XCTK2 forms a gradient of association with importin α/β from the poles to the chromatin. (A) Representative confocal fluorescence (CyPet, YPet, and MTs) and lifetime (FLIM) images of importin α-CyPet + nontagged XCTK2 (importin α-CyPet), importin α-CyPet + YPet-XCTK2-ΔNLS, and importin α-CyPet + YPet-XCTK2 spindle assembly reactions. Scale bar: 10 µm. (B) YPet fluorescence line scans of spindles assembled in A of importin α-CyPet with YPet-XCTK2-ΔNLS or YPet-XCTK2 normalized to percentage of spindle length (25 bins). YPet fluorescence percentage spindle length is graphed as the mean ± SEM (n = 30 YPet-XCTK2-ΔNLS + importin α-CyPet and 58 YPet-XCTK2 + importin α-CyPet spindles from three independent experiments). (C) Lifetime line scans of spindles imaged in A and B, where lifetimes are represented as the amplitude averaged lifetime (τAV/AMP), and lifetimes per normalized percentage spindle length are graphed as the mean ± SEM (n = 38 importin α-CyPet + XCTK2 spindles, 30 YPet-XCTK2-ΔNLS + importin α-CyPet, and 58 YPet-XCTK2 + importin α-CyPet spindles). (D) Line scans of the lifetimes from spindles with YPet-XCTK2 + importin α-CyPet relative to the fluorescence of the spindle MTs and the YPet fluorescence plotted as in B and C. (E) Chromatin and pole lifetime differences for each spindle analyzed in C with the mean ± SD indicated (D’Agostino and Pearson normality test and Brown–Forsythe’s and Welch’s with Dunnett’s multiple comparisons test: ****, P < 0.0001).
