Microtubule–kinetochore interactions are perturbed in the presence of constitutively active MPS1. (A) MPS1^WT or GFP-MPS1^DD mitotic cells were stained for DNA (gray), MAD1, and CENP-C (red in the merged image). MPS1 was visualized by GFP fluorescence. An enlarged image of kinetochore pairs (attached in WT, one unattached in DD), indicated by a dashed box, is shown on the righthand side of the images. (B and C) The x–y distance of individual kinetochores from the cell center in 23 (MPS1^WT) or 19 (MPS1^DD) cells (B) and the proportion of misaligned, MAD1-positive or MPS1-positive kinetochores were plotted (C; mean ± SEM). (D and E) GFP-MPS1^WT or MPS1^DD cells were filmed after Monastrol washout (D), and the fate of individual cells was tracked (E). Each horizontal bar represents a single cell. (F) GFP-MPS1 cells were released from Monastrol arrest into MG132 for 60 min in the presence or absence of 2 µM MPS1i. DNA (gray), microtubules (red), and GFP-MPS1 (green) are shown. Mean proportion ± SD of cells exhibiting aligned metaphase plates is plotted in the bar graphs. (G) KNL1-KNL1 interkinetochore distance is shown in GFP-MPS1^WT or GFP-MPS1^DD cells treated as in A. Each dot represents a kinetochore pair, and the gray bar represents the mean of 234 (WT) and 290 (DD) kinetochore pairs across 19 cells per condition from two independent experiments. ns, not significant (P > 0.05); *, P ≤ 0.05; **, P ≤ 0.001; ****, P ≤ 0.0001. (H) Cells as in A were cold treated before fixation. DNA is shown in gray and microtubules in red. The mean proportions ± SD of cells (282 WT and 331 DD cells from three independent experiments) with aligned metaphase plates or <10 stable microtubule–kinetochore attachments are plotted.