Figure 2.
Direct observation of augmin and γ-TuRC during the process of branching nucleation. (A) TIRF microscopy images of branching MT nucleation in the presence of native augmin–γ-TuRC complex (7.5 nM augmin; 0.15 nM γ-TuRC) purified from the nocodazole-arrested mitotic HAUS8-GFP-Strep/GCP2-SNAP double knock-in HeLa cells. Arrows denote the branching sites. Green and yellow circles indicate all visible augmin-GFP–positive puncta (green) and γ-TuRC-SNAP/AF647–positive puncta (yellow) along the MTs. White circles indicate the augmin-GFP and γ-TuRC-SNAP/AF647 double-positive puncta that initiate the branching nucleation. (B) Quantification of augmin-GFP–positive puncta (augmin+), γ-TuRC-SNAP/AF647–positive puncta (γ-TuRC+), double-positive puncta (+/+) and branching events for the experiments shown in A. Numbers in the circles were averaged from six experiments. The indicated ratios above circles represent mean ± SD. (C) Quantification of the total number of branched MTs per fan-like structure at indicated time points for the experiments shown in A. n = 10 experiments. (D) Schematic representation of indicated classes of branching events. Dashed lines in the roundtrip pattern represent the diffusion step of augmin+ γ-TuRC+ puncta (1b) and augmin+ puncta (2b). Blue arrows indicate the time point when γ-TuRC was captured by augmin (2a and 2b). (E) Quantification of indicated classes of branching events. 2: 2a + 2b. n = 8 experiments. (F–H) TIRF microscopy images and kymographs showing class 1a (F), class 1b (G), and class 2 (H) branching events. The red arrows indicate the puncta that eventually initiate branching nucleation. The yellow dashed line with two-direction arrowheads indicates the time point of augmin and γ-TuRC landing (in F and G) or augmin landing (in H). The red dashed line with two-direction arrowheads indicates the time point of branching nucleation. The inset shows an enlargement of the boxed area. (I and J) Distribution of fluorescence intensities for single molecules of purified GFP, EB3-GFP, and augmin-GFP (I) as well as SNAP, EB3-SNAP, and γ-TuRC-SNAP (J). The means of Gaussian distribution fitting are indicated. For all conditions, n = 5,000 molecules from three experiments. (K and L) The plot of the number of augmin-GFP (K) or GCP2-SNAP (L) molecules at the time point of landing and branching nucleation. The values were obtained by dividing the intensity of augmin-GFP (K) or γ-TuRC-SNAP (L) at branch sites by the fitted mean intensity of GFP (K) or SNAP (L), respectively, in parallel chambers. n = 20 branching events. Horizontal scale bar, 2 μm; vertical scale bar, 1 min. Data represent mean ± SD.

Direct observation of augmin and γ-TuRC during the process of branching nucleation. (A) TIRF microscopy images of branching MT nucleation in the presence of native augmin–γ-TuRC complex (7.5 nM augmin; 0.15 nM γ-TuRC) purified from the nocodazole-arrested mitotic HAUS8-GFP-Strep/GCP2-SNAP double knock-in HeLa cells. Arrows denote the branching sites. Green and yellow circles indicate all visible augmin-GFP–positive puncta (green) and γ-TuRC-SNAP/AF647–positive puncta (yellow) along the MTs. White circles indicate the augmin-GFP and γ-TuRC-SNAP/AF647 double-positive puncta that initiate the branching nucleation. (B) Quantification of augmin-GFP–positive puncta (augmin+), γ-TuRC-SNAP/AF647–positive puncta (γ-TuRC+), double-positive puncta (+/+) and branching events for the experiments shown in A. Numbers in the circles were averaged from six experiments. The indicated ratios above circles represent mean ± SD. (C) Quantification of the total number of branched MTs per fan-like structure at indicated time points for the experiments shown in A. n = 10 experiments. (D) Schematic representation of indicated classes of branching events. Dashed lines in the roundtrip pattern represent the diffusion step of augmin+ γ-TuRC+ puncta (1b) and augmin+ puncta (2b). Blue arrows indicate the time point when γ-TuRC was captured by augmin (2a and 2b). (E) Quantification of indicated classes of branching events. 2: 2a + 2b. n = 8 experiments. (F–H) TIRF microscopy images and kymographs showing class 1a (F), class 1b (G), and class 2 (H) branching events. The red arrows indicate the puncta that eventually initiate branching nucleation. The yellow dashed line with two-direction arrowheads indicates the time point of augmin and γ-TuRC landing (in F and G) or augmin landing (in H). The red dashed line with two-direction arrowheads indicates the time point of branching nucleation. The inset shows an enlargement of the boxed area. (I and J) Distribution of fluorescence intensities for single molecules of purified GFP, EB3-GFP, and augmin-GFP (I) as well as SNAP, EB3-SNAP, and γ-TuRC-SNAP (J). The means of Gaussian distribution fitting are indicated. For all conditions, n = 5,000 molecules from three experiments. (K and L) The plot of the number of augmin-GFP (K) or GCP2-SNAP (L) molecules at the time point of landing and branching nucleation. The values were obtained by dividing the intensity of augmin-GFP (K) or γ-TuRC-SNAP (L) at branch sites by the fitted mean intensity of GFP (K) or SNAP (L), respectively, in parallel chambers. n = 20 branching events. Horizontal scale bar, 2 μm; vertical scale bar, 1 min. Data represent mean ± SD.

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