N-WASP通过polyPro和VCA结构域调控大脑皮层神经元迁移

doi:10.16288/j.yczz.18-066

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

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()

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

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

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)

摘要/Abstract

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

沈秀莲, 逯宜超, 甲芝莲, 吴强. N-WASP通过polyPro和VCA结构域调控大脑皮层神经元迁移[J]. 遗传, 2018, 40(5): 390-401.

Shen Xiulian, Lu Yichao, Jia Zhilian, Wu Qiang. N-WASP regulates cortical neuron migration through its polyPro and VCA domains[J]. Hereditas(Beijing), 2018, 40(5): 390-401.

参考文献

[1]	Wang S, Xu ZH . Progress in the study of molecular mechanisms of developmental cortex malformations. Chin J Cell Biol, 2011,33(8):837-846.
[1]	王硕, 许执恒 . 大脑皮层发育畸形及分子遗传机理研究进展. 中国细胞生物学学报, 2011,33(8):837-846.
[2]	Angevine JB Jr, Sidman RL . Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature, 1961,192:766-768.
[3]	Marín O, Rubenstein JL . Cell migration in the forebrain. Annu Rev Neurosci, 2003,26:441-483.
[4]	Marín O, Valiente M, Ge XC, Tsai LH . Guiding neuronal cell migrations. Cold Spring Harb Perspect Biol, 2010,2(2):a001834.
[5]	Ayala R, Shu TZ, Tsai LH . Trekking across the brain: the journey of neuronal migration. Cell, 2007,128(1):29-43.
[6]	Huang Z . Molecular regulation of neuronal migration during neocortical development. Mol Cell Neurosci, 2009,42(1):11-22.
[7]	Tissir F, Goffinet AM . Shaping the nervous system: role of the core planar cell polarity genes. Nat Rev Neurosci, 2013,14(8):525-535.
[8]	LoTurco JJ, Bai JL . The multipolar stage and disruptions in neuronal migration. Trends Neurosci, 2006,29(7):407-413.
[9]	Lui JH, Hansen DV, Kriegstein AR . Development and evolution of the human neocortex. Cell, 2011,146(1):18-36.
[10]	Tabata H, Nakajima K . Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex. J Neurosci, 2003,23(31):9996-10001.
[11]	Li W, Wu Q . Protocadherin and the diversity of neurons. Sci Technol Vision, 2013, ( 27):14.
[11]	李伟, 吴强 . 原钙粘蛋白分子与神经元的多样性. 科技视界, 2013, ( 27):14.
[12]	Ying GX, Wu S, Hou RQ, Huang W, Capecchi MR, Wu Q . The protocadherin gene Celsr3 is required for interneuron migration in the mouse forebrain. Mol Cell Biol, 2009,29(11):3045-3061.
[13]	Peek SL, Mah KM, Weiner JA . Regulation of neural circuit formation by protocadherins. Cell Mol Life Sci, 2017,74(22):4133-4157.
[14]	Wu Q, Li W. From human genome to the development of brain: the regulation and function of protocadherin in the development of brain. In: Qiao ZD, He L, ed. The Enlightment of the Frontier Life. Beijing: Science Press, 2016: 461-477.
[14]	吴强, 李伟 . 从人类基因组到大脑发育: 原钙粘蛋白在脑发育中的调控与功能研究. 见: 乔中东, 贺林. 前沿生命的启迪. 北京: 科学出版社, 2016: 461-477.
[15]	Garrett AM, Schreiner D, Lobas MA, Weiner JA . γ-protocadherins control cortical dendrite arborization by regulating the activity of a FAK/PKC/MARCKS signaling pathway. Neuron, 2012,74(2):269-276.
[16]	Suo L, Lu HN, Ying GX, Capecchi MR, Wu Q . Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase. J Mol Cell Biol, 2012,4(6):362-376.
[17]	Lefebvre JL, Kostadinov D, Chen WV, Maniatis T, Sanes JR . Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature, 2012,488(7412):517-521.
[18]	Jia ZL, Guo Y, Tang YX, Xu Q, Li BJ, Wu Q . Regulation of the protocadherin Celsr3 gene and its role in globus pallidus development and connectivity. Mol Cell Biol, 2014,34(20):3895-3910.
