Figure S3.
Construction of pTT5Multi vector-based plasmids for coexpression of the 8 components of augmin and 11 components of γ-TuRC. (A) Schematic diagram of the empty pTT5Multi vector modified from the original pTT5 vector (see Materials and methods for detailed information). Note that SpeI, AvrII, and MluI are three important restriction sites for the cloning strategy. (B) Scheme for the construction of the pTT5Multi plasmid that contains multiple gene cassettes. First, Gene1 and Gene2 were individually cloned into the MCSs of the empty pTT5Multi vector, generating two plasmids, pTT5Multi-Gene1 and pTT5Multi-Gene2. Then, pTT5Multi-Gene1 was double digested with AvrII and MluI, while pTT5Multi-Gene2 was double digested with SpeI and MluI. After double digestion, as AvrII and SpeI were isocaudomers, the backbone containing Gene1 cassette was ligated with the fragment containing Gene2 cassette to give rise to the plasmid pTT5Multi-Gene1-Gene2. Since the plasmid pTT5Multi-Gene1-Gene2 still contains the SpeI, AvrII, and MluI sites, this process can be repeated to add more gene cassettes. (C) Schematic diagram of two pTT5Multi plasmids that contain indicated components of human augmin and three pTT5Multi plasmids that contain indicated components of human γ-TuRC. H, HAUS. Tags for augmin in this study: GFP-Strep, mCherry-Strep, SNAP-Strep, and GFP-Bio. Tags for γ-TuRC in this study: GFP and GFP-Strep. NEDD1 was highlighted with a dashed box because it was removed for experiments with γ-TuRCΔNEDD1. (D) Coomassie blue–stained gel with recombinant augmin-GFP complex purified from nocodazole-arrested mitotic HEK293T cells coexpressing pTT5Multi-H6-GFP-Strep/H2/H8/H3 and pTT5Multi-H7/H5/H4/H1. (E) Coomassie blue–stained gel with recombinant γ-TuRC-GFP complex purified from nocodazole-arrested mitotic HEK293T cells coexpressing pTT5Multi-GCP3-GFP-Strep/GCP2/γ-tubulin/γ-tubulin, pTT5Multi-GCP4/GCP6/GCP5, and pTT5Multi-MZT2A/MZT2B/MZT1/NME7/NEDD1. (F) TIRF microscopy images of de novo nucleation of MTs (red) from solution in the presence of recombinant γ-TuRC-GFP complex (green) at indicated concentrations. Images were acquired 10 min after imaging. (G) Quantification of the total MT number per 1,000 μm2 for experiments shown in F. n = 3 experiments. (H) Plots of the total MT number per 1,000 μm2 at indicated time points for experiments shown in F. n = 3 experiments. (I and J) Quantification of intensities of augmin-mCherry (I) and γ-TuRC-GFP (J) along MTs for the experiments shown in Fig. 3 G. The values were normalized to the intensity of augmin-mCherry alone (I) or γ-TuRC-GFP alone (J). n = 60 MTs from three experiments. (K) Coomassie blue–stained gel with recombinant complete augmin–γ-TuRC complex copurified from nocodazole-arrested mitotic HEK293T cells coexpressing augmin-mCherry-Strep and γ-TuRC-GFP. (L) TIRF microscopy images showing robust branching MT nucleation in the presence of recombinant complete augmin-mCherry-Strep-γ-TuRC-GFP complex (10 nM augmin; 0.62 nM γ-TuRC). (M) Quantification of the total number of branched MTs per fan-like structure at indicated time points for the experiments shown in L. n = 3 experiments. (N) Distribution of branch angles for the experiments shown in L. n = 300 branch angles from four experiments. (O) Coomassie blue–stained gels with recombinant complete augmin–γ-TuRC complexes copurified from nocodazole-arrested mitotic HEK293T cells expressing augmin-mCherry-Strep and γ-TuRC-GFP, together with or without untagged CDK1/cyclin B, HA-PLK1, or both. Scale bars, 2 μm. Data represent mean ± SD.

