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Fig. 7. Morphological analysis of chromosomes assembled in the absence of Pds5A and Pds5B. (A) Representative images of individual chromosomes assembled in mock-depleted (left), cohesin-depleted (center) and Pds5-depleted (both Pds5A and Pds5B) extracts (right) and stained with DAPI, anti-CAP-G (condensin I), and anti-CAP-H2 (condensin II). The signals of condensin I and II appear in green and red, respectively, in the merged images shown in the bottom panels. The arrowheads indicate the enrichment of CAP-H2 at the centromeric region. (B) Mitotic chromosomes were assembled in extracts that had been mock-depleted (top) or depleted of Pds5A and Pds5B (bottom) as in A, and co-stained with DAPI, anti-SA1 (cohesin) and anti-CAP-E (condensin). A mass of entangled chromosomes is shown. (C) Mitotic chromosomes assembled in extracts that had been mock-depleted (left), depleted of cohesin (middle) or depleted of both Pds5A and Pds5B (right) were co-stained with anti-INCENP (green), anti-CENP-E (red) and DAPI (blue). Merged images of DAPI and CENP-E (whole chromosome) or CENP-E and INCENP (inset) are shown. To better compare the organization of the pericentromeric region of these chromosomes, we show a chromosome from the cohesin-depleted sample with a less severe cohesion defect than the chromosome presented in Fig. 6A, center. The defect is nonetheless obvious, as judged by the increased separation between the sister chromatids in the upper part of the chromosome as well as between the sister kinetochores. Bar, 5 µm.
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