Figure 4.

Localization of cohesin complexes in nuclei and chromosomes assembled in Xenopus egg extracts. (A) Xenopus sperm chromatin was incubated in interphase or mitotic HSS. The assembled interphase chromatin (I) and mitotic chromosomes (M) were fixed, isolated through a glycerol cushion, and immunostained with anti-XSA1 (e and f, green) or anti-XSA2 (g and h, green). The DNA was counterstained with DAPI (a–d, red). Merged images are also shown (i–l). (B) Xenopus sperm chromatin was incubated at 22°C for 90 min in interphase LSS, where it was replicated. Half of this interphase mixture (I) was fixed and processed for immunostaining with anti-XSA1 (a, f, and k) or anti-XSA2 (d, i, and n). The other half was driven into mitosis (M) by the addition of a mitotic LSS, incubated for another 2 h, fixed, and immunostained. A mass of entangled chromosomes (b, g, l, e, j, and o) and an individual chromosome (c, h, and m) are shown. (a–e, red) DNA; (f–h, green) anti-XSA1; (i and j, green) anti-XSA2; (k–o) merged images. Stronger staining with anti-XSA1 was often observed on a bent region of the chromosome, most likely corresponding to the pericentromeric region (arrowhead). The images of antibody staining were captured using different exposure times to visualize weak signals on the mitotic chromosomes and, therefore, the label intensities cannot be compared quantitatively. (C) Xenopus sperm nuclei were incubated in an interphase LSS depleted of x-cohesins and supplemented with h-cohesin^SA1 and h-cohesin^SA2. After incubation at 22°C for 90 min, the nuclei were fixed, and hSA1 and hSA2 were detected with anti-XSA1 and anti-XSA2, respectively. (a and b, red) DNA; (c and d, green) antibody staining; (e and f) merged images. Bars, 5 μm.