Overexpression of Flag-TUBA1B ^E77A in Ate1 ^+/+ cells phenocopies microtubule defects observed in Ate1 ^−/− cells. (A) mCherry-EB3 signal (top) and tracks color-coded by mean straight-line speed ranging from 0 to 0.4 μm/s (bottom) from movies of wild-type cells overexpressing Flag-TUBA1B^WT, Flag-TUBA1B^E77A, TUBB5^WT-Flag, or TUBB5^D74A-Flag and mCherry-EB3 (Videos 3 and 4). Scale bar = 20 μm. (B) Quantification of the EB3 comet velocity. Mean ± SEM, N = 3 (n = 12 cells per condition), Flag-TUBA1B^WT versus Flag-TUBA1B^E77A: **P = 0.003, two-tailed paired t test. Symbol colors indicate paired average measurements. The violin plot represents the distribution of average EB3 comet velocity for individual cells. (C) Immunofluorescence images of wild-type cells overexpressing Flag-TUBA1B^WT, Flag-TUBA1B^E77A, TUBB5^WT-Flag, or TUBB5^D74A-Flag treated with 0.5 μM nocodazole for 0 or 10 min, extracted, and stained for Flag (magenta), ⍺-tubulin (green), and DAPI (blue). Scale bar = 10 μm. (D) Quantification of the average microtubule-positive area per cell normalized to t = 0. Mean ± SEM, N = 3 (n = 124–197 cells per condition), 10 min Flag-TUBA1B^WT versus Flag-TUBA1B^E77A: **P = 0.004, two-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. The truncated violin plot represents the distribution of the average microtubule-positive area for individual cells.