Rescue of ensheathed chromosomes by the induced relocalization of ER. (A) Stills from live-cell imaging of ER clearance experiments. FKBP-GFP-Sec61β (green), Stargazin-mCherry-FRB (red), and SiR-DNA (gray) are shown. Insets are 2× expansions of the ROI shown. Scale bars, 10 µm; 1 µm (insets). See Video 7. (B) Semiautomated 4D tracking of misaligned chromosome location is used to monitor congression. Two tracks from the cells in A are shown. The shortest Euclidean distance from the centroid of the misaligned chromosome to the edge of the main chromosome plate is plotted as a function of time. (C) Fate of misaligned chromosomes in control or rapamycin-treated cells. Rescue of misaligned chromosomes was detected in 26 of 30 rapamycin-treated cells. Coloring in B and C is with the color scale shown. Tracks terminate at 90 min or when the chromosome merges with the plate. Median termination time was 93 min (control, n = 36) and 45 min (rapamycin, n = 30); P = 7.1 × 10−9, Wilcoxon rank test. Rapamycin is applied after the first frame (T = 0).
