MPS1 activity is required for mitotic elongation and cell division in acentrosomal cells.

(a) Immunofluorescence staining of control and acentriolar RPE-1 TP53-/- or U2OS cells. Acentriolar cells were created by treatment with 125 nM centrinone (CNONE) or by deletion of SASS6. DAPI is shown in blue, γ-tubulin in green, and centrin-3 in magenta. Insets are shows for cells in which centrosomes were present. (a’) Quantification of a. Graphed are means and S.E.M. Significance was determined through a Fisher’s exact test. n=100 cells per condition. (b) Live phase contrast imaging showing example mitosis in U2OS cells pre-treated with DMSO or centrinone (CNONE) for 10 days. Green bars below cells indicate the duration of nuclear envelope breakdown (NEBD) through the onset of anaphase, while magenta bars below cells indicate the duration of the completion of anaphase, telophase, and cytokinesis. (b’) Mitotic durations from NEBD through metaphase (green) and anaphase, telophase, and cytokinesis (magenta) as in (b). Time is in minutes. Points represent individual cells; boxplots represent mean and interquartile range. Significance was determined through Welch’s t-test. n20 cells per condition. (c) Live phase contrast imaging showing example mitosis in U2OS cells pre-treated with DMSO or centrinone (CNONE) for 10 days before being imaging in 1 μM CFI-402257. (c’) Quantification of mitotic duration in cells of indicated genotype or treatment in DMSO (grey) or CFI-402257 (blue). Time is in minutes. Points represent individual cells; boxplots represent mean and interquartile range. Significance was determined through Welch’s t-test. n25 cells per condition. (d) Quantification of daughter cell fate of cells of the given pre-treatment imaged in DMSO or CFI-402257 (1 μM). Shown are the percentages for each fate of the total mitotic observations. Significance was determined through a Fisher’s exact test. (e) Confocal timelapse imaging of U2OS cells pretreated for 10 d with 125 nm centrinone and imaged in 1 μM CFI-402257. Shown are endogenously tagged α-tubulin (GFP-TUBA1B) and DNA (Sir-Hoechst). (f) Quantification of daughter cells fates of cells of the given pre-treatment imaged in DMSO or CFI-402257 (1 μM) with concurrent treatment with proTAME (12 μM for RPE1 cells, 24 uM for U2OS cells). Shown are the percentages for each fate of the total mitotic observations. Significance was determined through a Fisher’s exact test. n40 cells per condition. In all cases, not significant (n.s.) denotes p>0.05, * denotes p<0.05, **denotes p<0.01, and *** denotes p<0.001. All scale bars: 10 μm.

Division failure induced by Mps1 inhibition in acentriolar cells is time-dependent.

(a) Example mitoses in RPE1 TP53-/- or RPE1 TP53-/-; SASS6-/- cells treated with 50nM AZD1152 with or without 1μM CFI-402257. (a’) Quantification of mitotic duration in cells of indicated genotype or treatment in DMSO (grey), 50nM AZD1152 (green), or 50nM AZD1152 and 1μM CFI-402257 (purple). Points represent individual cells; boxplots represent mean and interquartile range. Significance was determined through Welch’s t-test. n≥20 cells per condition. (a’’) Quantification of daughter number of cells of the given pre-treatment imaged in DMSO, 50nM AZD1152 or 50nM AZD1152 and 1μM CFI-402257. Shown are the percentages for each fate of the total mitotic observations. n≥20 cells per condition. Significance was determined through a Fisher’s exact test. (b) Example mitoses in RPE1 TP53-/- or RPE1 TP53-/-; SASS6-/- cells treated with 1μM CFI-402257 and proTAME (12 μM for RPE1 cells, 24 μM for U2OS cells). (b’) Quantification of mitotic duration in cells of indicated genotype or treatment in 1μM CFI-402257 (blue) or 1 μM CFI-402257 with 12 μM (RPE1 cells) or 24 μM (U2OS cells) proTAME. Points represent individual cells; boxplots represent mean and interquartile range. Significance was determined through Welch’s t-test. n≥20 cells per condition. (b’’) Quantification of daughter number of cells of the given pre-treatment imaged in 1μM CFI-402257 (blue) or 1 μM CFI-402257 with 12 μM (RPE1 cells) or 24 μM (U2OS cells) proTAME after a mitotic arrest of greater than six hours. Shown are the percentages for each fate of the total mitotic observations. n≥20 cells per condition. Significance was determined through a Fisher’s exact test. In all cases not significant (n.s.) denotes p>0.05 and *** denotes p<0.001. All scale bars: 10 μm.

Polyploidy is not sufficient to confer MPS1-inhibition-sensitive division failure.

(a) Metaphase spreads from RPE1 TP53-/- or RPE1 TP53-/-; SASS6-/- cells. Indicated beneath example images are the mean and standard errors of chromosome pairs for the two genotypes. Chromosomes were stained with DAPI. (b) Schematic of experimental setup. REP1 TP53-/-cells were treated with 50 μM blebbistatin for 12h, washed, and then treated with 1μM CFI-402257 and imaged live via phase microscopy. (b’) Example mitoses in mononucleate or binucleate cells treated with 1 μM CFI-402257 after washout (WO) of blebbistatin. (b’’) Quantification of mitotic outcome of RPE1 TP53-/- or U2OS cells imaged in DMSO, 5 μM STLC, 1 μM CFI-402257, or 5 μM STLC and 1 μM CFI-402257 together. Shown are the percentages for each fate of the total mitotic observations. n=40 cells per condition Significance was determined through a Fisher’s exact test. In all cases, not significant (n.s.) denotes p>0.05 and *** denotes p<0.001. All scale bars: 10 μm.

Division failure due to MPS1 inhibition occurs with monopolar spindles, but not polyploidy.

(a) Confocal timelapse imaging of U2OS cells pretreated for 10d with either DMSO or centrinone (125 nM). Shown are endogenously tagged α-tubulin (GFP-TUBA1B) and DNA (Sir-Hoechst). Time indicates minutes before (-) or after (+) NEBD. (a’) Proportion of cells with 2 foci of microtubules before NEBD based on experiments as in (a). (b) Live phase imaging of RPE1 TP53-/- or U2OS cells imaged in 1μM CFI-402257 or 5 μM STLC and 1μM CFI-402257 together. (b’) Quantification of mitotic outcome of RPE1 TP53-/- or U2OS cells imaged in DMSO, 1μM CFI-402257, 5 μM STLC, or 5 μM STLC and 1μM CFI-402257 together. Shown are the percentages for each fate of the total mitotic observations. Significance was determined through a Fisher’s exact test. n=50 cells per condition (c) Confocal timelapse imaging of U2OS cells imaged in 5μM STLC with 1μM CFI-402257. Shown are endogenously tagged α-tubulin (GFP-TUBA1B) and DNA (Sir-Hoechst). Asterisk indicated extruded chromosomes. Arrow indicated midbody-like α-tubulin structure. (d) Graphical summary of results. In all cases, not significant (n.s.) denotes p>0.05 and *** denotes p<0.001. All scale bars: 10 μm.