Construction of pTT5Multi vector-based plasmids for coexpression of the 8 components of augmin and 11 components of γ-TuRC. (A) Schematic diagram of the empty pTT5Multi vector modified from the original pTT5 vector (see Materials and methods for detailed information). Note that SpeI, AvrII, and MluI are three important restriction sites for the cloning strategy. (B) Scheme for the construction of the pTT5Multi plasmid that contains multiple gene cassettes. First, Gene1 and Gene2 were individually cloned into the MCSs of the empty pTT5Multi vector, generating two plasmids, pTT5Multi-Gene1 and pTT5Multi-Gene2. Then, pTT5Multi-Gene1 was double digested with AvrII and MluI, while pTT5Multi-Gene2 was double digested with SpeI and MluI. After double digestion, as AvrII and SpeI were isocaudomers, the backbone containing Gene1 cassette was ligated with the fragment containing Gene2 cassette to give rise to the plasmid pTT5Multi-Gene1-Gene2. Since the plasmid pTT5Multi-Gene1-Gene2 still contains the SpeI, AvrII, and MluI sites, this process can be repeated to add more gene cassettes. (C) Schematic diagram of two pTT5Multi plasmids that contain indicated components of human augmin and three pTT5Multi plasmids that contain indicated components of human γ-TuRC. H, HAUS. Tags for augmin in this study: GFP-Strep, mCherry-Strep, SNAP-Strep, and GFP-Bio. Tags for γ-TuRC in this study: GFP and GFP-Strep. NEDD1 was highlighted with a dashed box because it was removed for experiments with γ-TuRCΔNEDD1. (D) Coomassie blue–stained gel with recombinant augmin-GFP complex purified from nocodazole-arrested mitotic HEK293T cells coexpressing pTT5Multi-H6-GFP-Strep/H2/H8/H3 and pTT5Multi-H7/H5/H4/H1. (E) Coomassie blue–stained gel with recombinant γ-TuRC-GFP complex purified from nocodazole-arrested mitotic HEK293T cells coexpressing pTT5Multi-GCP3-GFP-Strep/GCP2/γ-tubulin/γ-tubulin, pTT5Multi-GCP4/GCP6/GCP5, and pTT5Multi-MZT2A/MZT2B/MZT1/NME7/NEDD1. (F) TIRF microscopy images of de novo nucleation of MTs (red) from solution in the presence of recombinant γ-TuRC-GFP complex (green) at indicated concentrations. Images were acquired 10 min after imaging. (G) Quantification of the total MT number per 1,000 μm2 for experiments shown in F. n = 3 experiments. (H) Plots of the total MT number per 1,000 μm2 at indicated time points for experiments shown in F. n = 3 experiments. (I and J) Quantification of intensities of augmin-mCherry (I) and γ-TuRC-GFP (J) along MTs for the experiments shown in Fig. 3 G. The values were normalized to the intensity of augmin-mCherry alone (I) or γ-TuRC-GFP alone (J). n = 60 MTs from three experiments. (K) Coomassie blue–stained gel with recombinant complete augmin–γ-TuRC complex copurified from nocodazole-arrested mitotic HEK293T cells coexpressing augmin-mCherry-Strep and γ-TuRC-GFP. (L) TIRF microscopy images showing robust branching MT nucleation in the presence of recombinant complete augmin-mCherry-Strep-γ-TuRC-GFP complex (10 nM augmin; 0.62 nM γ-TuRC). (M) Quantification of the total number of branched MTs per fan-like structure at indicated time points for the experiments shown in L. n = 3 experiments. (N) Distribution of branch angles for the experiments shown in L. n = 300 branch angles from four experiments. (O) Coomassie blue–stained gels with recombinant complete augmin–γ-TuRC complexes copurified from nocodazole-arrested mitotic HEK293T cells expressing augmin-mCherry-Strep and γ-TuRC-GFP, together with or without untagged CDK1/cyclin B, HA-PLK1, or both. Scale bars, 2 μm. Data represent mean ± SD.

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