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Study on cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy

清水, 優太朗 東京大学 DOI:10.15083/0002006720

2023.03.24

概要

論文審査の結果の要旨
氏名

清水 優太朗

本論文は 4 章からなる。第 1 章はイントロダクション、第 4 章は総合考察である。研
究内容は、第 2 章および第3章に記述されている。
第 1 章では、本研究の背景と目的が述べられている。真核生物の細胞内には膜で囲まれ
た多種多様な細胞小器官が存在しており、各細胞小器官が独自の役割を果たすことで正常
な細胞活動が維持されている。トランスゴルジ網(trans-Golgi network: TGN)は、小胞体
で新規に合成され、ゴルジ体で修飾されたさまざまな積荷タンパク質を受け取り、それら
を仕分けて最終目的地に向けて送り出す役割を担っている。古典的な細胞生物学的手法、
生化学的手法、遺伝学的手法を用いた研究により、細胞膜/細胞外や液胞などの最終目的
地に運ばれる積荷タンパク質が TGN において選別されることが明らかにされており、
TGN が複数の輸送経路を制御するハブとして働くと考えられている。しかしながら、
TGN が異なる目的地へ輸送される積荷タンパク質をどのようにして選別しているのか、
その実態は明らかにされていない。本研究では、細胞内の膜交通システムに関与する因子
を可視化し、超解像ライブイメージング顕微鏡により観察することから、TGN における
選別輸送の動態を解明した。
第2章では、本研究で使用した材料と実験方法について述べられている。細胞小器官を
可視化するために、蛍光タンパク質を融合させたオルガネラマーカーを発現するシロイヌ
ナズナ形質転換植物を多数作成した。超解像ライブイメージング顕微鏡の詳細についても
この章に述べられている。
第3章では、本研究の結果について述べられている。TGN から細胞膜へ運ばれる積荷
タンパク質 VAMP721 と液胞へ運ばれる積荷タンパク質 VAMP727 を、異なる色の蛍光タ
ンパク質で可視化したシロイヌナズナをもちいて超解像ライブイメージングをおこなっ
た。その結果、両積荷タンパク質が同一の TGN 上で分離して局在することを明らかにし
た。VAMP721、VAMP727 の局在解析にくわえ、TGN 上で積荷タンパク質の選別に関与す
る AP-1 複合体と AP-4 複合体の局在も観察した。興味深いことに、これらも TGN 上で相
互排他的に局在することを明らかにした。さらに、TGN 上の積荷タンパク質 VAMP と選
別因子 AP 複合体の共局在解析を行い、細胞膜へ輸送される VAMP721 と AP-1 と、液胞膜
へ輸送される VAMP727 と AP-4 とは、同一の TGN の異なるゾーンに局在することを明ら
かにした。これらの結果から、TGN 上に細胞膜への輸送(分泌輸送)のためのゾーンと
液胞輸送のためのゾーンが別々に形成されていると結論づけた。
次に、TGN における代表的な輸送小胞であるクラスリン被覆小胞を形作るタンパク質
(クラスリン軽鎖: CLC)を可視化し、クラスリンが分泌輸送ゾーンに局在することを明
1

らかにした。また、積荷タンパク質の輸送がどのように行われているのか明らかにするた
めに、各輸送ゾーンに局在するタンパク質の動態を観察した。その結果、分泌輸送ゾーン
を構成する積荷タンパク質(VAMP721)と選別を担うタンパク質(AP-1・クラスリン)
は共局在するだけでなく挙動をともにすること、また、TGN から遊離することを発見し
た。これらの観察結果から、TGN 上の分泌輸送ゾーンから生じた小胞塊によって、細胞
膜への輸送が行われていると結論づけた。一方、興味深いことに、液胞輸送ゾーンに局在
し、TGN から積荷を運び出す役割が想定される AP-4 が TGN から遊離するような特徴的
な現象は観察されなかった。
第4章は、総合考察にあてられている。本研究では植物細胞の TGN を観察対象とした
が、酵母及び動物の TGN に関する知見との比較から、植物 TGN を研究する重要性が述べ
られている。また、これまでに報告されている植物 TGN に局在する因子と今回発見した
TGN における選別輸送ゾーンの関係についても考察している。今後は、より解像度の高
いライブイメージング技術を構築し、TGN を形成する膜脂質に注目することで、さらな
る植物 TGN の理解が進むと述べられている。
本研究は、ライブイメージングにより、細胞膜と液胞という異なる目的地に輸送される
積荷タンパク質の選別が、TGN 上の独立したゾーンで行われていることを明らかにした
研究である。これまでモデルとして提唱されてきた TGN の輸送選別ゾーンを、生きた細
胞で観察した最初の研究であり、独創性が高い。本論文の研究は、中野明彦博士、植村知
博博士らをはじめとする多くの研究者と共同で行われたが、論文提出者が主体となって実
験、観察および論証を行ったもので、論文提出者の寄与は十分である。
したがって、博士(理学)の学位を授与できると認める。

2

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