Fig. 7: Incomplete spindle elongation compromises passage across the pole for polar chromosomes located in the danger zone.

a Time-lapse images of RPE1 cells stably expressing CENP-A-GFP and Centrin1-GFP (colorful, confocal) and treated with Mps1 inhibitor AZ3146. The images show maximum intensity projections of the imaged cells. The upper panel represents cells without unaligned chromosomes and the lower panel represents cells with unaligned chromosomes. Kinetochores are color-coded for depth with the brgbcmyw LUT in ImageJ throughout 14–21 z-planes, corresponding to 5.6–8.4 µm. b Percentage of polar (left bar) and non-polar (right bar) kinetochore pairs, as determined at NEBD, that segregated normally (no error, light blue) or ended up as lagging (green), misaligned (dark blue) or unaligned (yellow) kinetochores in anaphase. Each group of kinetochores is depicted on the scheme below the graph. N = 52 polar kinetochore pairs in 7 cells from 4 experiments. N = 54 non-polar kinetochore pairs in 7 cells from 4 experiments. Statistical test: two-sided Fisher’s exact test. c Spindle length in time for cells treated with AZ3146 divided into those that have (orange) or do not have (blue) unaligned chromosomes at anaphase onset. Values are shown as mean (dark line) and SEM (shaded areas). N = 3 cells with unaligned chromosomes from 2 experiments. N = 4 cells without unaligned chromosomes from 2 experiments. d RPE1 cell imaged using lattice light-sheet microscopy at the end of centripetal movement. The dashed oval denotes the approximate position of the nucleus in the preceding interphase (see Supplementary Fig. 6). The straight white line denotes the position of the long nuclear axis in the preceding interphase, the blue line is the spindle axis in prometaphase at the end of the centripetal movement, and β is the angle between them. The image is a maximum intensity projection of 42 z-planes covering the whole cell, corresponding to 6.1 µm. e Scheme showing the angle β as in d, grey areas denote β < 45°. f Percentage of cells with β < 45° (yellow) and β > 45° (blue). Error bars represent SEM. N = 24 cells from 8 experiments. g Images show nuclei in interphase in fixed RPE1 cells stably expressing dCas9-3xGFP (orange) and stained with anti-α-tubulin (gray) and DAPI (blue). Cells are transduced with lentiviruses containing sgRNA for Chr1-telo or Chr9-cen, as indicated. Images are maximum intensity projections of 30 z-planes, corresponding to 15 µm. The diagram on the right illustrates the “danger zone,” which consists of two nuclear caps. Each cap covers one-third of the nuclear long axis. Chromosomes positioned in these caps during interphase are more likely to end up behind the centrosome in early mitosis. h Percentage of cells with chromosome 1 or 9 located in the nuclear danger zone (yellow) or outside the nuclear danger zone (blue). N = 266 chromosomes in 133 cells from 4 experiments for chromosome 1. N = 72 chromosomes in 36 cells from 3 experiments for chromosome 9. Statistical test: two-sided Chi-squared test. All scale bar, 2 µm.