Fig 2: Mps1 C-tail is required for the functional integrity of SAC. (A and C) Representative immunofluorescence images of HeLa cells transfected with shMps1 and different shMps1-resistant GFP-Mps1 constructs as indicated. After 36 h of transfection, cells were treated with nocodazole plus MG132 for 1 h. Then cells were fixed and co-stained for pMETL-Knl1 (red, A) or Mad2 (red, C), DNA (blue), and ACA (in gray scale). Scale bar, 10 µm. (B and D) Bar graphs illustrating kinetochore intensity of pMETL-Knl1 as in A and Mad2 as in C. Bars represent the mean intensity (±SD) normalized to the values of Mps1WT group. Each dot represents one cell (>18 cells from three independent experiments). Student’s t-test was used to calculate P-values. (E) Bar graph illustrating percentage of cells treated as indicated with anaphase lagging chromosomes. Cells were co-transfected with shMps1, mCherry-H2B, and different GFP-Mps1 plasmids, respectively. Bars indicate mean±SD (>30 cells from three independent experiments). Student’s t-test was used to calculate P-values. (F) Scatter plot of the time from NEBD to anaphase beginning in cells co-transfected with shMps1, different GFP-Mps1, and mCherry-H2B. Bars indicate mean±SD. Each dot represents one cell (>30 cells from three independent experiments). Student’s t-test was used to calculate P-values.

Fig 3: Impact of ensheathed chromosomes on cell division. (A) Mitotic timing of RPE-1 cells. Cumulative frequencies for NEB to metaphase (NEB-Meta) and metaphase to anaphase (Meta-Ana) are shown. RPE-1 stably expressing GFP-Sec61ß were treated with 150 nM GSK923295 for 3 h before washout. Three classes of metaphase were seen: all chromosomes aligned (Aligned, n = 29), cells with one or more free chromosomes (Free, n = 11), and cells with one or more ensheathed chromosome (Ensheathed, n = 107). Timing of untreated parental (Parental, n = 69) and stable RPE-1 (Control, n = 52) cells is also shown. Inset in Meta-Ana shows same data on an expanded time scale. Comparison of NEB-Meta and Meta-Ana timing distributions for ensheathed vs. control, P = 1.9 × 10-57 and 7.8 × 10-23, Kolmogorov–Smirnov test. (B) Micrographs of immunofluorescence experiments to detect Bub1 or Mad2 (SAC, red) at kinetochores (CENP-C, blue) in cells stably expressing GFP-Sec61ß (green); DAPI-stained DNA is shown in gray. Scale bars, 10 µm; 2 µm (insets). (C) Quantification of Bub1 and Mad2 immunofluorescence at kinetochores marked by CENP-C. Ensheathed chromosomes were classified using the GFP-Sec61ß signal. Dots represent kinetochores, boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles (Bub1: nA = 132, nF = 30, nE = 37; (Mad2: nA = 103, nF = 20, nE = 31). (D) Stills from live-cell imaging experiments to track Mad2 levels at kinetochores of ensheathed chromosomes. A GSK923295-pretreated RPE-1 cell is shown, stably coexpressing GFP-Mad2 (green) and mCherry-Sec61ß (red); DNA is stained using SiR-DNA (blue). Time relative to anaphase is shown in minutes. Insets show 2× zoom of the indicated ROI. Scale bars, 10 µm; 2 µm (insets). (E) Quantification of live Mad2 imaging experiments. Kaplan–Meier plot to show congression times of the last misaligned chromosome to align. Measurement of mCherry-Sec61ß (mean ± SD) and GFP-Mad2 is shown for the misaligned that congressed and those that were missegregated (misseg). A linear regression fit with 95% confidence intervals is shown for GFP-Mad2. All plots are shown in time (minutes) relative to anaphase onset. Total cells with misaligned chromosomes, n = 72; cells where all chromosomes congressed, n = 56; and where there was missegregation, n = 16.

Fig 4: The Molecular Landscape Required to Establish Functional Kinetochore-Microtubule Attachments Is Conserved in Indian Muntjac(A) Live-cell imaging of Indian muntjac fibroblasts stably expressing H2B-GFP to visualize the chromosomes (green) and treated with 50 nM SiR-tubulin to label spindle microtubules (magenta). Ndc80, Mad2, Mps1, Clasp1, and Survivin were knocked down by RNAi. Scale bars, 5 µm. Time, hr:min.(B) Mitotic timing of Indian muntjac fibroblasts stably expressing H2B-GFP with or without addition of 50 nM SiR-tubulin. There is no statistically significant difference in mitotic timing from NEB to anaphase onset (ANA) in the presence or absence of SiR-tubulin (Mann-Whitney rank-sum test, p = 0.591). n.s., not significant.(C) Protein lysates obtained after RNAi were immunoblotted with an antibody specific to each protein of interest. GAPDH was used as loading control.See also Video S1.

Fig 5: miR-493-3p levels negatively associate with Mad2 gene expression in ovarian cancer cell linesQuantification of A. miR-493-3p expression, B. Mad2 mRNA and C. Mad2 protein in OVCAR-8 and CAOV-3 cell lines. A representative Western blot is shown. D. Quantification of Mad2 mRNA and protein levels 48 h after the transfection of OVCAR-8 and CAOV-3 cells with miR-control or miR-493-3p. Representative Western blots are shown. E. Quantification of mitotic duration (NEBD-to-anaphase) in OVCAR-8 and CAOV-3 cells transfected with miR-control or miR-493-3p (n = 300 cells per group). F. Quantification of miR-493-3p and Mad2 protein levels 48 h after the transfection of OVCAR-8 and CAOV-3 cells with miR-control or anti-miR-493-3p. Representative Western blots are shown. All data is mean +/- s.d. from 3 independent experiments. The asterisks denote statistical significance (* = p = 0.05, ** = p = 0.01, *** = p = 0.001).