Phospho-null TPX2 inhibits MT flux on metaphase spindles. (A–D) U2OS cells were cotransfected with photoactivatable GFP-tagged α-tubulin (PAGFP-tubulin) and mCherry-TPX2, -2A, or -2D. GFP signal in a rectangular region near the MT plus ends was activated (time point 0, arrows) and tracked every 15 s. Representative time-course images are shown in A–C and fluorescence intensity profiles are plotted at the bottom. Bars, 5 µm. The x coordinate of the maximum intensity from each frame (≥ time 0) is shown in D. (E and F) The mean velocity of MT flux is defined by the ratio between distance relative to the equator and time. n in F: 18, 25, and 16. Error bars indicate SEM. ***, P < 0.001.