Figure 2.
Induction of misaligned chromosomes in stably diploid RPE1 cells by pretreatment with a CENP-E inhibitor. (A) Polar, misaligned chromosomes can be induced by treatment with CENP-E inhibitor GSK923295 (150 nM, 3 h) and subsequent washout (1 h). (B) Confocal micrographs to show that these misaligned chromosomes (SiR-DNA, red) are either outside the exclusion zone delineated by GFP-Sec61β (green), termed ensheathed, or at the boundary and inside the exclusion zone, termed free. Scale bars, 10 µm; 1 µm (inset). (C) Spatially averaged 3D view of all CENP-C–positive kinetochores in the dataset; see Materials and methods). Small gray points represent kinetochores at the metaphase plate. Colored points represent misaligned chromosomes that were ensheathed (orange) and those that were not (free, blue). Spindle poles are shown in black. (D) Box plot to show the relative position of each kinetochore relative to the exclusion zone boundary. Chromosome misalignment was induced by pretreatment with GSK923295 (150 nM). Ratio of kinetochores within the exclusion zone are <0 and those within the ER are >0 on a log2 scale. Dots represent kinetochore ratios from 31 RPE-1 cells at metaphase. Boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. Inset: Schematic diagram to show how the position of kinetochores relative to the exclusion zone boundary was calculated. C is the centroid of aligned kinetochores, P is a kinetochore, and Q is the point along the 3D path (CP) that intersects the exclusion zone boundary. The ratio of CP to CQ is taken for each kinetochore (aligned kinetochores, gray; free, blue; and ensheathed, orange). (E) Single SBF-SEM image showing an ensheathed chromosome. Boxed region is shown expanded and modeled (zoom). Single slice and a 3D model (bottom right) of slices 87–126 are shown. Scale bar, 2 µm (black) and 500 nm (white). (F) Modeled substacks from SBF-SEM images showing a chromosome outside the exclusion zone, ensheathed in ER. Slices shown and angles and axes of rotation are indicated (see Video 3). Scale bar, 2 µm.

Induction of misaligned chromosomes in stably diploid RPE1 cells by pretreatment with a CENP-E inhibitor. (A) Polar, misaligned chromosomes can be induced by treatment with CENP-E inhibitor GSK923295 (150 nM, 3 h) and subsequent washout (1 h). (B) Confocal micrographs to show that these misaligned chromosomes (SiR-DNA, red) are either outside the exclusion zone delineated by GFP-Sec61β (green), termed ensheathed, or at the boundary and inside the exclusion zone, termed free. Scale bars, 10 µm; 1 µm (inset). (C) Spatially averaged 3D view of all CENP-C–positive kinetochores in the dataset; see Materials and methods). Small gray points represent kinetochores at the metaphase plate. Colored points represent misaligned chromosomes that were ensheathed (orange) and those that were not (free, blue). Spindle poles are shown in black. (D) Box plot to show the relative position of each kinetochore relative to the exclusion zone boundary. Chromosome misalignment was induced by pretreatment with GSK923295 (150 nM). Ratio of kinetochores within the exclusion zone are <0 and those within the ER are >0 on a log2 scale. Dots represent kinetochore ratios from 31 RPE-1 cells at metaphase. Boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. Inset: Schematic diagram to show how the position of kinetochores relative to the exclusion zone boundary was calculated. C is the centroid of aligned kinetochores, P is a kinetochore, and Q is the point along the 3D path (CP) that intersects the exclusion zone boundary. The ratio of CP to CQ is taken for each kinetochore (aligned kinetochores, gray; free, blue; and ensheathed, orange). (E) Single SBF-SEM image showing an ensheathed chromosome. Boxed region is shown expanded and modeled (zoom). Single slice and a 3D model (bottom right) of slices 87–126 are shown. Scale bar, 2 µm (black) and 500 nm (white). (F) Modeled substacks from SBF-SEM images showing a chromosome outside the exclusion zone, ensheathed in ER. Slices shown and angles and axes of rotation are indicated (see Video 3). Scale bar, 2 µm.

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