核膜孔複合体を介した物質輸送機構解明のための1分子解析学的アプローチ

• Project/Area Number: 17687012
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Single-year Grants
• Research Field: Functional biochemistry
• Research Institution: Kyoto University
• Principal Investigator: 吉村 成弘 Kyoto University, 生命科学研究科, 准教授 (90346106)
• Project Period (FY): 2005 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥30,940,000 (Direct Cost: ¥23,800,000、Indirect Cost: ¥7,140,000); Fiscal Year 2007: ¥5,980,000 (Direct Cost: ¥4,600,000、Indirect Cost: ¥1,380,000); Fiscal Year 2006: ¥5,980,000 (Direct Cost: ¥4,600,000、Indirect Cost: ¥1,380,000); Fiscal Year 2005: ¥18,980,000 (Direct Cost: ¥14,600,000、Indirect Cost: ¥4,380,000)
• Keywords: 核輸送 / 核膜孔複合体 / インポーティン / 一分子観察 / 一分子力測定 / 原子間力顕微鏡 / 核内輸送 / 1分子蛍光観察技術 / 1分子計測 / 1分子蛍光

Research Abstract

(1)原子間力顕微鏡を用いたインポーティンβ-ヌクレオポリン間相互作用の一分子力測定
インポーティンβをカンチレバーに結合させ、ガラス基板上に吸着させたヌクレオポリン(Nup153)との間の相互作用を原子間力顕微鏡を用いた1分子力測定により解析した。この結果、インポーティンβには少なくとも4つの性質の異なる結合ポケットが存在すること、これらのポケットはRanGTPの結合により異なる影響を受けること、を明らかにした。
(2)FGヌクレオポリンと輸送担体とのアフィニティー測定
マイクロウェルプレートアッセイ、および表面プラズモン共鳴を用いて、FGリピートを含む10種のヌクレオポリンと輸送担体との解離定数および速度定数を測定した。この結果、インポーティンβの場合、ほぼ全てのヌクレオポリンで解離定数は数nMから数10nMの範囲であった。核膜孔複合体の外側と内側のヌクレオポリンで解離定数に大きな差は得られなかった。
(3)二光路顕微鏡による核輸送の1分子4次元観察
量子ドットにストレプトアビジンを共有結合させ、これに末端をビオチン化した核輸送シグナルペプチドを結合させた。ジギトニン処理して細胞膜と細胞質を取り除いたHeLa細胞にこの量子ドットとインポーティンβなどの輸送に必要なタンパク質を加え、量子ドットが核膜孔を通過して核内に輸送される様子を二光路蛍光顕微鏡により観察した。この顕微鏡システムはx,y方向に3nm、z方向に10nmの空間解像度で毎秒1000枚のイメージを取得することができる。つまり1つの量子ドットの輝点を1ミリ秒の時間分解能および数nmの空間分解能で追跡することができる系が確立できた。

