Figure 1. Reconstitution of branching MT... | Rockefeller University Press

Figure 1.

$Reconstitution of branching MT nucleation using purified native augmin–γ-TuRC complex. (A) StrepTactin pull-down assay with extracts of HAUS8-GFP-Strep knock-in HeLa cells without or with nocodazole treatment, analyzed by Western blotting with indicated antibodies. (B) TIRF microscopy images of branching MT nucleation both from GMPCPP seeds (denoted by arrows) and from solution (denoted by arrowhead) in the presence of native augmin–γ-TuRC complex (green; 10 nM augmin; 0.35 nM γ-TuRC) purified from the nocodazole-arrested mitotic HAUS8-GFP-Strep knock-in HeLa cells. (C) TIRF microscopy images of individual branching events occurred from a single GMPCPP seed. Circled numbers indicate primary, secondary, and tertiary branches. (D) Quantification of the total number of branched MTs per fan-like structure at indicated time points for the experiments shown in B. n = 20 experiments. (E) Schematic representation of an ordered series of branching events that occurred from a single GMPCPP seed. θ denotes the branch angle. (F) Quantification of the number of daughter, granddaughter, and great-granddaughter MTs per fan-like structure at the 25-min time point for the experiments shown in B. n = 20 experiments. (G) Distribution of branch angles. n = 300 branch angles from 10 experiments. (H) Distribution of fractional distance. Three types of fractional distance were obtained by dividing the corresponding distance as illustrated in E by the total length of the mother MT at the time of branching. From left to right, n = 232, 46, and 131 from 10 experiments. Scale bars, 2 μm. Data represent mean ± SD.$

Reconstitution of branching MT nucleation using purified native augmin–γ-TuRC complex. (A) StrepTactin pull-down assay with extracts of HAUS8-GFP-Strep knock-in HeLa cells without or with nocodazole treatment, analyzed by Western blotting with indicated antibodies. (B) TIRF microscopy images of branching MT nucleation both from GMPCPP seeds (denoted by arrows) and from solution (denoted by arrowhead) in the presence of native augmin–γ-TuRC complex (green; 10 nM augmin; 0.35 nM γ-TuRC) purified from the nocodazole-arrested mitotic HAUS8-GFP-Strep knock-in HeLa cells. (C) TIRF microscopy images of individual branching events occurred from a single GMPCPP seed. Circled numbers indicate primary, secondary, and tertiary branches. (D) Quantification of the total number of branched MTs per fan-like structure at indicated time points for the experiments shown in B. n = 20 experiments. (E) Schematic representation of an ordered series of branching events that occurred from a single GMPCPP seed. θ denotes the branch angle. (F) Quantification of the number of daughter, granddaughter, and great-granddaughter MTs per fan-like structure at the 25-min time point for the experiments shown in B. n = 20 experiments. (G) Distribution of branch angles. n = 300 branch angles from 10 experiments. (H) Distribution of fractional distance. Three types of fractional distance were obtained by dividing the corresponding distance as illustrated in E by the total length of the mother MT at the time of branching. From left to right, n = 232, 46, and 131 from 10 experiments. Scale bars, 2 μm. Data represent mean ± SD.