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分割蛍光タンパク質再構成法を用いた生理機能イメージングの実践応用研究

Publicly Offered Research

Project AreaMutli-dimensional fluorescence live imaging of cellular function and molecular activity
Project/Area Number 25113704
Research Category

Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionThe University of Tokyo

Principal Investigator

小澤 岳昌  東京大学, 理学(系)研究科(研究院), 教授 (40302806)

Project Period (FY) 2013-04-01 – 2015-03-31
Project Status Completed (Fiscal Year 2014)
Budget Amount *help
¥20,020,000 (Direct Cost: ¥15,400,000、Indirect Cost: ¥4,620,000)
Fiscal Year 2014: ¥10,010,000 (Direct Cost: ¥7,700,000、Indirect Cost: ¥2,310,000)
Fiscal Year 2013: ¥10,010,000 (Direct Cost: ¥7,700,000、Indirect Cost: ¥2,310,000)
Keywordsイメージング
Outline of Annual Research Achievements

研究課題1では,平成25年度に作製したトランスジェニックゼブラフィッシュを用いて,アポトーシス細胞のイメージングを行った.実体蛍光顕微鏡下でゼブラフィッシュを観察したところ,コントロールに対してUV照射したゼブラフィッシュからは,多くの緑色の輝点が観察された.この輝点が死細胞であることを確認するために, TUNEL法によりアポトーシス細胞の存在を確認した.TUNEL法により染色された細胞とプローブの緑色蛍光の局在が良い一致を示したことから,緑色蛍光輝点が確かに死細胞をしめしていることが解った.ゼブラフィッシュ生体の局所をUV刺激すると,生きたゼブラフィッシュで死細胞を確認できることを実証した.
研究課題2では,Bak凝集体を超解像で可視化検出するために,紫外線を照射してから細胞を固定し全反射蛍光顕微鏡で観察を行った.細胞内のミトコンドリア上で多くのBak凝集体を示す点状蛍光が観察された. 得られた画像の空間分解能を点状蛍光の半値全幅(FWHM)で評価したところ, 通常のイメージング法では330 nmであったのに対し, 本分析法では79 nmであった. 次に本分析法で得られた超解像画像を用いてBak凝集体のサイズ評価を行ったところ, 直径が100-1000 nm, 凝集体あたりのBak分子数が50-1700と広い分布を示した. この結果から, ミトコンドリア上で形成されるBak凝集体の7割以上が光学限界以下の直径であり, それらのうちの多くが100 nm程度と非常に小さいサイズであることが明らかとなった. さらに, アポトーシスを引き起こすBak凝集体に含まれるBak分子数はこれまで70-250程度と考えられてきたが, 実際にはより多くのBak分子が凝集体を形成していることが明らかとなった.その分子密度はBak分子数にかかわらず一定であることを実証した.

Research Progress Status

26年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

26年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2014 Annual Research Report
  • 2013 Annual Research Report
  • Research Products

    (21 results)

All 2015 2014 2013

All Journal Article (3 results) (of which Peer Reviewed: 3 results,  Open Access: 1 results,  Acknowledgement Compliant: 2 results) Presentation (18 results) (of which Invited: 18 results)

  • [Journal Article] Simultaneous time-lamination imaging of protein association using a split fluorescent timer protein2015

    • Author(s)
      A. Takamura, M. Hattori, H. Yoshimura and T. Ozawa
    • Journal Title

      Anal. Chem.

      Volume: 87 Issue: 6 Pages: 3366-3372

    • DOI

      10.1021/ac504583t

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Methods of split-reporter reconstitution for the analysis of biomolecules.2014

    • Author(s)
      H. Yoshimura and T. Ozawa
    • Journal Title

      Chem. Record

      Volume: 14 Issue: 3 Pages: 492-501

    • DOI

      10.1002/tcr.201402001

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Folding coupled with assembly in split green fluorescent proteins studied by structure-based molecular simulations.2013

    • Author(s)
      M. Ito, T. Ozawa and S. Takada
    • Journal Title

      J. Phys. Chem.

      Volume: 117 Issue: 42 Pages: 13212-13218

    • DOI

      10.1021/jp4032817

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Presentation] Imaging and analysis of biomolecules in living cells2015

    • Author(s)
      T. Ozawa,
    • Organizer
      PITTCON 2015
    • Place of Presentation
      New Orleans (USA)
    • Year and Date
      2015-03-10
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Luminescent sensors for single cell analysis2015

    • Author(s)
      T. Ozawa
    • Organizer
      Symposium on Multi-dimensional Fluorescence Live Imaging of Cellular Functions and Molecular Activities
    • Place of Presentation
      Kyoto
    • Year and Date
      2015-01-28
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Luminescent sensors for single cell analysis2014

