Loss of KIF4A decreases MT flux. (A) DIC and fluorescence images show representative control and KIF4A-depleted cells in late prometaphase/metaphase. Images were captured before (preconversion) and at various time points (indicated in minutes and seconds) after photo-conversion of mEos-α-tubulin. MT flux was measured by photo-conversion of regions from both half-spindles in U2OS-mEos-tubulin cells and photo-converted mEos-tubulin was tracked over a time interval of 3 min (arrows). Bottom panels of each merged frame show separated green and red channels. Bar, 5 µm. (B) Representative fluorescence intensity curves of control vs. KIF4A-depleted U2OS-mEos-tubulin cell created immediately after photo-conversion and 120 s later. Fluorescence intensity peaks are generated by manual estimation of the peak region, followed by fitting of the intensity data points to a parabola. The maximum of the parabola was used as the effective peak position. (C) Both KIF4A and KIF4A/hKID double-knockdown late prometaphase/metaphase U2OS-mEos-tubulin cells display significant reduction of kinetochore-MT poleward flux in comparison with controls (P < 0.001). Error bars represent SEM derived from three independent experiments each containing 11–26 cells.