Figure 2
NuSAP binds with MCAK and modulates the dynamics of MCAK.
(A) NuSAP immunoprecipitates contain MCAK and importin-β. FLAG and FLAG-NuSAP immunoprecipitates from HEK 293T cell lysates were analysed by Coomassie blue staining. The indicated protein bands were identified by mass spectrometry and confirmed by western blotting using anti-MCAK, anti-importin-β and anti-NuSAP antibodies. (B) Identification of the MCAK-binding domain on NuSAP. Immunoprecipitated proteins were detected with the use of anti-HA and anti-FLAG antibodies. Blotting of proteins in whole cell lysates (WCL) are also shown as controls. (C) Representative images and kymographs representing MCAK dynamics at the kinetochore region in metaphase HeLa cells expressing mCherry-vector (control), mCherry-NuSAP, mCherry-NuSAP1–233, mCherry-NuSAP233–441, or mCherry-NuSAPdelMCBD. Yellow squares represent the 1 × 1-μm photobleaching region. Kymographs were generated from the photobleaching kinetochore region. Scale bar, 5 μm. (D) Normalised signal recovery curves of FRAP assays. Solid lines represent the fit values of each group and dots indicate the mean values. Turnover half-life was calculated using a single constrained exponential curve. Data were collected from three independent experiments and “n” indicates the number of mitotic spindles analysed. Error bars represent ± SD. *p < 0.001. (E) Representative images and kymographs of MCAK dynamics at kinetochore region in metaphase HeLa cells treated with DMSO or 10 μM Taxol for 1 hour. Scale bar, 5 μm. (F) Normalised signal recovery curves of FRAP assays. Error bars represent ± SD. *p < 0.001. (G) Representative images of spindle microtubule signal loss in MCAK siRNA transfected HeLa cells and HeLa cells expressing GFP-NuSAP with FLIP assays. Scale bar, 5 μm. (H) Normalised signal decreasing curves of mCherry-α-tubulin signal intensity at the metaphase spindle region in FLIP assays. Error bars represent ± SD. *p < 0.001.
