Chromosome congression, timely SAC satisfaction, and chromosome segregation fidelity relies on both CLASP2 KT- and MT-binding properties. (A) Live-cell imaging of U2OS PA–GFP–α-tubulin cells (without and with CLASPs RNAi) and expressing the different mRFP-CLASP2γ (red) constructs. DNA (blue) and SiR-tubulin (magenta) are also depicted. NEB = time 00:00. The anaphase panel also shows the H2B-GFP signal alone to highlight the segregation errors. Time is in hours:minutes. Scale bar is 5 µm. (B) Quantification of NEB-to-anaphase onset duration. First column represents parental U2OS PA–GFP–α-tubulin cells. The remaining columns represent CLASPs RNAi. Each data point represents an individual cell. Bars represent mean and SD. Parental without RNAi = 11 cells, pool of three independent experiments; Parental with RNAi = 12 cells, pool of three independent experiments; WT = 28 cells, pool of six independent experiments; 2ea-3eeaa = 32 cells, pool of 14 independent experiments; IP12 = 33 cells, pool of 13 independent experiments; 2ea-IP12 = 39 cells, pool of 10 independent experiments; IP12-3eeaa = 19 cells, pool of seven independent experiments; ΔC = 23 cells, pool of nine independent experiments; ΔC-Gcn4 = 15 cells, pool of five independent experiments; ΔC-Spc25 = 41 cells, pool of eight independent experiments; C-term = 52 cells, pool of seven independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; t test. (C) Quantification of the NEB-to-metaphase duration for the same dataset as in B. (D) Quantification of chromosome segregation errors for the same dataset as in B and C. Discrimination of the rates for each error type is represented.