EB binding is necessary for MCAK to concentrate on microtubule tips. (A) Live imaging of a HeLa cell cotransfected with EB3-RFP and GFP-wtMCAK. Black arrows indicate the position of the polymerizing microtubule tip at a time of 0 s and later (white arrows). (B) A live cell cotransfected with EB3-RFP and GFP-IPNN-MCAK. (C) EB interaction correlates with less microtubule polymer in fixed interphase cells transfected with GFP-wtMCAK versus GFP-IPNN-MCAK or GFP. Error bars correspond to the SEM of the cell pair means of the tubulin ratios between a transfected and untransfected cell in the same field. (D) Fixed HeLa cells transfected with RFP–CENP-B (to mark centromeres; red) and EGFP-wtMCAK or EGFP-IPNN (green) and stained for α-tubulin. EGFP-wtMCAK concentrates at centrosomes (asterisks), centromeres, the spindle midzone (arrows), and microtubule tips (boxes and magnified images). (E) EGFP-IPNN localizes to centrosomes (asterisks), centromeres, and the spindle midzone (arrows) but not at microtubule tips (shown by the boxes and magnified images). Bars: (A, B, D, and E) 10 µm; (D and E, insets) 9 µm.