Research Products

• [Journal Article] Dynamic structures of Bacillus subtilis RecN-DNA complexes. (2008)
  • Author(s): S. H. Yoshimura
  • Journal Title: Nuc. Acids Res. 36 Pages: 110-120
  • Peer Reviewed
• [Journal Article] Following translation by single ribosomes one codon at a time (2008)
  • Author(s): S. H. Yoshimura
  • Journal Title: Nature 452 Pages: 598-603
  • Peer Reviewed
• [Journal Article] Nuclear Architectures and Chromatin Dynamics Revealed by Atomic Force Microscopy in Combination with Biochemistry and Cell Biology (2008)
  • Author(s): S. H. Yoshimura
  • Journal Title: Pflugers Arch. 456 Pages: 139-153
  • Peer Reviewed
• [Journal Article] Scanning probe microscope revealed mechanical properties of plasma membrane and nuclear envelope in living cells (2008)
  • Author(s): S. H. Yoshimura
  • Journal Title: J. Microscopy (印刷中)
  • Peer Reviewed
• [Journal Article] Atomic force microscopy dissects the hierarchy of genome architecture in eukaryote, prokaryote and chloroplast. (2007)
  • Author(s): S. H. Yoshimura
  • Journal Title: Microsc. Microanal. 13 Pages: 3-12
  • Peer Reviewed
• [Journal Article] Single-molecule detection of phosphorylation-induced plasticity changes during ezrin activation. (2007)
  • Author(s): S. H. Yoshimura
  • Journal Title: FEBS Lett. 581 Pages: 3536-3571
  • Peer Reviewed
• [Journal Article] Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and loooing. (2007)
  • Author(s): S. H. Yoshimura
  • Journal Title: Proc. Natl. Acad. Sci. USA. 104 Pages: 12755-12760
  • Peer Reviewed
• [Journal Article] Covalent attachment of protein to the tip of a multiwalled carbon nanotube without sidewall decoration. (2007)
  • Author(s): S. H. Yoshimura
  • Journal Title: J. Appl. Phys. 102 Pages: 94701-94701
  • Peer Reviewed
• [Journal Article] Atomic force microscopy dissects the hierarchy of genome architecture in eukaryote, prokaryote and chloroplast. (2007)
  • Author(s): S.H.Yoshimura 他
  • Journal Title: Microsc.Microanal. 13・1 Pages: 3-12
• [Journal Article] Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy (2006)
  • Author(s): S.H.Yoshimura 他
  • Journal Title: FEBS Letter 580・16 Pages: 3961-3965
• [Journal Article] Fast-scanning stomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL (2006)
  • Author(s): S.H.Yoshimura 他
  • Journal Title: EMBO J. 25・19 Pages: 4567-4576
• [Journal Article] Fast-scanning atomic force microscopy revealed the molecular mechanism of DNA cleavage by ApaI endonuclease. (2006)
  • Author(s): S.H.Yoshimura 他
  • Journal Title: IEE Proc. Nonobiotech. 153・4 Pages: 60-66
• [Journal Article] Comparative structural biology of the genome : Nano-scale imaging of single nucleus from different kingdoms reveals the chromatin built up (2006)
  • Author(s): S.H.Yoshimura 他
  • Journal Title: J.Electr.Microsc. 55・1 Pages: 31-40
• [Journal Article] Human ObgH1 and ObgH2 participate in the maintenance of mitochondria and nucleolar architectures. (2006)
  • Author(s): S.H.Yoshimura 他
  • Journal Title: Genes Cells 11 Pages: 1295-1304
• [Journal Article] Comparative structural biology of the genome : Nano-scale imaging of single nucleus from different kingdoms reveals the chromatin built up on a 40 nm (2006)
  • Author(s): S.H.Yoshimura 他
  • Journal Title: Journal of Electron Microscopy (印刷中)
• [Journal Article] Fast-scanning atomic force microscopy revealed the molecular mechanism of DNA cleavage by Apal endonuclease. (2006)
  • Author(s): S.H.Yoshimura 他
  • Journal Title: IEE Proc.Nanobiotech. (印刷中)
• [Journal Article] Phase transition in reconstituted chromatin. (2005)
  • Author(s): S.H.Yoshimura 他
  • Journal Title: Europhys.Lett. 69・6 Pages: 1024-1030
• [Journal Article] Linker histone H1 per se can induce three-dimensional folding of chromatin fiber. (2005)
  • Author(s): K.Hizume, S.H.Yoshimura, 他
  • Journal Title: Biochemistry 44・39 Pages: 2978-12989
• [Presentation] Nano-scale imaging and single molecule manipulation techniques for the study of nuclear dynamics. (2008)
  • Author(s): S.H. Yoshimura
  • Organizer: The 9th AEARU Workshop on Molecular Biology and Biotechnology
  • Place of Presentation: Hong Kong, China
  • Year and Date: 2008-03-26
• [Presentation] Advances in Single-molecular Research for Biology & Nanoscience (2008)
  • Author(s): S.H. Yoshimura
  • Organizer: Xth Annual Linz Winter Workshop
  • Place of Presentation: Linz, Austria
  • Year and Date: 2008-02-17
• [Presentation] Nano-scale Imaging and single-molecule manipulation techniques for uncovering the nuclear architecture and dynamics. (2008)
  • Author(s): S.H. Yoshimura
  • Organizer: Xth Asian Conference on Transcription and Transcription Assembly
  • Place of Presentation: Bangarol, India
  • Year and Date: 2008-01-15
• [Presentation] Nano-biology of cell nucleus (2007)
  • Author(s): S. H. Yoshimura
  • Organizer: 第30回日本分子生物学会第80回日本生化学会合同大会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-12
• [Presentation] Sub-nuclear structures and their dynamics revealed by a set of nuclear antibodies (2007)
  • Author(s): S. H. Yoshimura
  • Organizer: American Society for Cell Biology-47th annual meeting
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2007-12-03
• [Presentation] Single-molecule force measurement revealed the characteristics of protein transport through Nuclear Pore Complex (2007)
  • Author(s): S. H. Yoshimura
  • Organizer: American Society for Cell Biology-47th annual meeting
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2007-12-03
• [Presentation] Single-molecule imaging, force measurement and fluorescence observation reveal protein and chromosome dynamics around the nuclear envelope (2007)
  • Author(s): S. H. Yoshimura
  • Organizer: International Symposium on Micro-NanoMechatronics and Human Science
  • Place of Presentation: Nagoya, Japan
  • Year and Date: 2007-11-12
• [Presentation] 1分子観察・計測・操作技術とバイオ操作 (2007)
  • Author(s): 吉村成弘
  • Organizer: 日本機会学会先端技術フォーラム
  • Place of Presentation: 大阪
  • Year and Date: 2007-10-13
• [Presentation] LEMタンパク質MAN1の細胞周期依存的リン酸化とその機能 (2007)
  • Author(s): 吉村成弘
  • Organizer: 第7回核ダイナミクス研究会
  • Place of Presentation: 札幌
  • Year and Date: 2007-09-26
• [Presentation] 核膜孔複合体を介した物質輸送の1分子解析 (2007)
  • Author(s): 吉村成弘
  • Organizer: 第7回核ダイナミクス研究会
  • Place of Presentation: 札幌
  • Year and Date: 2007-09-26
• [Presentation] The tudor domain of LBR plays an important role for its mobility in the nuclear envelope (2007)
  • Author(s): S. H. Yoshimura
  • Organizer: EMBO Conference Series on Nuclear Structure and Dynamics
  • Place of Presentation: Montpellier, France
  • Year and Date: 2007-09-03
• [Book] Nuclear Dynamics (2006)
  • Author(s): S.H.Yoshimura
  • Total Pages: 280
  • Publisher: Springer
• [Book] Nuclear Dynamics (2006)
  • Author(s): S.H.Yoshimura
  • Publisher: Springer Verlag(未定)