitored by spindle morphology and septation (sept.) index. (c) Residual chromosomal cohesin during anaphase. Chromosome spreads from an exponentially growing culture were stained against Rad21-Pk9. The kinetochore marker Mis6-GFP was used to identify chromosomes in anaphase.