The proportion of straight oligomers was increased by the Y222F mutation in β-tubulin (sequential numbering, with Y222 corresponding to Y224 in the numbering used in Nawrotek et al. Both GTP and GDP tubulin assemble into single-stranded oligomers of various lengths and curvatures, but it is only with GTP tubulin that a minor population of straight oligomers appears. Our analysis shows that a GTP-dependent curvature shift occurs in single-stranded oligomers in the very early stage of nucleation. We overcome this difficulty by using rapid flush negative stain EM ( Frado and Craig, 1992), where we quickly dilute the sample to the concentration optimal for EM observation (approximately submicromolar). Because the nucleation intermediates that indeed grow to MTs appear only at high tubulin concentrations (∼10 µM), their visualization is impossible by ordinary imaging techniques, which require much less concentrated samples.
In this work, we characterized the oligomers, and in particular their curvature, that form before MT assembly. However, when and how straightening occurs have been a subject of long debate ( Bennett et al., 2009 Brouhard and Rice, 2014 Rice et al., 2008). Therefore, during some step in polymerization, GTP tubulin is expected to change its conformation. GTP tubulin is known to assume a curved conformation in solution ( Manuel Andreu et al., 1989 Rice et al., 2008) and a straight conformation when integrated in the MT lattice ( Nogales et al., 1999 Wang and Nogales, 2005). As a first step toward the understanding of the mechanism of nucleation in vivo, we clarify the basic scheme of nucleation using a simple in vitro system composed only of tubulin. In other words, nucleation in cells and nucleation in vitro should have a fundamental scheme in common ( Roostalu and Surrey, 2017). However, the kinetics of the templated assembly show a kind of time lag between the onset of the reaction and the start of MT growth from the template ( Wieczorek et al., 2015 Woodruff et al., 2017), suggesting that, even in the presence of a template, the initial phase of assembly is thermodynamically unfavorable until the nucleation intermediate reaches a critical size.
This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.Ĭompared with the rapidly growing interest among cell biologists in the mechanism of in vivo nucleation mediated by the template of the γ-tubulin ring complex (γTuRC) and other cellular factors ( Brunet et al., 2004 Flor-Parra et al., 2018 Kollman et al., 2011 Roostalu et al., 2015 Schatz et al., 2003 Thawani et al., 2018 Zheng et al., 1995), the interest in the mechanism of spontaneous nucleation in vitro has been scarce. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. With tubulin having the Y222F mutation in the β subunit, the proportion of straight oligomers increases and nucleation accelerates. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers.
Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization.