Figure 7: Microtubule-binding activity of Cep57 contributes to checkpoint silencing.
From: Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1–Mad2
(a) Microtubule-binding assays in vitro. GST-Cep57 (195–500 amino acids; 0.1 μM) expressed in E. coli and Flag-Mad1 (0.05 μM) expressed in HEK293T cells were purified and incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. After centrifugation, supernatant (S) and pellet (P) were separated and used for Coomassie blue staining (top), and western blotting with anti-Flag antibody (bottom). (b) GST-Cep57 (195–500 amino acids; 0.1 μM)-coupled Glutathione Sepharose 4B beads were incubated with taxol-stabilized microtubules and purified Flag-Mad1 (0.05 μM) in BRB80 buffer at room temperature. The bead-bound proteins were analysed by western blotting with anti-Flag and anti-tubulin antibodies. GST-Cep57 (195–500 amino acids) was detected by Coomassie blue staining. (c) Microtubule-binding assays in vitro. Flag-Cep57 (0.05 μM) and Flag-Cep57-12A (0.05 μM) expressed in HEK293T cells and purified, and were incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. Samples were separated by centrifugation, and analysed by western blotting with anti-Flag antibody (top) and Coomassie blue staining (bottom). 12A: K432A, K434A, K435A, K438A, K441A, K442A, K467A, R469A, K473A, R474A, R475A and K476A. (d) Immunostaining of α-tubulin (red) in HeLa cells expressing Cep57-GFP or Cep57-12A-GFP. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. (e) Immunostaining of Flag-Cep57 (green) and Mad1 (red) in metaphase HeLa cells expressing RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A after transfection with Cep57-siRNA. DNA was stained with DAPI (blue). Scale bars, 5 μm. (f) Quantification of the percentage of metaphase cells with Mad1 signals at kinetochores from (e). Fifty cells were measured. (g) Quantification of the percentage of kinetochores with Mad1 signals in metaphase cells from (e). Greater than 100 kinetochores from 10 cells were measured. (h) Quantification of the percentage of metaphase cells in negative control (NC) or Cep57-depleted prometaphase and metaphase HeLa cells that expressed RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A. Mitotic stages were quantified by the morphology of DNA and spindles. Greater than 100 cells were measured. For f,g and h, the experiment was repeated three times. Data are mean±s.e.m. **P<0.01 (unpaired two-tailed Student’s t-test).
