Augmin interacts both with γ-TuRC and MTs via complementary subcomplexes. (A) 150 nM augmin complexes were mixed with GMPCPP-MT seeds and visualized by TIRF microscopy. Augmin is displayed in green and MTs in red. Bar, 10 µm. (B) In vitro pull-down assays were performed using purified γ-TuRC and four augmin complexes and analyzed by Western blots. Because the GFP-tag is common among the four complexes, anti-GFP antibody (αGFP) was used for pull-down assays. The results demonstrate that augmin holocomplex, octamer(H6ΔC), and T-III directly interact with γ-TuRC; however, T-II does not. Input is the 1:5 dilution of purified γ-TuRC sample used for pull-down assays. X denotes negative control in which no augmin is used as bait. (C) Negative-stain analysis of augmin. 2D class averages of augmin complexes generated by Relion-2. Two representative class averages for both holocomplex (holo) and T-III are shown. Bar, 10 nm. Each class average for the holo and T-III contains ∼1,000 and 2,000 raw particle images, respectively. The class averages appear fuzzy at the distal ends, suggesting some level of flexibility for the complex. (D) Representative images of augmin holocomplex on GMPCPP-MTs. Bar, 20 nm. (E) A model for augmin-mediated localization of γ-TuRC to MT for branching MT nucleation.