Figure 3.
Impact of ensheathed chromosomes on cell division. (A) Mitotic timing of RPE-1 cells. Cumulative frequencies for NEB to metaphase (NEB-Meta) and metaphase to anaphase (Meta-Ana) are shown. RPE-1 stably expressing GFP-Sec61β were treated with 150 nM GSK923295 for 3 h before washout. Three classes of metaphase were seen: all chromosomes aligned (Aligned, n = 29), cells with one or more free chromosomes (Free, n = 11), and cells with one or more ensheathed chromosome (Ensheathed, n = 107). Timing of untreated parental (Parental, n = 69) and stable RPE-1 (Control, n = 52) cells is also shown. Inset in Meta-Ana shows same data on an expanded time scale. Comparison of NEB-Meta and Meta-Ana timing distributions for ensheathed vs. control, P = 1.9 × 10−57 and 7.8 × 10−23, Kolmogorov–Smirnov test. (B) Micrographs of immunofluorescence experiments to detect Bub1 or Mad2 (SAC, red) at kinetochores (CENP-C, blue) in cells stably expressing GFP-Sec61β (green); DAPI-stained DNA is shown in gray. Scale bars, 10 µm; 2 µm (insets). (C) Quantification of Bub1 and Mad2 immunofluorescence at kinetochores marked by CENP-C. Ensheathed chromosomes were classified using the GFP-Sec61β signal. Dots represent kinetochores, boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles (Bub1: nA = 132, nF = 30, nE = 37; (Mad2: nA = 103, nF = 20, nE = 31). (D) Stills from live-cell imaging experiments to track Mad2 levels at kinetochores of ensheathed chromosomes. A GSK923295-pretreated RPE-1 cell is shown, stably coexpressing GFP-Mad2 (green) and mCherry-Sec61β (red); DNA is stained using SiR-DNA (blue). Time relative to anaphase is shown in minutes. Insets show 2× zoom of the indicated ROI. Scale bars, 10 µm; 2 µm (insets). (E) Quantification of live Mad2 imaging experiments. Kaplan–Meier plot to show congression times of the last misaligned chromosome to align. Measurement of mCherry-Sec61β (mean ± SD) and GFP-Mad2 is shown for the misaligned that congressed and those that were missegregated (misseg). A linear regression fit with 95% confidence intervals is shown for GFP-Mad2. All plots are shown in time (minutes) relative to anaphase onset. Total cells with misaligned chromosomes, n = 72; cells where all chromosomes congressed, n = 56; and where there was missegregation, n = 16.

Impact of ensheathed chromosomes on cell division. (A) Mitotic timing of RPE-1 cells. Cumulative frequencies for NEB to metaphase (NEB-Meta) and metaphase to anaphase (Meta-Ana) are shown. RPE-1 stably expressing GFP-Sec61β were treated with 150 nM GSK923295 for 3 h before washout. Three classes of metaphase were seen: all chromosomes aligned (Aligned, n = 29), cells with one or more free chromosomes (Free, n = 11), and cells with one or more ensheathed chromosome (Ensheathed, n = 107). Timing of untreated parental (Parental, n = 69) and stable RPE-1 (Control, n = 52) cells is also shown. Inset in Meta-Ana shows same data on an expanded time scale. Comparison of NEB-Meta and Meta-Ana timing distributions for ensheathed vs. control, P = 1.9 × 10−57 and 7.8 × 10−23, Kolmogorov–Smirnov test. (B) Micrographs of immunofluorescence experiments to detect Bub1 or Mad2 (SAC, red) at kinetochores (CENP-C, blue) in cells stably expressing GFP-Sec61β (green); DAPI-stained DNA is shown in gray. Scale bars, 10 µm; 2 µm (insets). (C) Quantification of Bub1 and Mad2 immunofluorescence at kinetochores marked by CENP-C. Ensheathed chromosomes were classified using the GFP-Sec61β signal. Dots represent kinetochores, boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles (Bub1: nA = 132, nF = 30, nE = 37; (Mad2: nA = 103, nF = 20, nE = 31). (D) Stills from live-cell imaging experiments to track Mad2 levels at kinetochores of ensheathed chromosomes. A GSK923295-pretreated RPE-1 cell is shown, stably coexpressing GFP-Mad2 (green) and mCherry-Sec61β (red); DNA is stained using SiR-DNA (blue). Time relative to anaphase is shown in minutes. Insets show 2× zoom of the indicated ROI. Scale bars, 10 µm; 2 µm (insets). (E) Quantification of live Mad2 imaging experiments. Kaplan–Meier plot to show congression times of the last misaligned chromosome to align. Measurement of mCherry-Sec61β (mean ± SD) and GFP-Mad2 is shown for the misaligned that congressed and those that were missegregated (misseg). A linear regression fit with 95% confidence intervals is shown for GFP-Mad2. All plots are shown in time (minutes) relative to anaphase onset. Total cells with misaligned chromosomes, n = 72; cells where all chromosomes congressed, n = 56; and where there was missegregation, n = 16.

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