[19]	Yu Y, Suo L, Wu Q . Protocadherin α gene cluster is required for myelination and oligodendrocyte development. Zool Res, 2012,33(4):362-366.
[19]	于钰, 索伦, 吴强 . PCDHα在髓鞘形成和少突胶质细胞发育中的作用. 动物学研究, 2012,33(4):362-366.
[20]	Chen WV, Nwakeze CL, Denny CA , O'Keeffe S, Rieger MA, Mountoufaris G, Kirner A, Dougherty JD, Hen R, Wu Q, Maniatis T. Pcdhαc2 is required for axonal tiling and assembly of serotonergic circuitries in mice. Science, 2017,356(6336):406-411.
[21]	Chen BY, Brinkmann K, Chen ZC, Pak CW, Liao YX, Shi SY, Henry L, Grishin NV, Bogdan S, Rosen MK . The WAVE regulatory complex links diverse receptors to the actin cytoskeleton. Cell, 2014,156(1/2):195-207.
[22]	Derry JM, Ochs HD, Francke U . Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. Cell, 1994,78(4):635-644.
[23]	Thrasher AJ . WASp in immune-system organization and function. Nat Rev Immunol, 2002,2(9):635-646.
[24]	Aldrich RA, Steinberg AG, Campbell DC . Pedigree demonstrating a sex-linked recessive condition characterized by draining ears, eczematoid dermatitis and bloody diarrhea. Pediatrics, 1954,13(2):133-139.
[25]	Takenawa T, Suetsugu S . The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nat Rev Mol Cell Biol, 2007,8(1):37-48.
[26]	Alekhina O, Burstein E, Billadeau DD . Cellular functions of WASP family proteins at a glance. J Cell Sci, 2017,130(14):2235-2241.
[27]	Kessels MM, Schwintzer L, Schlobinski D, Qualmann B . Controlling actin cytoskeletal organization and dynamics during neuronal morphogenesis. Eur J Cell Biol, 2011,90(11):926-933.
[28]	Kawauchi T, Hoshino M . Molecular pathways regulating cytoskeletal organization and morphological changes in migrating neurons. Dev Neurosci, 2008,30(1/3):36-46.
[29]	Miki H, Miura K, Takenawa T . N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases. EMBO J, 1996,15(19):5326-5335.
[30]	Padrick SB, Rosen MK . Physical mechanisms of signal integration by WASP family proteins. Annu Rev Biochem, 2010,79(1):707-735.
[31]	Moreau V, Frischknecht F, Reckmann I, Vincentelli R, Rabut G, Stewart D, Way M . A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization. Nat Cell Biol, 2000,2(7):441-448.
[32]	Carlier MF, Ducruix A, Pantaloni D . Signalling to actin: the Cdc42-N-WASP-Arp2/3 connection. Chem Biol, 1999,6(9):R235-R240.
[33]	Kim AS, Kakalis LT, Abdul-Manan N, Liu GA, Rosen MK . Autoinhibition and activation mechanisms of the Wiskott- Aldrich syndrome protein. Nature, 2000,404(6774):151-158.
[34]	Miki H, Sasaki T, Takai Y, Takenawa T . Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature, 1998,391(6662):93-96.
[35]	Rohatgi R, Ma L, Miki H, Lopez M, Kirchhausen T, Takenawa T, Kirschner MW . The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell, 1999,97(2):221-231.
[36]	Carlier MF, Nioche P , Broutin-L'Hermite I, Boujemaa R, Le Clainche C, Egile C, Garbay C, Ducruix A, Sansonetti P, Pantaloni D. GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASp) with actin-related protein (ARP2/3) complex. J Biol Chem, 2000,275(29):21946-21952.
[37]	Rohatgi R, Ho HY, Kirschner MW . Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate. J Cell Biol, 2000,150(6):1299-1310.
[38]	Okrut J, Prakash S, Wu Q, Kelly MJS, Taunton J . Allosteric N-WASP activation by an inter- SH3 domain linker in Nck. Proc Natl Acad Sci USA, 2015,112(47):E6436-E6445.
