GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation.

Rie Ayukawa,Ken Sekimoto,Benoît Gigant,Seigo Iwata,Tsukasa Makino,Mikako Shirouzu,Hiroshi Imai,Kien Xuan Ngo,Shinji Kamimura,Masahito Hayashi,Hideki Shigematsu,Etsuko Muto,Itsushi Minoura,Seiichi Uchimura

doi:10.1083/jcb.202007033

Abstract

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. 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. 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. With tubulin having the Y222F mutation in the β subunit, the proportion of straight oligomers increases and nucleation accelerates. 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. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.

Highlights

In eukaryotes, dynamic arrangement of microtubule (MT) organization in proper timing and location is critical for various cellular functions, such as cell shape determination and chromosome segregation
Structure and kinetics of WT tubulin and of the Y222F mutant It has been proposed that GTP binding promotes MT assembly by fostering a conformational switch of the β-tubulin T5 loop, a loop involved in tubulin–tubulin longitudinal contacts (Fig. 1, A and B; Nawrotek et al, 2011)
We demonstrate here the linkage between GTP-dependent movement of the T5 loop, formation of straight oligomers of critical size, and nucleation of MTs

Summary

Introduction

Dynamic arrangement of microtubule (MT) organization in proper timing and location is critical for various cellular functions, such as cell shape determination and chromosome segregation. Understanding the molecular mechanism of MT dynamics is important for developing an effective treatment of these diseases. While the catastrophic transition to depolymerization has been well characterized as “dynamic instability” (Brouhard, 2015; Mitchison and Kirschner, 1984), the inverse process involving the nucleation of MTs from tubulin is still poorly understood. Nucleation in general is a stochastic process in which the thermodynamically unfavorable growth of precritical molecular composites turns into favorable growth via the formation of critical nuclei (Burton, 1977; Langer, 1969). We solve a long-standing important question: How does GTP tubulin nucleate MTs in vitro? The question remained unresolved for decades because the nucleation intermediates around the critical nuclei exist only transiently, so capturing them is a major challenge We solve a long-standing important question: How does GTP tubulin nucleate MTs in vitro? Despite its central importance, the question remained unresolved for decades because the nucleation intermediates around the critical nuclei exist only transiently, so capturing them is a major challenge

Methods

Results

Conclusion