PIASγ Is Required for Faithful Chromosome Segregation in Human Cells
Figure 6
Sister chromatids cannot separate in PIASγ-depleted cells lacking Topoisomerase II activity.
Metaphase arrested cells depleted of PIASγ were collected as described in Figure 4, then the Aurora B inhibitor ZM447439 and the Topoisomerase II inhibitor ICRF-193 were added and samples taken for cytological analysis. (A) metaphase; (B,C,D) failed sister disjunction during anaphase and subsequent exit from mitosis. In B and C sister chromatids can still be observed to be cohered while the chromatin is largely decondensing (double arrows). For comparison, small inserted panels show metaphase (left-most panel) and anaphase (two panels on the right) in the presence of ZM447439 alone. (E) Frequency of cells in which sisters were able to disjoin upon addition of ZM447439, with or without ICRF-193. (F–I) Immunostaining of Topoisomerase IIα in HeLa cells expressing H2B-GFP (inserts show a slight enlargement of a selected region from the same micrograph). (F–I) Merge - Topoisomerase IIα (red) and H2B-GFP (green). (F′–I′) Topoisomerase IIα staining only. (F′′–I′′) H2B-GFP only. (F–G) Control mitotic cells showing Topoisomerase IIα localized to cores and concentrated at centromere regions; (H) PIASγ-depleted mitotic cell with diffusely localized Topoisomerase IIα on the chromatin; (I) PIASγ-depleted mitotic cell with Topoisomerase IIα localized to cores but not strongly enriched at centromere regions. (J) Classification of Topoisomerase IIα and INCENP immunostaining patterns in control and PIASγ-depleted mitotic cells. The “Kinetochore” and “Core & Kinetochore” categories required that Topoisomerase IIα was strongly concentrated at the centromere regions.