[39]	Padrick SB, Cheng HC, Ismail AM, Panchal SC, Doolittle LK, Kim S, Skehan BM, Umetani J, Brautigam CA, Leong JM, Rosen MK . Hierarchical regulation of WASP/WAVE proteins. Mol Cell, 2008,32(3):426-438.
[40]	Lommel S, Benesch S, Rottner K, Franz T, Wehland J, Kühn R . Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells. EMBO Rep, 2001,2(9):850-857.
[41]	Jain N, Lim LW, Tan WT, George B, Makeyev E, Thanabalu T . Conditional N-WASP knockout in mouse brain implicates actin cytoskeleton regulation in hydrocephalus pathology. Exp Neurol, 2014,254:29-40.
[42]	Sheldon M, Rice DS , D'Arcangelo G, Yoneshima H, Nakajima K, Mikoshiba K, Howell BW, Cooper JA, Goldowitz D, Curran T. Scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice. Nature, 1997,389(6652):730-733.
[43]	Suetsugu S, Tezuka T, Morimura T, Hattori M, Mikoshiba K, Yamamoto T, Takenawa T . Regulation of actin cytoskeleton by mDab1 through N-WASP and ubiquitination of mDab1. Biochem J, 2004,384(Pt 1):1-8.
[44]	The ENCODE Project Consortium . An integrated encyclopedia of DNA elements in the human genome. Nature, 2012,489(7414):57-74.
[45]	Tsuchiya D, Kitamura Y, Takata K, Sugisaki T, Taniguchi T, Uemura K, Miki H, Takenawa T, Shimohama S . Developmental expression of neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP family verprolin- homologous protein (WAVE)-related proteins in postnatal rat cerebral cortex and hippocampus. Neurosci Res, 2006,56(4):459-469.
[46]	Abe T, Kato M, Miki H, Takenawa T, Endo T . Small GTPase Tc10 and its homologue RhoT induce N-WASP- mediated long process formation and neurite outgrowth. J Cell Sci, 2003,116(Pt 1):155-168.
[47]	Pommereit D, Wouters FS . An NGF-induced Exo70-TC10 complex locally antagonises Cdc42-mediated activation of N-WASP to modulate neurite outgrowth. J Cell Sci, 2007,120(Pt 15):2694-2705.
[48]	Wegner AM, Nebhan CA, Hu L, Majumdar D, Meier KM, Weaver AM, Webb DJ . N-wasp and the arp2/3 complex are critical regulators of actin in the development of dendritic spines and synapses. J Biol Chem, 2008,283(23):15912-15920.
[49]	You JJ, Lin-Chao S . Gas7 functions with N-WASP to regulate the neurite outgrowth of hippocampal neurons. J Biol Chem, 2010,285(15):11652-11666.
[50]	Mohamed AM, Boudreau JR, Yu FPS, Liu J, Chin-Sang ID . The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance. PLoS Genet, 2012,8(2):e1002513.
[51]	Tsai JW, Chen Y, Kriegstein AR, Vallee RB . LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages. J Cell Biol, 2005,170(6):935-945.
[52]	Bai JJ, Ramos RL, Ackman JB, Thomas AM, Lee RV , LoTurco JJ. RNAi reveals doublecortin is required for radial migration in rat neocortex. Nat Neurosci, 2003,6(12):1277-1283.
[53]	Xiao F, Wang XF, Li JM, Xi ZQ, Lu Y, Wang L, Zeng Y, Guan LF, Yuan J . Overexpression of N-WASP in the brain of human epilepsy. Brain Res, 2008,1233:168-175.
[54]	Kovacs EM, Verma S, Ali RG, Ratheesh A, Hamilton NA, Akhmanova A, Yap AS . N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway. Nat Cell Biol, 2011,13(8):934-943.
[55]	Luan Q, Zelter A , MacCoss MJ, Davis TN, Nolen BJ. Identification of Wiskott-Aldrich syndrome protein (WASP) binding sites on the branched actin filament nucleator Arp2/3 complex. Proc Natl Acad Sci USA, 2018,115(7):E1409-E1418.

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N-WASP通过polyPro和VCA结构域调控大脑皮层神经元迁移

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

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