Uncoordinated Loss of Chromatid Cohesion Is a Common Outcome of Extended Metaphase Arrest (original) (raw)
Figure 4
The spindle assembly checkpoint is reactivated upon loss of cohesion and is required to maintain the scattered state.
(A) Immunofluorescence images of metaphase-aligned and scattered cells expressing Cyclin B Δ86 stained for DNA, Mad1 and ACA. No Mad1 is seen on fully aligned chromosomes, whereas Mad1 relocalizes to kinetochores of single chromatids upon scattering. Scale bar, 5 µm; magnified image 1 µm. (B and C) Selected images from time-lapse imaging sequences of cells expressing histone H2b:mRFP for (B) HeLa FRT and hTERT-RPE1 cells treated with a CENP-E inhibitor and (C) HeLa FRT cells expressing either Cyclin B Δ86 or the Spindly motif mutant. The scheme for each set of experiments is depicted above the images. Time (in mins) is indicated on the lower right of each panel; time 0 is NEBD. Scale bar, 5 µm. (D) Determination and quantification of the endpoint of cells in the scattered state. All error bars represent SD. (E) Selected images from time-lapse imaging sequences of cells expressing histone H2b:mRFP. HeLa FRT cells were imaged for 5 hours in the presence of the CENP-E inhibitor prior to the addition of 0.5 µM reversine. The red arrowhead indicates the frame after which reversine was added for that cell. Time (in mins) is indicated in the lower right of each panel; time 0 is directly after drug addition. Scale bar, 5 µm. (F) Quantification of the results for (E). Each mark represents one cell and the black line indicates the average.