遗传 ›› 2018, Vol. 40 ›› Issue (5): 390-401.doi: 10.16288/j.yczz.18-066

• 研究报告 • 上一篇    下一篇

N-WASP通过polyPro和VCA结构域调控大脑皮层神经元迁移

沈秀莲,逯宜超,甲芝莲,吴强()   

  1. 上海交通大学系统生物医学研究院比较生物医学研究中心,系统生物医学教育部重点实验室,上海 200240
  • 收稿日期:2018-03-15 修回日期:2018-04-19 出版日期:2018-05-20 发布日期:2018-05-04
  • 通讯作者: 吴强 E-mail:qiangwu@sjtu.edu.cn
  • 作者简介:沈秀莲,硕士研究生,专业方向：发育神经生物学。E-mail: shenxiulian92@163.com
  • 基金资助:
    国家自然科学基金资助(91519302);国家自然科学基金资助(31171015)

N-WASP regulates cortical neuron migration through its polyPro and VCA domains

Xiulian Shen,Yichao Lu,Zhilian Jia,Qiang Wu()   

  1. Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China
  • Received:2018-03-15 Revised:2018-04-19 Online:2018-05-20 Published:2018-05-04
  • Contact: Wu Qiang E-mail:qiangwu@sjtu.edu.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China(91519302);Supported by the National Natural Science Foundation of China(31171015)

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摘要：

在大脑皮层发育过程中,神经元迁移是一个动态的复杂过程,与细胞骨架构建和重塑的调控息息相关。N-WASP蛋白是Wiskott-Aldrich综合征蛋白家族(WASP-WAVE family)的一个重要成员,又名WAS-like蛋白(WASL),直接参与细胞骨架中肌动蛋白丝状分支的动态调控。本研究通过蛋白免疫印迹检测发现N-WASP表达于小鼠胚胎发育时期(E12.5~E18.5)的大脑皮层中,并且其表达水平随着发育逐渐降低。利用在体子宫内胚胎电转实验,结果发现过表达或者敲低N-WASP均会造成不同程度的大脑皮层神经元迁移障碍,说明N-WASP在大脑皮层神经元迁移中起到关键作用。N-WASP蛋白主要包含4个结构域：WH1、GBD、polyPro和VCA。为进一步研究N-WASP各结构域在神经元迁移中的调控功能,设计了一系列的显性负性突变实验。通过过表达结构域删除的N-WASP蛋白,发现ΔpolyPro、ΔVCA和ΔWH1均能造成神经元迁移障碍。但是,过表达不能结合Cdc42的N-WASP蛋白(H208D突变体)却不能造成明显的神经元迁移障碍。另外,单独过表达N-WASP的结构域polyPro或VCA能够造成神经元迁移障碍,而过表达WH1结构域却不能影响迁移。最后,通过过表达polyPro和VCA结构域同时删除的N-WASP (WH1-GBD),发现WH1-GBD结构域对神经元迁移没有明显影响。上述结果表明N-WASP蛋白主要是通过polyPro和VCA两个结构域调控大脑皮层神经元的迁移过程。

关键词: 大脑皮层神经元迁移, N-WASP, 子宫内胚胎电转技术, 肌动蛋白细胞骨架动态

Abstract:

Cortical neuron migration in the developing mouse forebrain is a complex process, which contains several steps related to cytoskeleton dynamics and remodeling. Neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP-WAVE family, regulates actin cytoskeleton reorganization through the binding of its VCA domain to the Arp2/3 complex. Here we report expression patterns of N-WASP gene in the mouse developing embryonic cortex (E12.5~ E18.5) and find its expression levels are decreased during embryonic development. By using in utero electroporation (IUE) method, we find that either N-WASP overexpression or knockdown impairs cortical neuron migration, and the defects of cortical neuron migration caused by N-WASP overexpression are much more severe than that by its knockdown. N-WASP protein contains four domains: WH1, GBD, polyPro, and VCA. We generated a series of dominant negative N-WASP mutants by modifying these domains. Overexpression of N-WASP mutant lacking domain polyPro, VCA, or WH1, impairs cortical neuron migration. However, overexpression of N-WASP with the H208D point mutation, which abolishes the Cdc42 binding to N-WASP, causes only a marginal defect of cortical neuron migration. Finally, overexpression of the individual domain polyPro or VCA, but not WH1, can recapitulate the defects by N-WASP overexpression. However, overexpression of WH1-GBD fragment has no apparent effect on cortical neuron migration. In conclusion, our data demonstrate that N-WASP regulates cortical neuron migration mainly through its polyPro and VCA domains.

Key words: cortical neuron migration, N-WASP, in utero electroporation, actin cytoskeleton dynamics