Characterization of the contribution of NEDD1 WD to γ-TuRC’s nucleation activity and the synergistic MT-binding activities between NEDD1 and augmin. (A) Coomassie blue–stained gel with purified γ-TuRC^{StN/CDK1/PLK1}-GFP and γ-TuRC^{ΔWD/StN/CDK1/PLK1}-GFP. (B) TIRF microscopy images of de novo nucleation of MTs from solution in the presence of γ-TuRC^{StN/CDK1/PLK1}-GFP (FL) or γ-TuRC^{ΔWD/StN/CDK1/PLK1}-GFP (ΔWD) at indicated concentrations. (C) Quantification of intensities of γ-TuRC^{StN/CDK1/PLK1}-GFP (FL) and γ-TuRC^{ΔWD/StN/CDK1/PLK1}-GFP (ΔWD) along MTs for experiments shown in B. The values were normalized to the intensity of 50 nM γ-TuRC^{StN/CDK1/PLK1}-GFP. n = 60 MTs from three experiments. (D) Quantification of the total MT number per 1,000 μm² for experiments shown in B. n = 3 experiments. (E) TIRF microscopy images of MTs (red) grown in the presence of 10 nM augmin^CDK1/PLK1-SNAP (blue), 10 nM NEDD1^CDK1/PLK1-GFP (green), 10 nM NEDD1 ΔWD^CDK1/PLK1-GFP (green), either alone or combined in pairs. (F and G) Quantification of intensities of augmin^CDK1/PLK1-SNAP (F) as well as NEDD1^CDK1/PLK1-GFP and NEDD1 ΔWD^CDK1/PLK1-GFP (G) along MTs for experiments shown in E. The values were normalized to the intensities of the augmin^CDK1/PLK1-SNAP and NEDD1^CDK1/PLK1-GFP pair. N.D., not detected. n = 50 MTs from three experiments. (H) Schematic diagram showing the MT-binding activity of different versions of augmin (augmin^CDK1/PLK1 and augmin^RO/BI) and NEDD1 (NEDD1^CDK1/PLK1 and NEDD1^RO/BI), either alone or combined in pairs. Scale bars, 2 μm. Data represent mean ± SD. ***, P < 0.001, two-tailed t test.