Figure 3
NuSAP stabilises kinetochore microtubules through its regulation on MCAK.
(A) Representative kymographs and normalized signal recovery curves of photoactivation assays in metaphase HeLa cells expressing mCherry-vector (n = 12), mCherry-NuSAP (n = 12), mCherry-NuSAP1–233 (n = 14), mCherry-NuSAP233–441 (n = 13), or mCherry-NuSAPdelMCBD (n = 14). Solid lines represent the fit values of each group calculated by double exponential regression analysis, R2 > 0.99. (B) Bar chart representing the half-life of kinetochore microtubules calculated according to a double exponential regression curve. Error bars represent ± SD. (C) HeLa cells transfected with control siRNA or NuSAP siRNA together with GFP vector, GFP-NuSAP, or GFP-NuSAPdelMCBD were cold-treated with monastrol. Kinetochores were labelled with ACA, spindle poles with anti-γ-tubulin and DNA with Hoechst 333342. Scale bar, 5 μm. (D) Bar chart represents average kinetochore microtubule length in HeLa cells transfected with control siRNA or NuSAP siRNA together with GFP vector, GFP-NuSAP, or GFP-NuSAPdelMCBD following cold-treatment with monastrol. Error bars represent ± SD. *p < 0.001. (E) Different concentrations of NuSAP or NuSAPdelMCBD and 20 nM MCAK proteins were incubated with 1.5 μM tubulin at 37 °C for 10 min. Scale bar, 20 μm.
