CLASP2 C-term, but not self-association, is required and sufficient for KT localization. (A) Schematic representation of the RNAi-resistant mRFP-CLASP2γ constructs. Mutations in TOG2 and TOG3 domains and SxIP motifs are indicated. (B) Localization of the different CLASP2 constructs at unattached KTs (+Nocodazole) upon endogenous CLASP depletion by RNAi. U2OS cells were immunostained with ACAs (green), and mRFP signal (red) was acquired directly. Magnified images of mRFP and ACA colocalization are shown. Scale bar is 5 µm. Scale bar in inset is 0.5 µm. (C) Quantification of KT fluorescence intensity (F.I.) of mRFP/ACA signals for each cell line. Each data point represents an individual KT. Bars represent mean and SD. Quantifications from a pool of three independent experiments. WT = 26 cells, 260 KTs; 2ea-3eeaa = 27 cells, 267 KTs; IP12 = 27 cells, 269 KTs; 2ea-IP12 = 10 cells, 101 KTs; IP12-3eeaa = 26 cells, 253 KTs; ΔC = 26 cells, 253 KTs; ΔC-Gcn4 = 18 cells, 196 KTs; ΔC-Spc25 = 10 cells, 100 KTs; C-term = 10 cells, 100 KTs. ****, P < 0.0001; t test.