Figure 2.

Depletion of separase does not block cohesion fatigue. (A) Upper Panels: Live cell imaging confirms loss of separase activity mediated by RNAi; cells fail to appropriately segregate sister chromatids during anaphase and cytokinesis results in a cut phenotype. Lower Panels: Separase depletion does not prevent scattering in cells arrested at metaphase by proteasome inhibition (MG132). (B) Chromosome spreads and cell extracts were prepared from arrested mitotic cells that had been transfected with non-targeting, separase-targeting, or SKA3-targeting siRNA duplexes. These cells had arrested in mitosis due to proteasome inhibition and/or SKA3 depletion. Graphically represented are the percentages of chromosome spreads classified as separated. Chromosome spreads reveal that separase depletion does not prevent cohesion fatigue in cells arrested by proteasome inhibition or SKA3 depletion. The number of spreads tallied for each condition ranged between 164 and 270. Western blotting indicates successful depletion of separase and SKA3 by RNAi. (C) Separase is required for early but not late chromatid separation induced by overexpression of degradation-resistant cyclin B. Upper panels: HeLa H2B-mCherry/R42A-CyclinB-GFP cells delay at metaphase for durations less than 1.6 hours, separate sister chromatids, and remain arrested in mitosis. Lower panels: HeLa H2B-mCherry/R42A-CyclinB-GFP cells lacking separase delay for several hours at metaphase and subsequently experience cohesion fatigue. HeLa H2B-mCherry/R42A-CyclinB-GFP cells separated sister chromatids with an average metaphase to scatter duration of 1.6 ± 1.1 hours. In contrast, 46% of HeLa H2B-mCherry/R42A-CyclinB-GFP cells lacking separase maintained metaphase alignment of chromosomes; the remaining 54% separated sister chromatids after an extended metaphase arrest. Bars = 10 μm, Time = hr:min, Error bars = standard deviation (see also Figure S2B, S2C and Movies S2, S3, and S4)