Spatially distinct residues in TACC3 dictate mitotic duration and fidelity.

<p>(A) Spindle length measured in 3D using Volocity. Number of cells analysed is indicated in graph (n). Student t-test (**** P < 0.0001). (B) Mitotic spindle morphologies (as described in main text) observed during time-lapse microscopy of GFP-tubulin-expressing TACC3 mutant cell lines. Representative still frames are shown on left. Scale bar = 5 μm. (C) Durations of NEBD to anaphase onset obtained from time-lapse microscopy performed on GFP-tubulin-expressing TACC3 mutant cell lines. F543A_1 and F543A_2 cells represent independently derived clones. Note that we observed no correlation between GFP levels and mitotic timing, or between mitotic timing and time of NEBD with respect to duration of filming in these experiments. Box plot shows 10–90 percentiles for each genotype. Number of cells analysed is indicated in graph (n). Mann Whitney nonparametric t-test (**** P < 0.0001, *** P < 0.001 and n.s. stands for ‘no significance’). (D) Durations of NEBD to anaphase onset obtained from time-lapse microscopy performed on EB3-GFP-expressing cells. Box plot shows 10–90 percentiles. Number of cells analysed is indicated in the graph (n). Mann Whitney nonparametric t-test. (**** P < 0.0001). (E) Analysis of chromosome segregation. Top table shows percentage of lagging chromatids seen during anaphase in fixed cells. Bottom table shows results from metaphase chromosome (chr) spreads. Number of autosomes 1, 2, 3, 4 and the sex chromosome, Z was analysed in cells. Frequencies of loss or gain of a single copy of individual chromosomes are indicated. Note that WT DT40 cells are trisomic for chromosome 2, whilst all the TACC3 mutants are diploid. Therefore, cells with two or three copies of chromosome 2’s are marked with ‘*’ or ‘#’, respectively. (F) Plot depicts time between NEBD and bipolar spindle appearance in time-lapse microscopy from Fig 7B. Cells with multipolar spindles and cells in which one of the two spindle poles was outside of the Z stack range for these time points were excluded from analysis. Number of cells analysed is indicated in graph (n). Mann Whitney nonparametric t-test (* P = 0.04, **** P < 0.0001).