Laboratory for Molecular Biophysics, RIKEN Center for Brain Science, Saitama, Japan.
Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Tokyo, Japan.
J Cell Biol. 2021 Apr 5;220(4). doi: 10.1083/jcb.202007033.
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.
微管（MTs）的成核对于细胞活动至关重要，但由于聚合早期稀有随机事件的捕捉难度，其机制尚不清楚。在这里，我们结合快速冲洗负染色电子显微镜（EM）和动力学分析，证明了关键大小的直寡聚体的形成对于成核至关重要。GDP 和 GTP 微管蛋白都形成具有广泛曲率的单链寡聚体，但在成核后，GTP 寡聚体的曲率分布会发生偏移，从而产生一小部分直寡聚体。当β亚基中的 Y222F 突变存在于微管蛋白中时，直寡聚体的比例增加，成核加速。我们的结果支持这样一种模型，即 GTP 结合产生一小部分与侧向缔合和进一步生长到 MTs 兼容的直寡聚体。这项研究表明，参与成核的细胞因子通过稳定直寡聚体来促进成核。
