Fig. 3

TACC3 S558A mutation affects recruitment of γ-TuRC proteins to the centrosomes. a. The polymerization of αβ-tubulin (15 μM) in the presence of recombinant TACC3 (500–838) WT (red line), TACC3 (500–838) S558A (blue line), and TACC3 (500–838) S558D (pink line) was measured by turbidity assay. TACC3 proteins used were 15 μM, each. The curves represent the amount of turbidity resulting from microtubule polymerization under each condition. Polymerization curve of tubulin by 10% DMSO as polymerization inducer is also shown (black line). b. Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (S558A)-TACC3 shRNA (middle panel) or GFP-TACC3 (S558D)-TACC3 shRNA (bottom panel)-transfected (48 h) mitotic metaphase synchronized HeLa Kyoto cells showing the localization of γ-tubulin. Enlarged images of γ-tubulin localization are shown in insets. Scale bar, 5 μm. γ-tubulin (red) was stained with rabbit polyclonal anti-γ-tubulin antibody. c. Quantification of intensities (sum of both poles) of γ-tubulin fluorescence at the centrosomes/spindle poles in GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (S558A)-TACC3 shRNA, and GFP-TACC3 (S558D)-TACC3 shRNA transfected (48 h) metaphase cells. The bars represent mean +/− S.E. (three independent experiments). Statistical analysis between WT and S558A mutant data is shown. d. Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (S558A)-TACC3 shRNA (middle panel) or GFP-TACC3 (S558D)-TACC3 shRNA (bottom panel) transfected (48 h) mitotic metaphase synchronized HeLa Kyoto cells showing the localization of GCP6. Enlarged images of centrosomal GCP localization are shown in insets. Scale bar, 5 μm. GCP6 (red) was stained with rabbit polyclonal anti-GCP6 antibody. e. Quantification of intensities (sum of both poles) of GCP6 fluorescence at the centrosomes/spindle poles in GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (S558A)-TACC3 shRNA, and GFP-TACC3 (S558D)-TACC3 shRNA transfected (48 h) metaphase cells. The bars represent mean +/− S.E. (three independent experiments). n refers to number of cells analyzed