Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure

Abstract

Highlights d The g-tubulin ring complex (gTuRC) nucleates microtubules and caps their minus ends d Microtubule nucleation from purified gTuRC is highly cooperative, yet inefficient d A partly open, asymmetric structure of gTuRC explains inefficient nucleation d Actin and MZT2 stabilize the closed part of the gTuRC structure

Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons

“…Our mechanistic models are strongly supported by published data obtained through in vitro studies of MTs that match extremely well with virtually all our findings. Key examples are: (1) the complementary binding preferences of EB1 and Tau for GTP-/GDP-tubulin (Castle et al, 2020, Zanic, Stear et al, 2009; (2) the mutual enhancement of Eb1 and XMAP215/Msps originally suggested in Xenopus extracts and then demonstrated in reconstitution essays with purified proteins from fly and vertebrates (Kronja, Kruljac-Letunic et al, 2009, Li et al, 2012, Zanic et al, 2013; (3) the correlation of GTP cap size with comet velocity (Roostalu et al, 2020); (4) the reduction of comet numbers but not dynamics ( Fig.1-S1) upon depletion of α1tubulin or Stathmin (a prominent regulator of tubulin availability; Duncan et al, 2013, Manna, Grenningloh et al, 2007 which are consistent with in vitro observations that MT nucleation is far more sensitive to tubulin levels than polymerisation (Consolati, Locke et al, 2020). This list of similarities is in stark support of our model and a clear indication that many in vitro observation seem to apply in cellular contexts.…”

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

“…~95% of microtubule nucleation events were observed to initiate from -TuRC mini -GFP puncta ( Figure S1F). The growth rates and catastrophe frequencies of microtubules nucleated by -TuRC mini -GFP matched published values for those of plus ends grown off of GMPCPP seeds (Brouhard et al, 2008;Gardner et al, 2011) , axonemes (Walker et al, 1988) , and native -TuRCs (Consolati et al, 2020;Thawani et al, 2020) ( Figure S1G-H). Moreover, the position of fluorescent-tubulin "speckles" along the growing microtubule's length did not change relative to the position of microtubule end-bound complexes (Figure S1I-K;(Waterman-Storer et al, 1998) ), indicating that -TuRC mini -GFP nucleates microtubules that grow from their distal plus ends.…”

“…The microtubule nucleating activity of the native human -TuRC has been found to be surprisingly low (<1% nucleation efficiency; (Consolati et al, 2020) ). We also found that many -TuRC mini -GFPs (>95%) did not nucleate microtubules during the time course of our experiments.…”

“…In vertebrates, the -TuRC contains at least 8 proteins, including the tubulin-like GTPase -tubulin, -tubulin ring complex proteins (GCPs) 2-6, and the mitotic-spindle organizing proteins associated with a ring of -tubulin (MZT1 and MZT2) (Murphy et al, 2001;Teixidó-Travesa et al, 2010;Hutchins et al, 2010) . The stoichiometry, location, and structures of these proteins in the context of the native -TuRC were recently identified using high-resolution cryo-electron microscopy (cryo-EM) (Wieczorek et al, 2019;Liu et al, 2019;Wieczorek et al, 2020;Consolati et al, 2020) . These studies showed that the -TuRC is an asymmetric, cone-shaped structure, in which GCP2-6 orient 14 -tubulin molecules in a helical arrangement that is poised to nucleate microtubules.…”

“…We use electron microscopy to show that this subcomplex adopts a semi-conical shape organizing ~8 copies of -tubulin into an arrangement that mimics the native -TuRC, but which lacks an ordered "overlap" region and lumenal bridge. Despite its partial structure, this subcomplex can nucleate individual microtubules in a manner analogous to native -TuRCs (Consolati et al, 2020) .…”