Figure 1
NuSAP stabilises kinetochore microtubules during metaphase.
(A) Representative images of spindle microtubule signal loss in mCherry-α-tubulin stable metaphase HeLa cells and HeLa cells expressing GFP-NuSAP, GFP-NuSAP1–233, or GFP-NuSAP233–441 analysed by FLIP assay. Scale bar, 5 μm. (B) Normalised signal-decreasing curves of mCherry-α-tubulin signal intensity at the metaphase spindle region in FLIP assays. Dotted grey lines represent each individual measurement and black lines represent the mean value of each group. Turnover half-life was calculated by linear regression analysis. Data were collected from three independent experiments and “n” indicates the total number of mitotic spindles analysed. Error bars represent ± SD. *p < 0.001. (C) Kinetochore microtubules in cold-treated metaphase HeLa cells expressing GFP-NuSAP, GFP-NuSAP1–233, GFP-NuSAP233–441, or GFP vector (control). Cells were stained with anti-α-tubulin antibody and DNA labelled with Hoechst 333342. Scale bar, 5 μm. Arrows indicate kinetochore bundles. (D) Bar chart representing average α-tubulin immunofluorescence intensity on metaphase spindles stained as C in cells expressing GFP-NuSAP, GFP-NuSAP1–233, GFP-NuSAP233–441 and GFP vector only (control). Data were collected across three independent experiments. The number of cells quantified was 37, 36, 35 and 39 for GFP, GFP-NuSAP, GFP-NuSAP1–233 and GFP-NuSAP233–441, respectively. Error bars represent ± SD. *p < 0.001. (E) Purified microtubules (1.5 μM) were incubated either alone or with 1 μM recombinant NuSAP for 10 min and fixed for electron microscopy. In the presence of NuSAP, microtubule bundles were detectable. (F) GMPCPP microtubules (2 μM) were incubated either alone or with 1 μM NuSAP for 10 min. Scale bar, 20 μm. (G) Different concentration of NuSAP was incubated with 20 μM tubulin for 10 min. Scale bar, 20 μm.
