Integrated Model of the Vertebrate Augmin Complex

Abstract

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which exponentially amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but understanding how augmin promotes branching has been hindered by a lack of structural information about the complex. Here, we report an integrated model of vertebrate augmin, combining cryo-electron microscopy, advanced protein structural prediction, and the visualization of fused bulky tags via negative stain electron microscopy. This strategy allowed us to identify the location and orientation of each subunit within the structure. Evolutionary analysis of augmin's structure reveals that it is highly conserved across diverse eukaryotes, and that augmin contains a previously-unidentified microtubule binding site. Moreover, we identify homology with the kinetochore-localized NDC80 complex. This new model of the augmin complex provides insight towards the mechanism and evolution of branching microtubule nucleation.

RanGTP regulates the augmin complex

“…Based on our binding experiments with the two tetrameric subcomplexes, we suspect that this second binding site must be located somewhere within T-II, since importins do not bind T-III. However, we could not determine the location of this second importin-α binding region due to the lack of obvious predicted NLS sequences and because the entwined structure of the remaining T-II subunits prevents any other augmin fragments from folding correctly and thus retaining function (Gabel et al, 2022; Travis, 2022; Zupa, 2022).…”

“…Later, characterization of augmin in vitro demonstrated that augmin has binds to the universal MT nucleator, γ-TuRC, at one end of the complex known as tetramer III (T-III), while the other end of the complex, tetramer II (T-II) recognizes and binds MTs (Hsia et al, 2014; Song et al, 2018) (Figure 1a). Within T-II, there are two MT binding sites (Hsia et al, 2014; Travis et al, 2022a; Wu et al, 2008). The primary MT binding site was localized to the augmin subunit Haus8, within its intrinsically-disordered N-terminus (Hsia et al, 2014; Wu et al, 2008), while a second, minor MT binding site was very recently located within the Haus6 subunit (Travis et al, 2022a; Zupa et al, 2022).…”

“…Within T-II, there are two MT binding sites (Hsia et al, 2014; Travis et al, 2022a; Wu et al, 2008). The primary MT binding site was localized to the augmin subunit Haus8, within its intrinsically-disordered N-terminus (Hsia et al, 2014; Wu et al, 2008), while a second, minor MT binding site was very recently located within the Haus6 subunit (Travis et al, 2022a; Zupa et al, 2022). Thus, augmin promotes branching MT nucleation by recruiting γ-TuRC to the side of the MT (Alfaro-Aco et al, 2020; Song et al, 2018; Tariq et al, 2020; Zhang et al, 2022).…”

Microtubule binding of the human HAUS complex is directly controlled by importins and Ran-GTP

“…The eight subunits of the augmin complex have been reported to form two biochemically distinct subcomplexes, called tetramer T-II (containing HAUS2, 6, 7, and 8) and tetramer T-III (containing HAUS1, 3, 4, and 5) (Lawo et al 2009). Recent cryo-electron microscopy and single particle analysis studies showed that both tetramers together form an interconnected, highly flexible Y-shaped octameric complex (with a V-shaped head and a filamentous tail) (Gabel et al 2022, Zupa et al 2022, Travis et al 2022). The primary microtubule binding site has been localized to the intrinsically-disordered N-terminus of HAUS8 (Hsia et al 2014, Wu et al 2008), while a second, minor microtubule binding site was recently located within the HAUS6 subunit (Travis et al 2022, Zupa et al 2022), locating both binding sites to the V-shaped head.…”

“…Recent cryo-electron microscopy and single particle analysis studies showed that both tetramers together form an interconnected, highly flexible Y-shaped octameric complex (with a V-shaped head and a filamentous tail) (Gabel et al 2022, Zupa et al 2022, Travis et al 2022). The primary microtubule binding site has been localized to the intrinsically-disordered N-terminus of HAUS8 (Hsia et al 2014, Wu et al 2008), while a second, minor microtubule binding site was recently located within the HAUS6 subunit (Travis et al 2022, Zupa et al 2022), locating both binding sites to the V-shaped head. In vitro experiments suggest that several augmin complexes together recruit γTURC (Zhang et al 2022), but how the binding of augmin to microtubules is controlled remains unknown.…”