Figure 3.
Reconstitution of branching MT nucleation using overexpressed recombinant augmin–γ-TuRC complex. (A) TIRF microscopy images and kymographs of MTs (red) grown in the presence of recombinant augmin-GFP (green) at indicated concentrations. Yellow arrows track one augmin-GFP punctum, the intensity of which progressively increases over time. (B–D) (Ba) Fluorescence intensity profiles along the MT (denoted by the horizontal dashed line in A) for indicated concentrations of augmin-GFP (green) and tubulin channel (red), showing stronger augmin-GFP intensity on older GDP-MT lattice. The origin of the x-axis represents the plus end of MT. Intensity profiles obtained from 20 different MTs were averaged and plotted in C, and the values were normalized to the maximum intensity of 20 nM augmin. (Bb) Mean intensity of indicated concentrations of augmin-GFP (green) and tubulin channel (red) along a 2-μm-long segment of GDP-MT (between two vertical dashed lines in A) over time, showing time-dependent accumulation of augmin-GFP. The mean intensity of indicated concentrations of augmin-GFP over time from 20 different MTs was averaged and plotted in D, and the values were normalized to the maximum intensity of 20 nM augmin. (E) Quantification of intensities of indicated concentrations of augmin-GFP along GDP lattice after 10 min of GDP-MT growth from seed. The values were normalized to the intensity of 20 nM augmin. n = 60 MTs from three experiments. (F) TIRF microscopy images and kymographs showing nucleation of an MT (red) from a single γ-TuRC-GFP (green) dot (arrows). (G) TIRF microscopy images of MTs (red) grown in the presence of 10 nM augmin-mCherry (blue; upper panels), 10 nM γ-TuRC-GFP (green; middle panels), or their combination (lower panels). Recombinant augmin-mCherry and γ-TuRC-GFP were separately purified from HEK293T cells. (H) TIRF microscopy images of branching MT nucleation in the presence of recombinant complete augmin–γ-TuRC complex (10 nM augmin; 0.62 nM γ-TuRC) copurified from nocodazole-arrested mitotic HEK293T cells expressing augmin-mCherry-Strep and γ-TuRC-GFP. Circled numbers indicate primary and secondary branches. (I) Western blot analysis of the separately purified recombinant augmin-mCherry and γ-TuRC-GFP as well as the copurified complete augmin–mCherry–γ-TuRC–GFP complex with indicated antibodies. The red asterisk indicates the up-shifted band of NEDD1. Horizontal scale bar, 2 μm; vertical scale bar, 2 min. Data represent mean ± SD.

Reconstitution of branching MT nucleation using overexpressed recombinant augmin–γ-TuRC complex. (A) TIRF microscopy images and kymographs of MTs (red) grown in the presence of recombinant augmin-GFP (green) at indicated concentrations. Yellow arrows track one augmin-GFP punctum, the intensity of which progressively increases over time. (B–D) (Ba) Fluorescence intensity profiles along the MT (denoted by the horizontal dashed line in A) for indicated concentrations of augmin-GFP (green) and tubulin channel (red), showing stronger augmin-GFP intensity on older GDP-MT lattice. The origin of the x-axis represents the plus end of MT. Intensity profiles obtained from 20 different MTs were averaged and plotted in C, and the values were normalized to the maximum intensity of 20 nM augmin. (Bb) Mean intensity of indicated concentrations of augmin-GFP (green) and tubulin channel (red) along a 2-μm-long segment of GDP-MT (between two vertical dashed lines in A) over time, showing time-dependent accumulation of augmin-GFP. The mean intensity of indicated concentrations of augmin-GFP over time from 20 different MTs was averaged and plotted in D, and the values were normalized to the maximum intensity of 20 nM augmin. (E) Quantification of intensities of indicated concentrations of augmin-GFP along GDP lattice after 10 min of GDP-MT growth from seed. The values were normalized to the intensity of 20 nM augmin. n = 60 MTs from three experiments. (F) TIRF microscopy images and kymographs showing nucleation of an MT (red) from a single γ-TuRC-GFP (green) dot (arrows). (G) TIRF microscopy images of MTs (red) grown in the presence of 10 nM augmin-mCherry (blue; upper panels), 10 nM γ-TuRC-GFP (green; middle panels), or their combination (lower panels). Recombinant augmin-mCherry and γ-TuRC-GFP were separately purified from HEK293T cells. (H) TIRF microscopy images of branching MT nucleation in the presence of recombinant complete augmin–γ-TuRC complex (10 nM augmin; 0.62 nM γ-TuRC) copurified from nocodazole-arrested mitotic HEK293T cells expressing augmin-mCherry-Strep and γ-TuRC-GFP. Circled numbers indicate primary and secondary branches. (I) Western blot analysis of the separately purified recombinant augmin-mCherry and γ-TuRC-GFP as well as the copurified complete augmin–mCherry–γ-TuRC–GFP complex with indicated antibodies. The red asterisk indicates the up-shifted band of NEDD1. Horizontal scale bar, 2 μm; vertical scale bar, 2 min. Data represent mean ± SD.

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