Misaligned chromosomes outside the exclusion zone are ensheathed in endomembranes. (A) Confocal image of a mitotic RPE-1 cell stably coexpressing GFP-Sec61β (green) Histone H3.2-mCherry (DNA, red) and stained with SiR-Tubulin (gray). Scale bar, 10 µm. (B) SBF-SEM imaging of mitotic cells and subsequent segmentation reveals the endomembranes (ER, blue) and mitochondria (Mito, orange) beyond the exclusion zone boundary (EZ, pink), with the chromosomes (DNA, gray) within. Angle of rotation about y axis is shown. Scale bar, 2 µm. (C) Confocal image of an untreated HeLa cell coexpressing GFP-Sec61β (green) and Histone H2B-mCherry (magenta) with a spontaneously occurring ensheathed chromosome. (D) SBF-SEM imaging of an untreated HeLa cell with a spontaneously occurring ensheathed chromosome. Model shows the position of two ensheathed chromosomes (red) away from the metaphase plate; height of slice 232 is indicated. Scale bar, 2 µm. Segmentation shows endomembranes (green and lilac surrounding the chromosome marked with a star), rendered in 3D (reconstruction). Scale bars, 1 µm. See Videos 1 and 2.