GTP-dependent formation of straight oligomers leads to nucleation of microtubules
Résumé
Microtubule (MT) nucleation is essential for cellular activities, but its mechanism is not known because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. In cells, MTs are nucleated at tubulin concentrations significantly lower than those required for spontaneous nucleation in vitro. The high efficiency of nucleation is due to the synergistic effects of various cellular factors, but the underlying mechanism has not been clarified yet. Here, combining negative stain electron microscopy and kinetic analysis, we demonstrate that the formation of single-stranded straight oligomers with critical size is essential for nucleation in vitro. While the single-stranded oligomers of GTP-tubulin that form prior to MT nucleation show variable curvatures including a few straight oligomers, only curved oligomers are observed among the GDP-bound counterparts. The Y222F mutation in β-tubulin increases the proportion of straight oligomers and drastically accelerates MT nucleation. Our results support a model in which GTP binding causes a small shift in the distribution of oligomer curvature, generating a minor population of straight oligomers compatible with lateral association and further growth to MTs. Our study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.
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