TTBK2 regulates cell migration via KIF2A. (A) Still images during wound healing of HeLa cells transfected with control or TTBK2 siRNA. TTBK2 depletion inhibited the migration of HeLa cells. (B) Migratory tracks of representative control, TTBK2-depleted, KIF2A-depleted, or TTBK2- and KIF2A-depleted cells. (C) Three parameters (migration distance, velocity, and directionality) were measured. For the control, TTBK2 depletion, and TTBK2/KIF2A double depletion, ≥150 cells from more than four independent experiments were analyzed. For KIF2A depletion or rescue with TTBK2-WT, ≥100 cells or ≥60 cells from three independent experiments were analyzed, respectively. ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (D) MT organization in migrating HeLa cells during wound healing. The boxes in the top panels are enlarged in the bottom panels. MT density at the cell front was measured as the mean intensity in the region indicated. TTBK2 depletion decreased MT intensity at the cell front. The decrease in MT intensity was reversed by co-depletion of KIF2A. Single-plane images focused on the cell periphery were used for the analysis. The fluorescence intensity was quantified in >20 cells for each condition and is shown as a ratio to the control cell value. The data represent the mean ± SD of three independent experiments (total of >60 cells for each condition). ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (E) The effects of TTBK2 depletion on the migration of cerebellar granule neurons in vivo. Granule neurons expressing control shRNA migrated toward the IGL through the ML, but neurons expressing TTBK2 shRNA exhibited only minimal migration. The graph shows the quantitative results. Error bars indicate the SEM. ***, P < 0.001 (one-way ANOVA). The data used for the statistical analysis were obtained from 12 slices of three brains (>300 cells). Bars: (A and B) 100 µm; (D) 10 µm; (D, magnification) 5 µm; (E) 50 µm.