Figure 1.
NuSAP regulates spindle length control and microtubule flux. (A) Control and NuSAP-knockout HeLa cells were stained for γ-tubulin (green), NuSAP (red), and DNA (blue). (B) NuSAP knockout efficiency of HeLa cells was analyzed by Western blotting. (C) Spindle length (indicated by white lines in A) was quantified by γ-tubulin immunofluorescence in control or NuSAP-knockout metaphase cells. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.0006. (D) HeLa cells expressing GFP or GFP-NuSAP were stained for γ-tubulin (red), α-tubulin (magenta), and DNA (blue). (E) Spindle length (indicated by white lines) was measured by γ-tubulin immunofluorescence in metaphase cells. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.0006. (F and G) NuSAP-knockout HeLa cells were transfected with photoactivatable GFP-tagged α-tubulin (PAGFP-α-tubulin, green). Microtubules were stained with SiR-tubulin (red), and the concentration of SiR-tubulin is 0.04 μM, which does not affect normal mitotic progression. 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. (H and I) Corresponding kymograph profiles of the photoactivated regions in F and G (red dotted lines highlight microtubule-flux slopes). Scale bars, 10 s. (J and K) The fluorescence intensity profiles at time points 0 and 120 s in F and G are shown, respectively. (L) The mean velocity was defined by the ratio of the distance that the PAGFP signal travels to 2 min it takes. ΔDistance: the distance between the two points with the strongest PAGFP fluorescence intensity at 0 and 120 s. ΔTime: 2 min. (M) The rates of microtubule flux shown in F and G were measured in control or NuSAP-knockout cells. Error bars indicate SD. Three independent replicates of 14 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P < 0.0001. ***, P < 0.001; ****, P < 0.0001. n = the number of independent experiments presented. Scale bars, 10 μm. Source data are available for this figure: SourceData F1.

NuSAP regulates spindle length control and microtubule flux. (A) Control and NuSAP-knockout HeLa cells were stained for γ-tubulin (green), NuSAP (red), and DNA (blue). (B) NuSAP knockout efficiency of HeLa cells was analyzed by Western blotting. (C) Spindle length (indicated by white lines in A) was quantified by γ-tubulin immunofluorescence in control or NuSAP-knockout metaphase cells. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.0006. (D) HeLa cells expressing GFP or GFP-NuSAP were stained for γ-tubulin (red), α-tubulin (magenta), and DNA (blue). (E) Spindle length (indicated by white lines) was measured by γ-tubulin immunofluorescence in metaphase cells. Error bars indicate SD. Three independent replicates of 30 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P = 0.0006. (F and G) NuSAP-knockout HeLa cells were transfected with photoactivatable GFP-tagged α-tubulin (PAGFP-α-tubulin, green). Microtubules were stained with SiR-tubulin (red), and the concentration of SiR-tubulin is 0.04 μM, which does not affect normal mitotic progression. 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. (H and I) Corresponding kymograph profiles of the photoactivated regions in F and G (red dotted lines highlight microtubule-flux slopes). Scale bars, 10 s. (J and K) The fluorescence intensity profiles at time points 0 and 120 s in F and G are shown, respectively. (L) The mean velocity was defined by the ratio of the distance that the PAGFP signal travels to 2 min it takes. ΔDistance: the distance between the two points with the strongest PAGFP fluorescence intensity at 0 and 120 s. ΔTime: 2 min. (M) The rates of microtubule flux shown in F and G were measured in control or NuSAP-knockout cells. Error bars indicate SD. Three independent replicates of 14 cells per replicate were quantified (n = 3). Unpaired two-tailed t test: P < 0.0001. ***, P < 0.001; ****, P < 0.0001. n = the number of independent experiments presented. Scale bars, 10 μm. Source data are available for this figure: SourceData F1.

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