The RanGTP gradient promotes global and local targeting of XCTK2 within the spindle. (A) Representative MT confocal and Rango-2 FLIM images of spindles assembled with XB control buffer, 10 µM Ran, or 20 µM Ran addition. Lifetimes are represented as the amplitude averaged lifetime (τAV/AMP). (B) Line scans from pole to pole of the spindle lifetime images for the indicated conditions normalized for percent spindle length (25 bins). Lifetimes per spindle length are graphed as the mean ± SEM (n = 57–78 spindles per condition from three to four independent experiments). (C) Lifetime difference between the pole and chromatin regions for each spindle in each condition from B (D’Agostino and Pearson normality test and one-way ANOVA with Tukey’s multiple comparisons test compared with XB control: *, P < 0.05). (D) Representative wide-field fluorescence microscopy images of spindle assembly reactions from parallel experiments with XB control buffer or with added Ran that were stained with α-XCTK2. (E) Spindles from D were analyzed using a Cell Profiler pipeline that measured the mean total XCTK2 spindle fluorescence based on α-XCTK2 staining in the indicated conditions and plotted with the mean ± SD (n = 314–564 spindles per condition from five independent experiments). (F) Line scans of bipolar spindles from D were performed, normalized for percentage spindle length (101 bins), and graphed as the mean ± SEM (n = 168–248 spindles per condition from four independent experiments). (G) XCTK2 peak pole fluorescence plotted as the average fluorescence from the two poles per spindle analyzed in F. (H) XCTK2 chromatin fluorescence plotted as the center spindle position for each spindle analyzed in F. In G and H, the mean ± SD is indicated. (E–H) D’Agostino and Pearson normality tests and Kruskal–Wallis with Dunn’s multiple comparisons tests compared with XB control were performed: *, P < 0.05; ****, P < 0.0001. Scale bars: 10 µm.