Phosphorylation status of NuSAP by Aurora A regulates spindle microtubule flux. (A–C) NuSAP-knockout HeLa cells were transfected with photoactivatable GFP-tagged α-tubulin (PAGFP-α-tubulin, green) and mCherry-tagged NuSAP-WT, -S240A, or -240D (red) followed by live-cell imaging. GFP signal in a rectangular region near the microtubule plus ends was activated (time point 0, arrows) and tracked every 10 s. Representative time-course images are shown. Microtubules were stained with SiR-tubulin (magenta). (D–F) Corresponding kymograph profiles of the photoactivated regions in A–C (red dotted lines highlight microtubule-flux slopes). Scale bars, 10 s. (G–I) The fluorescence intensity profiles at time points 0 and 120 s in A–C are shown, respectively. (J) The rates of microtubule flux shown in A–C were measured (as described in the legend for Fig. 1 L). Error bars indicate SD. Three independent replicates of 13 or 14 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P < 0.0001 for GFP-NuSAP/GFP-S240A; P < 0.0001 for GFP-S240A/GFP-S240D; P = 0.2511 for GFP-NuSAP/GFP-S240D. (K) NuSAP-knockout HeLa cells were transfected with GFP-tagged NuSAP-WT, -S240A, or -S240D (green) for 48 h, followed by immunofluorescence labeling using anti-α-tubulin antibodies (red) and anti-γ-tubulin antibodies (magenta). (L) Quantification of spindle length in K. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.0002 for GFP-NuSAP/GFP-S240A; P = 0.0004 for GFP-S240A/GFP-S240D; P = 0.0550 for GFP-NuSAP/GFP-S240D. ns, not significant; ***, P < 0.001; ****, P < 0.0001. n = the number of independent experiments presented. Scale bars, 10 μm.