NEDD1 WD40 domain possesses MT-binding activity and is essential for branching nucleation. (A and B) TIRF microscopy images and quantification of NEDD1-GFP (green) binding at indicated concentrations to MTs (red). The values were normalized to the intensity of 200 nM NEDD1-GFP. n = 30 MTs from two experiments. (C and E) TIRF microscopy images and quantification of the binding of 100 nM C-terminally GFP-tagged NEDD1 or its indicated fragments (green) to MTs (red). The values were normalized to the intensity of full-length (FL) NEDD1-GFP. n = 30 MTs from two experiments. (D) Schematic overview of the domain organization of NEDD1 and the deletion mutants, and summary of their MT binding activity. (F) TIRF microscopy images of branching MT nucleation in the presence of 10 nM augmin^CDK1/PLK1-SNAP (blue) together with 10 nM γ-TuRC^{StN/CDK1/PLK1}-GFP or indicated concentrations of γ-TuRC^{ΔWD/StN/CDK1/PLK1}-GFP (green). Images were acquired 10 min after imaging. (G) Quantification of the total number of branched MTs per fan-like structure for the experiments shown in F. N.D., not detected. n = 3 experiments. (H and I) Quantification of intensities of augmin^CDK1/PLK1-SNAP (H) and γ-TuRC^{StN/CDK1/PLK1}-GFP or γ-TuRC^{ΔWD/StN/CDK1/PLK1}-GFP (I) along MTs for the experiments shown in F. The values were normalized to the intensity of the pair of 10 nM augmin^CDK1/PLK1 and 10 nM γ-TuRC^{StN/CDK1/PLK1}. n = 60 MTs from three experiments. Scale bars, 2 μm. Data represent mean ± SD.