Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules

Abstract

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin's architecture and function, we purified augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs.

Biochemical reconstitution of branching microtubule nucleation

“…The GFP-labeled eight-subunit X. laevis augmin holocomplex was co-expressed in insect cells and 102 co-purified (Song et al, 2018), the native 2.2 MDa g-TuRC was purified from Xenopus egg extract 103 (Thawani et al, 2018), and GFP-TPX2 was expressed from E. coli and purified (King and Petry, 104 2019). 105…”

“…The eight-subunit 43 protein complex augmin is required for branching microtubule nucleation in plant, human and 44 Drosophila cells, and meiotic Xenopus egg extract, where its depletion leads to reduced spindle 45 microtubule density, less kinetochore fiber tension, metaphase arrest, and cytokinesis failure 46 (David et al, 2019;Decker et al, 2018;Goshima et al, 2008;Hayward et al, 2014;Ho et al, 47 2011; Kamasaki et al, 2013;Lawo et al, 2009;Nakaoka et al, 2012;Petry et al, 2011;Uehara 48 et al, 2009). Augmin is necessary to recruit g-TuRC to spindle microtubules (Goshima et al, 49 2007), and following the recombinant expression of augmin (Hsia et al, 2014), this activity was 50 confirmed using purified proteins (Song et al, 2018). In meiotic Xenopus egg extract, the Ran-51 regulated protein TPX2 is released near chromatin (Gruss et al, 2001), where it stimulates 52 branching microtubule nucleation (Petry et al, 2013), potentially by activating g-TuRC (Alfaro-53 Aco et al, 2017).…”

“…Microtubules are nucleated from specific locations in the cell, and several of these microtubule 31 nucleation pathways converge to form a particular cytoskeletal architecture (Kollman et al, 2011;32 Lin et al, 2015; Lüders and Stearns, 2007). Importantly, microtubules in cells are nucleated by the 33 microtubule nucleator g-TuRC (Kollman et al, 2011;Zheng et al, 1995) and its co-nucleation 34 factor XMAP215 (Thawani et al, 2018). At the same time, each microtubule nucleation pathway 35 requires a unique set of nucleation effectors to recruit and regulate g-TuRC at distinct cellular 36 locations (Lin et al, 2015).…”

“…This implies that TPX2 directly regulates augmin's binding to microtubules, but does not 174 rule out additional regulation that could encompass other factors such as EML3(Luo et al, 2019). 175Although microtubule nucleation effectors alone can generate microtubules in vitro 176(Roostalu et al, 2015; Woodruff et al, 2017), g-TuRC is required for physiological microtubule 177 nucleation(Kollman et al, 2011;Thawani et al, 2018), and this reconstitution highlights the 178 importance of including it when studying microtubule nucleation. Localizing g-TuRC to a specific 179 location, from which it nucleates a microtubule in vitro as it occurs in the cell, serves as a 180 pioneering example for the biochemical reconstitution of other microtubule nucleation pathways 181 or microtubule organizing centers.…”

Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation

“…It exponentially increases MT numbers while preserving their polarity and is critical for rapid and accurate spindle assembly (Decker et al, 2018; Kaye et al, 2018; Petry et al, 2013). Branching MT nucleation requires the universal MT nucleator module, consisting of the γ-Tubulin Ring Complex (γ-TuRC) (Oegema et al, 1999; Zheng et al, 1995) and its recently discovered co-factor XMAP215 (Flor-Parra et al, 2018; Gunzelmann et al, 2018; Thawani et al, 2018), as well as the protein complex augmin that directly recruits γ-TuRC along the length of a pre-existing MT (Song et al, 2018). Branching MT nucleation is initiated by the microtubule associated protein TPX2 (Petry et al, 2013), which has been proposed to activate γ-TuRC via TPX2’s C-terminal domain (Alfaro-Aco et al, 2017; Scrofani et al, 2015).…”