Fig 2: Correcting erroneous kinetochore–microtubule attachments requires TAO1. (a) Schematic of assay to measure error-correction efficiency showing time-points for monastrol treatment and release prior to time-lapse imaging of HeLaHis2B-GFP;CherryTub cells treated with either TAO1-St1 or Control-St siRNA. (b) Time-lapse images of cells treated with siRNA, as indicated, undergoing anaphase following a release from monastrol treatment. Blue arrowhead marks lagging DNA and yellow arrowhead marks unaligned and mis-segregating chromosomes. Scale bar, 20 μm. (c) Cumulative distribution graph of anaphase onset time in Control or TAO1-depleted cells following a release from monastrol treatment, accrued from time-lapse movies as in (b). (d) Graph of mis-segregation severity quantifying the percentage of cells that exhibited normal segregation or mis-segregation with lagging DNA strands or lagging chromosomes or uncongressed chromosomes, assessed from time-lapse movies as in (b). n = number of cells analysed from three independent experiments. Error bars are s.e.m. values across three experimental repeats. p-Values representing significance level were derived using the proportion test. Asterisk (*) indicates significant difference.

Fig 3: TAO1-depleted cells display mis-segregating lagging chromosomes. (a–c) Time-lapse analysis of HeLaHis2B-GFP;CherryTub cells transfected with TAO1-St1 or Control-St siRNA. (a) Successive frames of fluorescent images from representative live-cell movies of HeLaH2BGFP;CherryTub cells treated with siRNA as indicated. Images were acquired once every 2 min. Uncongressed chromosomes are marked with blue arrowheads and mis-segregating lagging chromosomes are marked with yellow arrowheads. Scale bar, 20 μm. (b) Cumulative distribution graph showing the time consumed from NEBD to congression of chromosomes (alignment of last chromosome prior to anaphase) in cells treated with TAO1-St1 or Control-St siRNA. T50 represents chromosome congression time in 50% of mitotic cells. (c) Graph showing percentage of mitotic cells displaying normal, defective or no segregation within the first 3 h of mitosis. (d) Immunoblots of lysates of cells treated with siRNA as indicated showing the levels of Mad2, BubR1, Mad1, MPS1, TAO1, Aurora-B, Bub3 or γ-tubulin. γ-Tubulin is used as a loading control. (e) Graph showing percentage of mitotic cells displaying normal or uncongressed metaphase chromosomes and normal or mis-segregated anaphase chromosomes following a 1 h treatment with varying concentrations of 2ME2, as indicated. n = number of cells from three independent experiments. Error bars are s.e.m. values across three experimental repeats. p-Values representing significance level were derived using the proportion test. Asterisks (*) indicate significant difference.

Fig 4: Taxol or MPS1 inhibitor treatments in TAO1-depleted cells. (a) Graph of percentage of cells that underwent anaphase, within 6 h of mitosis onset (NEBD), in HeLaHis2B-GFP;CherryTub cells transfected with TAO1-St1 or Control-St siRNA and exposed to Taxol during imaging. n = number of cells that entered mitosis during imaging from three independent experiments. (b) Cumulative distribution graph of NEBD to anaphase onset times in Control-St or TAO1-St1 siRNA-treated HeLaHis2B-GFP;CherryTub cells that exited mitosis in the presence of Taxol. n = number of cells that underwent anaphase from two different experiments. (c) Time-lapse images of cells treated with siRNA, as indicated, in the presence or the absence of 200 nM MPS1 inhibitor. Yellow arrowhead marks lagging chromosomes in anaphase and blue arrowhead marks unaligned chromosomes. Scale bar, 40 μm. (d) Graph showing mitotic outcome and mis-segregation severity by quantifying the percentage of cells that exhibited normal segregation, no segregation or mis-segregation. Mis-segregating cells were binned as those displaying lagging chromosomes, uncongressed chromosomes at metaphase–anaphase transition or lagging DNA strands as assessed from time-lapse movies as in (c). n = number of cells analysed from three independent experiments. Error bars are s.e.m. values across three experimental repeats. p-Values representing significance level were derived using the proportion test. Asterisks (*) indicate significant difference.

Fig 5: TAO1-depleted cells display chromosome congression defects. (a) Evaluation of TAO1 depletion extent using Stealth siRNA oligos. Immunoblots of lysates of HeLa cells transfected with the indicated Stealth siRNA oligos for 72 h and then probed with anti-TAO1 or anti-β-tubulin antibodies as indicated. β-Tubulin was used as a loading control. (b) Graph showing percentage of mitotic cells displaying uncongressed chromosomes in cells treated with siRNA as indicated and then arrested in metaphase for 90 min using MG132 prior to immunostaining using α-tubulin antibody and CREST antisera and staining with a DNA dye, DAPI. A total of n < 150 cells from three experimental repeats shown in electronic supplementary material, figure S1. Average and s.e.m. values (error bars) were calculated from three independent repeats (two shown in electronic supplementary material, figure S1). p-Values calculated using the proportion test are indicated above the bars. (c) Images of metaphase cells, from cultures treated as in (b), showing uncongressed chromosomes (marked with yellow arrowheads). Scale bars, 5 μm.