TTBK2 phosphorylates KIF2A and removes it from MTs in an EB-dependent manner. (A) The distribution of KIF2A in HeLa cells transfected with scramble or TTBK2 siRNA. TTBK2 depletion increased KIF2A localization to MTs. (B) Rescue experiments for the distribution of KIF2A using resTTBK2-WT and its mutants. The expression of resTTBK2-WT canceled the extensive KIF2A binding to MTs, but its m1/2 and KN mutants did not. (C) Quantification of the colocalization between GFP-KIF2A and MTs. A correlation coefficient (Rr) was calculated for the region within 10 µm of the cell edge (see Calculation of the correlation coefficient in Materials and methods). TTBK2 depletion significantly increased the correlation of KIF2A with MTs. This increase in the correlation caused by TTBK2 depletion was partially rescued by resTTBK2-WT but not by resTTBK2-m1/2 or -KN mutants. Error bars indicate the SD (≥30 cells were analyzed for each condition). ***, P < 0.001 (one-way ANOVA, Tukey’s honest significant difference test [HSD]). (D) COS-7 cells were transfected with control HA or the indicated HA-TTBK2 mutants. The expression of TTBK2-WT increased the phosphorylation of KIF2A at S135, but that of TTBK2-m1/2 or -KN did not. The graph shows the relative band intensity (pS135-KIF2A/KIF2A) as the mean ± SD of three independent experiments. **, P < 0.01 versus control (one-way ANOVA, Tukey’s HSD). (E) Live imaging of HeLa cells expressing GFP-KIF2A and EB3-mRuby. The boxed regions were magnified and are shown as a time series. TTBK2 depletion altered the distribution of KIF2A. Arrowheads indicate the EB3-containing MT ends. The time is given in seconds. Also see Videos 1 and 2. (F) The intensity profiles of GFP-KIF2A and EB3-mRuby along growing MTs in living cells. Cells with mean GFP intensities of 6.0–8.5 A.U. were selected for analysis under both conditions. TTBK2 depletion increased the binding of KIF2A to MTs, especially at the EB3-containing MT ends. Error bars indicate the SEM (>50 MTs of five cells were analyzed for each condition). The horizontal axis was adjusted to 1.0 at the site of peak EB3 intensity. (G) The subcellular distribution of KIF2A-WT and its nonphosphorylatable mutant S135A. KIF2A-S135A extensively localized to the MTs in HeLa cells. (A, B, and G) The boxes in the left panels are enlarged in the right panels. (H) The effect of the SCA11-associated TTBK2 mutant on the distribution of KIF2A. The expression of the TTBK2 mutant increased the distribution of KIF2A along MTs. A.U., arbitrary unit. Bars: (A, B, and G; left) 10 µm; (A, B, and G magnified images) 5 µm; (E and H) 5 µm.