Ensheathed chromosomes do not have stable microtubule-kinetochore attachment. (A) Micrographs of RPE-1 cells stably expressing GFP-Sec61β (gray) pretreated with GSK923295 immunostained for tubulin (red) and CENP-C (green); DNA stained with DAPI. Examples show end-on attachments at aligned kinetochores and potential lateral kinetochore-MT contacts for ensheathed chromosomes. (B) Micrographs of RPE-1 cells stably expressing GFP-Sec61β (gray) pretreated with GSK923295 immunostained for kinastrin (red) and CENP-C (green); DNA stained by DAPI. Scale bars, 10 µm; 2 µm (insets). (C) Frequency distributions of the proximity of the nearest kinastrin punctum to each kinetochore (CENP-C punctum). Kinetochores (n, % with kinastrin <600 nm): aligned (3,124, 26.8%); free (74, 4.1%); ensheathed (227, 6.2%). (D and E) Still images from live-cell imaging experiments of RPE-1 cells stably expressing GFP-Sec61β (green) and Histone H3.2-mCherry (gray), pretreated with 150 nM GSK923295 and stained with SiR-Tubulin (red). Similar results were recorded in 25 cells with free chromosomes and 16 cells with ensheathed chromosomes. Scale bars, 10 µm; 2 µm (insets).