【学术前沿】鲍岚团队揭示α微管蛋白三甲基化修饰在微管功能调控和神经系统发育过程中的作用及其机制
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2021年7月5日，中国科学院分子细胞科学卓越创新中心（生物化学与细胞生物学研究所）鲍岚研究员团队在《Nature Communications》杂志在线发表了题为“α-TubK40me3 is required for neuronal polarization and migration by promoting microtbule formation”的研究论文。该研究揭示了α微管蛋白上新发现的三甲基化修饰可以促进微管的形成，从而调节神经元极性的建立和迁移。
微管作为细胞内一种主要的细胞骨架成分，在神经元的迁移过程中发挥着重要的作用。微管的动态性受多种微管蛋白翻译后修饰的调控。课题组前期的研究工作发现，α微管蛋白第40位赖氨酸上的乙酰化修饰在神经系统发育过程中发挥重要的作用，α微管蛋白乙酰化的缺失会引起皮层神经元迁移的缺陷，并导致神经元出现过度生长和分支的表型（Li et al, J Neurosci, 2012; Wei et al, Cereb Cortex, 2018）。已有研究表明，在α微管蛋白乙酰化修饰的同一位点上还存在着由SETD2催化的三甲基化修饰，但关于α微管蛋白三甲基化修饰对微管功能的调控机制及其在神经系统发育过程中的作用还知之甚少。
在本研究中，研究团队首先制备了特异性识别α微管蛋白三甲基化修饰的抗体，通过该抗体检测到微管蛋白在小鼠胚胎期第14天和第16天的大脑皮层中处于高甲基化状态。SETD2的敲减会影响皮层神经元由多极向双极状态的转换，从而导致神经元迁移的缺陷，在原代培养的神经元中敲减SETD2也会影响神经元极性的建立，以上表型可以通过表达细胞质定位的SETD2截短体和模拟三甲基化形式的微管蛋白突变体得到恢复，表明α微管蛋白三甲基化修饰在神经元的极化和迁移过程中发挥重要的作用。
在机制研究方面，发现三甲基化的α微管蛋白更倾向于分布在聚合状态的微管上，并可以通过促进微管蛋白的成核使神经元中产生足够数量的微管来完成极性的建立和转换。进一步的研究还发现，发生在同一位点上的α微管蛋白的三甲基化和乙酰化修饰之间存在竞争性的交互调节，α微管蛋白乙酰化缺失时三甲基化修饰水平升高，可能代偿了乙酰化修饰在大脑皮层发育过程中的作用。
本研究揭示了α微管蛋白三甲基化修饰在微管功能调控和神经系统发育过程中所发挥的作用及其机制，有助于拓展和加深对于神经元中微管组装和动态性调节的理解。
中科院分子细胞科学卓越创新中心鲍岚研究组的博士研究生谢玄和王少刚为本文共同第一作者，鲍岚研究员和冯文峰博士为共同通讯作者。该工作得到了国家基金委、中科院先导项目以及上海市科委的资助。
α-TubK40me3 is required for neuronal polarization and migration by promoting microtubule formation
During the development of central nervous system, formation of the six-layered structure of cerebral cortex is a tightly regulated process which is essential for the establishment of proper neuronal circuits and brain functions. Neuronal migration is a critical step in the development of cerebral cortex, which is highly dependent on the dynamic microtubule cytoskeleton. Recent studies are providing more insights into the importance of tubulin post-translational modifications (PTMs) in cortical development. According to the previous studies in our lab, impairment of α-tubulin acetylation at lysine 40 (α-TubK40ac) by knocking down the acetyltransferase MEC-17 causes migratory defects in the cortical projection neurons and interneurons, and perturbs the multipolar-to-unipolar/bipolar transition of projection neurons in the IZ region (Li et al, J Neurosci, 2012). Loss of α-tubulin acetylation results in axon overgrowth and overbranching (Wei et al, Cereb Cortex, 2017). A recent study reports that α-tubulin could be tri-methylated (α-TubK40me3) by SET domain containing 2 (SETD2) at the same residue undergoing acetylation (Park et al, Cell, 2016). However, the functions and mechanisms of α-TubK40me3 in microtubule properties and post-mitotic neurons are largely unclear.
This work entitled “α-TubK40me3 is required for neuronal polarization and migration by promoting microtubule formation” was published online in Nature Communications on July 5th , 2021. Xuan Xie, Shaogang Wang and colleagues, under the supervision of Prof. Lan Bao and Dr. Wenfeng Feng at the Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, found that α-TubK40me3 could promote microtubule formation and regulates neuronal polarization and migration.
In this study, using a specific homemade antibody against α-TubK40me3, researchers show that α-TubK40me3 is enriched in NPCs and neurons from embryonic day 14 (E14) to E16 in cerebral cortex. In utero knockdown of SETD2 at E14 leads to impaired morphological transition and thereby the migration of cortical neurons, which could be restored by overexpressing NES-SETD2(1469-1724), a cytoplasm-localized SETD2 truncation retaining enzyme activity or α-tubulinK40F, a tri-methylation mimicking mutant of α-tubulin. Mechanistically, α-TubK40me3 is preferably distributed on polymerized microtubules in cells and regulates neuronal polarization by promoting tubulin nucleation and microtubule formation. Interestingly, the duration of high-level α-TubK40me3 in the developing cerebral cortex is extended in MEC-17 knockout mice, and the impaired polarization and migration of α-TubK40ac-deficient neurons could be largely restored by overexpressing NES-SETD2(1469-1724) and α-tubulinK40F, implying a compensatory effect by α-TubK40me3. These findings greatly expand our knowledge about the functions and mechanisms of α-tubulin methylation in post-mitotic neurons.
This work was collaborated with Prof. Xu Zhang and Prof. Weiguo Zou. This work was funded by National Natural Science Foundation of China, the Strategic Priority Research Program of the Chinese Academy of Sciences, and the Shanghai Science and Technology Committee.
α-TubK40me3 is required for neuronal polarization and migration by promoting microtubule formation
Article links:https://www.nature.com/articles/s41467-021-24376-2