侯 旭濱

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博士学位論文の要旨及び審査結果の要旨 (Summary of Thesis(DR))othe

侯, 旭濱

305574

Tohoku University Repository (TOUR) / 東北大学機関リポジトリ

Role of actin binding protein Coactosin incell migration and axonal extension著者 侯 旭濱学位授与機関 Tohoku University学位授与番号 生博第172号URL http://hdl.handle.net/10097/51195ホウ　シュウビン氏名(本籍地)学　位　の　種　類学　位　記　番　号学位授与年月　日学位授与の要件研究科,専攻論　文　題　目博士論文審査委員侯　旭演博士(生命科学)生博第1 72号平成22年3月25日学位規程第4条第1項該当東北大学大学院生命科学研究科(博士課程)生命機能科学専攻Role of actin binding protein Coactosin ln cellmigration and axonal extension (アクチン結合タンパクcoactoslnの神経堤細胞移動･軸索伸長における役割)(主査)　教　授　仲　村　春　和教　授　八　尾　　　寛准教授　大　橋　一　正-　47　-論文内容の要旨Coactosin is a 17kDa actin binding protein that belongs to ADF/cofilin homology famlly. Cofilin,which belongs to the famlly, severs actjn filaments and promotes actin dynamics by accelerating thetreadmilllng Of actin filaments, a process in which monomers are removed from the pointed end offilaments and are added to the barbed end. On the other hand, it was reported that Coactosin inhibitsbarbed-end capplng Of actin filament, and is involved in actin polymerization in vitroIPreliminary examination by in situ hybridization showed that Coactosin was expressed in cephalicand trunk neural crest cells (NCCs), and their derivatives such as cranial ganglia and dorsal root ganglia･Coactosin was also expressed in the ceIIs that are formlng mesonephric duct, and endodermal ce"sISince these ce"s are highly motiLe ce"S, I decided to examine role ofCoactosin in ce" dynamics･ Forthestudy, l elaborated techniques. First, Hmproved the sagittal slice explant technique, which enables us toobserve neural crest cell mlgration nearly intact condition. Next, l developed conditional siRNAtechnique by modifying the system originally developed by Shinagawa and lshii (2003)A They developeda plasmid, PDECAP-RNAi expression vector, which contains the CMV promoter, a ribozyme cassette tocut ofrthe m7G cap structure, and a MAZ site for Po川pausln9･ The transcripts of long double strandRNAwithout cap and polyA tail stay ln the nucleus, and processed into siRNAby Dicer, which is presentin the nucleus. A ribozyme cassette and a MAZ site were put into BI vector that contains tet-on system･This system enables us to knock-down the target gene at desired stage by administrating doxycyclin, atetracycline derivative.lmmunocytochemistry with anti-Coactosin antibody showed that Coactosin is localized in thecytoplasm, and associated with actin stress fibers in cultured neural crest ceHsI Coactosin is alsoexpressed in the axon of oculomotor neⅣe and trigeminal neⅣe･ ln the growth cone of the oculomoto｢nerve axons, both Coactosin mRNA and protein were localized.Knockdown of Coactosin by electroporating shRNA expression vector resulted in impalrment Ofneural crest cell mlgration in vivo and in vitro. ln ceHs that were knocked down of Coactosin,arrangement of actin cytoskeleton was impaired; anchoring of neural crest ceHs at the focal adhesionsite by actin filament was destroyed･ The result indicates that mlgration defects in Coactosin-knockeddown ce"s may be caused by impalrment Of filopodial contact to the substrate or between neighboringNCCs. lncorporation of G-actin into F-actjn was interfered in Coactosin-knocked down ce"sI Time-lapseanalyses in sagittal slice explant