Abstract
Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed 'branching' MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts.
Highlights
Branching MT nucleation is dependent upon Augmin and g-Tubulin Ring Complex (g-TuRC) and generates the bulk of MTs required for both meiotic and mitotic spindle formation (Petry et al, 2011; Sanchez-Huertas and Luders, 2015; David et al, 2019) and has been visualised in vivo in Drosophila, Xenopus, plants, and humans (Petry et al, 2011; David et al, 2019; Hayward et al, 2014; Ho et al, 2011; Lawo et al, 2009; Goshima et al, 2008; Uehara et al, 2009; Prosser and Pelletier, 2017; Petry et al, 2013)
Understanding, and in vitro reconstitution of, this phenomenon has been hampered by methodological constraints relating to purification of functional protein complexes (Hsia et al, 2014; Song et al, 2018); Augmin is composed of 8 subunits, while the gTuRC is a ~ 2 MD protein complex containing multiple copies of at least six proteins, including 14 molecules of g-Tubulin (Zheng et al, 1995; Moritz et al, 1995; Kollman et al, 2011; Tovey and Conduit, 2018; Oegema et al, 1999)
Addition of Augmin-GFP dramatically enhanced g-TuRC-dependent nucleation of MTs, further reducing the x50 to 9.5 min (± 0.45 min) (Figure 2a,b). This effect was specific for the physical interaction between Augmin and g-TuRC, as addition of bacterially expressed and purified truncated Augmin subunits, Dgt3, Dgt5 and Dgt6, which we previously demonstrated interact directly with g-TuRC (Chen et al, 2017), resulted in nucleation/polymerisation curves indistinguishable to g-TuRC alone (Figure 2—figure supplement 1)
Summary
Branching MT nucleation is dependent upon Augmin and g-TuRC and generates the bulk of MTs required for both meiotic and mitotic spindle formation (Petry et al, 2011; Sanchez-Huertas and Luders, 2015; David et al, 2019) and has been visualised in vivo in Drosophila, Xenopus, plants, and humans (Petry et al, 2011; David et al, 2019; Hayward et al, 2014; Ho et al, 2011; Lawo et al, 2009; Goshima et al, 2008; Uehara et al, 2009; Prosser and Pelletier, 2017; Petry et al, 2013). To characterise the morphology of MTs generated in the presence of purified mitotic Augmin and g-TuRC, we initially took samples from the in vitro assays at t = 15 min, fixing and imaging them via fluorescence microscopy Control samples, or those containing purified Augmin, showed only very few, short MTs per field of view while, as expected, g-TuRC-containing samples possessed many individual MTs (Figure 3a). Samples taken at t = 2 and t = 5 min showed similar numbers of MTs to each other, per field of view, though the mean length of MTs, and proportion of MTs with branches, were significantly greater after 5 min, than after 2 min (4.01 mm versus 3.24 mm and 34% versus 24%, respectively) (Figure 3b; Figure 3—source data 1) This suggests that MT nucleation from purified g-TuRCs reaches maximal activity within the first few minutes of the assay. By isolating and combining purified, active proteins and protein complexes from any biological system of interest, "cl-AP TRAP" overcomes the limitations of traditional ‘bottom-up’ approaches, allowing exploration of the level of biological organisation between individual protein and biological process – the level at which emergence of cellular phenomena often occurs
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