a, Spindles of untreated (Control), MCAK-deficient (MCAK RNAi), and Kif2b-deficient (Kif2b RNAi) U2OS cells in the presence of monastrol (top panels) or after recovery from monastrol treatment (bottom panels). b, Number of cells with bipolar spindles that failed to align chromosomes with no treatment (blue) or following recovery from monastrol treatment (red). c, Spindles of untreated (Control), MCAK-deficient (MCAK RNAi), and Kif2b-deficient (Kif2b RNAi) cells following recovery from monastrol treatment in the presence of hesperadin (top panels) and then followed by recovery from hesperadin treatment (bottom panels). d, Number of bipolar cells that failed to align chromosomes after monastrol washout in the presence of hesperadin (blue) and then after recovery from hesperadin treatment (red). Bars in b and d represent mean ± s.e.m, n = 600, 3 experiments cells for b, 100 cells, 2 experiments for d. Scale bars, 5 μm. *, p < 0.0002, t-test.

a, Differential interference contrast (D.I.C.) and time-lapse fluorescent images of prometaphase spindles in untreated (Control) and Kif2b-deficient (Kif2b RNAi) U2OS cells before (Pre-PA) and at the indicated times (sec) after activation (Post-PA) of GFP-tubulin fluorescence. Scale bar, 5 μm. b, Normalized fluorescence intensity over time after photoactivation of spindles in untreated (filled squares), MCAK-deficient (filled circles), and Kif2b-deficient (white squares) prometaphase and metaphase cells. Datapoints represent mean ± s.e.m, n = 5 to 11 cells. c, Calculated kMT half-life under different conditions. Error bars represent S.E. from the regression analysis in b.

a, Anaphase spindles of both untreated and Kif2b-deficient U2OS cells containing CenpB-GFP to indicate kinetochores and stained for microtubules and DNA. White arrow highlights merotelic kinetochores. For clarity, a cell with only 2 lagging chromosomes was selected for this panel. b, Anaphase and pre-anaphase cells of both untreated and Kif2b-deficient RPE1 cells following recovery from nocodazole treatment stained for kinetochores with CREST, cyclin B, and DNA. c, d, Number of anaphase U2OS cells with lagging kinetochores (c) and the average number of laggards per anaphase (d) in untreated cells (blue) or cells following recovery from monastrol (green) or nocodazole (red) treatment. Bars represent mean ± s.e.m, n = 150 cells, 3 experiments. Scale bars, 5 μm.

Monopolar spindles induced with monastrol showing microtubules (red), GFP-Kif2b (green), and DNA (blue) in the presence of hesperadin and following removal of hesperadin. Scale bar, 5 μm.

a, b, Percent of anaphase cells with lagging kinetochores and average numbers of lagging chromosomes in anaphase of U2OS cells from representative experiments. Clone 5 and clone 8 are from Table 1. Bars represent mean ± s.e.m, n = 150 cells, 3 experiments (a) and bars represent values from 100 cells, 2 experiements (b), *, p < 0.05, t-test. c, Examples of a proper chromosome segregation in U2OS cells expressing GFP-MCAK (left panel) and mis-segregation event in U2OS cells expressing GFP-Kif2a (right panel) using FISH with probes for chromosomes 2 (green) and 3 (red) and DNA stained with DAPI (blue). Scale bars, 10 μm. d, Mis-segregation rates per chromosome per mitosis in untreated U2OS cells and U2OS cells overexpressing GFP-Kif2a, GFP-Kif2b, GFP-MCAK, or GFP-tubulin as indicated. Clone 5 and clone 8 are from Table 1. n > 660 cells. *, p < 0.05, Chi-square test. e, Model for temporal regulation of kMT dynamics. At low inter-kinetochore tension during prometaphase, the Aurora B kinase activity gradient recruits Kif2b to kinetochores and inhibits centromeric MCAK. The tension generated upon biorientation causes sister kinetochores to exceed the boundaries of the Aurora B kinase activity gradient releasing Kif2b from kinetochores and activating a subset of MCAK in the outer centromere. Kinetochores are shown in grey and microtubules are shown in green.