confirmed observations that knockdown of Coactosin impairedmlgration of neural crest cells.To exclude the possibility that above results are resulted from neural tube closure, conditionalknock-down of Coactosin was carried out, that is, Coactosin was knocked down a冊er they had left-　48　-neural tube by Teトon siRNA system. By this it was confirmed that neural crest cell m19ration wasimpaired by Coactosin-knock down.Coactosin protein and mRNA was localized in the oculomotor nerve. l wondered if Coactosin isinvolved in extension of the axon. Knockdown of Coactosin by Coactosin-siRNA resulted in impalrmentof oculomoter neⅣe elongation. To further investi9ate the function of Coactosin, NI E1 15 ce川ne wasused. The cells differentiate into neuron in the absence of serum, and we can obseⅣe neurite extension.Coactosin was localized at the tip offilopodia of the neuritis,Next, relation of Coactosin with Rho, Rac system was studied. Expression of dominant-positiveRacl or RhoA caused Coactosin to rapidly accumulate in lame"ipodia or fjlopodia. Coactosin waspartiaJly c0-localized with Racl and RhoA. OveraH, these findings suggest that in vertebratedevelopment, regulation of Coactosin is critical for neural crest ceH mlgration and cell shapemaintenance.Publication list:1. Directory related papersXubin Hou, Tatsuya Katahira, Jun Kimura and Harukazu Nakamura (2009)Expression of Chick Coactosin in ce"s in morphogenetic movement.Deve/op. Growth DI'qer. Volume 51 Issue 9, Pages 833 - 8402.OthersOdani, N., Hou. X., Nakamura, H.ln ovo electroporation as a strong tool to pursue molecular mechanisms of neural development in chick,Electroporation and Sonoporation in Developmental Biology. Springer JP, Tokyo-　49　-論文審査結果の要旨侯旭演君はアクチン結合タンパクCoactosinに着目し､その機能解析を行った｡ CoactosinはADF(actindepolymering factor) domainを持っており､アクチンと結合すること､アクチンの安定化に働く事は分かっていたが､その機能はよく分かっていなかった｡侯君はまずcoactosinのmRNAが､動眼神経などの神経軸素の成長円錐､神経堤細胞､中腎管の先端などの動きのある細胞に発現していることを明らかにした｡また､ Coactosinはアクチンおよびキヤツビングプロテインと結合することを示した｡そこで､神経堤細胞の移動､動眼神経の軸索伸長におけるCoactosinの役割を調べた｡培養神経堤細胞で､ Coactosinはアクチンと共局在しており､特にストレスファイバーと関連があることを示した｡ shRNAによるノックダウンにより､神経堤細胞の移動､動眼神経の軸索伸長が阻害された｡また､ GFP-G-actin発現ベクター､ Coactosin shRNA発現ベクターを発現させることにより､G-アクチンがF-アクチンに取り込まれるためにはCoactosinの存在が必要であることが示された｡細胞移動､軸索伸長においてはRho, Racのシグナルの下に糸状仮足､あるいは葉状仮足が作られるが､培養NIEll15細胞にCoactosinをトランスフェクトし､活性型Racで処理すると､糸状突起が活発に作られ､ Coactosinが糸状突起に集積した｡アクチン結合部位に変異を入れアクチンと結合できないCoactosinをトランスフェクトした場合は､ Racのシグナルがあっても糸状突起の形成は促進されず､またCoactosinが突起に集積することもなかった｡このことは､ CoactosinがRac等のシグナルの下流でアクチンと結合し糸状突起の形成に働き､細胞移動､突起伸展に大きな役割を果たしていることを示唆している｡侯君はこれらの研究を遂行するにあたり､いろいろな工夫を凝らしている｡神経堤細胞の移動の観察には体節などを残したまま培養できるsemi vivoの培養法を開発した｡その方法により､生体に近い条件での共焦点顕微鏡によるタイムラブス画像の解析を可能にした｡また､ Coactosinノックダウンにより､神経堤細胞の上皮間充織転換が阻害されるのか､あるいは移動そのものが阻害されるかについては､全く新しいsiRNAノックダウン法を開発し､世界で始めてsiRNAの発現の調節を可能にした｡この方法により､ Coactosinノックダウンにより阻害されるのは神経堤細胞の上皮間充織転換ではなく､移動そのものであることを示した｡侯旭演君は自立して研究活動を行うに必要な高度の研究能力と学識を有することを示しており､侯旭演君提出の論文は,博士(生命科学)の博士論文として合格と認める｡-　50　-

Role of actin binding protein Coactosin in cell migration and axonal extension

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アクチン結合タンパク Coactosin の神経堤細胞移動･軸索伸長における役割

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アクチン結合タンパク Coactosin の神経堤細胞移動 ･ 軸索伸長における役割

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アクチン結合タンパク Coactosin の神経堤細胞移動･軸索伸長における役割