    • Author(s)
      T. Ozawa,
    • Organizer
      Biomedical Molecular Imaging 2014
    • Place of Presentation
      Taipei (Taiwan)
    • Year and Date
      2014-11-08
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Luminescent sensors and optical switches for single cell analyses2014

    • Author(s)
      T. Ozawa
    • Organizer
      Swiss-Japan Chemical Biology Symposium 2014
    • Place of Presentation
      Bern (Swiss)
    • Year and Date
      2014-10-03
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Visualising and Manipulating Cell Stimulation2014

    • Author(s)
      T. Ozawa
    • Organizer
      8th Pituitary Meeting
    • Place of Presentation
      Edinburgh (UK)
    • Year and Date
      2014-09-22
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Imaging and Analysis of GPCRs using Protein Complementation Analysis2014

    • Author(s)
      T. Ozawa,
    • Organizer
      7th Summer School Medicinal Chemistry in Regensburg
    • Place of Presentation
      Regensburg (Germany)
    • Year and Date
      2014-09-18
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] タンパク質再構成法を利用する生理機能イメージング2014

    • Author(s)
      小澤岳昌
    • Organizer
      顕微鏡学会第70回記念学術講演会
    • Place of Presentation
      千葉,幕張メッセ
    • Year and Date
      2014-05-30
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] 13.Imaging of Dynamic Molecules and Signaling in Living Cells2014

    • Author(s)
      T. Ozawa
    • Organizer
      Asian International Symposium of CSJ
    • Place of Presentation
      Nagoya (Aichi)
    • Year and Date
      2014-03-28
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] タンパク質再構成法を利用する生理機能イメージング2014

    • Author(s)
      小澤岳昌
    • Organizer
      日本顕微鏡学会第70回記念学術講演会
    • Place of Presentation
      千葉
    • Year and Date
      2014-03-28
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] Protein Aggregation in Mitochondrial Apoptosis Revealed by Photoactivated Localization Microscopy2013

    • Author(s)
      T. Ozawa
    • Organizer
      SuperImaging
    • Place of Presentation
      Hamamatsu (Shizuoka)
    • Year and Date
      2013-12-02
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] 17.Fluorescence and Bioluminescence Imaging of Biological Functions Using Split-Reporter Reconstitution Analysis2013

    • Author(s)
      T. Ozawa,
    • Organizer
      The 9th International Forum on Post-Genome Technologies (9’IFPT)
    • Place of Presentation
      Shenzhen (China)
    • Year and Date
      2013-11-09
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] 生理機能を探る蛍光イメージングと新たな光分析法2013

    • Author(s)
      小澤岳昌
    • Organizer
      次世代マイクロ化学チップコンソーシアム第26回研究会
    • Place of Presentation
      東京
    • Year and Date
      2013-10-22
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] Luminescence Imaging of Biological functions Using Split-Reporter Reconstitution Analyses2013

    • Author(s)
      T. Ozawa, H. Yoshimura, M. Hattori
    • Organizer
      The second international conference on the frontier research in organic synthesis and materials
    • Place of Presentation
      Taiwan (China)
    • Year and Date
      2013-09-22
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] ルシフェラーゼ発光を利用した生理機能in vivoイメージング2013

    • Author(s)
      小澤岳昌
    • Organizer
      In vivoイメージングフォーラム2013
    • Place of Presentation
      東京工業大学蔵前工業会館(神奈川)
    • Year and Date
      2013-09-06
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] Imaging and Control of Biological Functions Using Protein-Fragment Reconstitution analysis2013

    • Author(s)
      T. Ozawa
    • Organizer
      RSC Tokyo International Conference 2013
    • Place of Presentation
      Chiba (Japan)
    • Year and Date
      2013-09-05
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] Imaging and Control of Biological Functions Using Protein-Fragment Reconstitution analysis2013

    • Author(s)
      T. Ozawa,
    • Organizer
      IEEE EMBC 2013 WorkShop
    • Place of Presentation
      Osaka (Japan)
    • Year and Date
      2013-07-03
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] Optical Control and Imaging of Biological Function in Live Cells2013

    • Author(s)
      T. Ozawa,
    • Organizer
      Systems Biology of Cellular Signaling
    • Place of Presentation
      Tokyo (Japan)
    • Year and Date
      2013-07-01
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] Imaging and Control of Biological Functions Using Split-Reporter Reconstitution Analysis,2013

    • Author(s)
      T. Ozawa,
    • Organizer
      10th Anniversary of Hwasun Optical Imaging Workshop & Symposium
    • Place of Presentation
      Gwanju (Korea)
    • Year and Date
      2013-05-28
    • Related Report
      2013 Annual Research Report
    • Invited

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Published: 2013-05-15   Modified: 2018-03-28  

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