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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技術の開発

Publicly Offered Research

Project AreaMutli-dimensional fluorescence live imaging of cellular function and molecular activity
Project/Area Number 23113505
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) 2011-04-01 – 2013-03-31
Project Status Completed (Fiscal Year 2012)
Budget Amount *help
¥22,100,000 (Direct Cost: ¥17,000,000、Indirect Cost: ¥5,100,000)
Fiscal Year 2012: ¥11,050,000 (Direct Cost: ¥8,500,000、Indirect Cost: ¥2,550,000)
Fiscal Year 2011: ¥11,050,000 (Direct Cost: ¥8,500,000、Indirect Cost: ¥2,550,000)
Keywords可視化 / ナノバイオ / バイオテクノロジー / 生理活性 / 1分子計測
Outline of Annual Research Achievements

研究1:タンパク質間相互作用の超分解蛍光イメージング法の開発: PALMイメージングに必要なフォトクロミック蛍光タンパク質(PA-FP)の2分割体再構成法を開発した.Rapamycin添加に伴うinducibleなFRB―FKBP相互作用により,2分割蛍光タンパク質の再構成効率の最適化を行った.蛍光タンパク質は,mEos2, mEos3, Dronpa, EYFPについて検討した.その結果,EYFPが最も蛍光強度が強く,次にmEosが効率よく蛍光回復できることが解った.この最適化したフラグメントを用いて,GPCRとβarrestinとの相互作用を蛍光顕微鏡で観察可能かどうかを検討した.共焦点レーザー走査型蛍光顕微鏡によりイメージングを行い,EYFPの分割体を利用した時に,細胞膜上で強い蛍光が観察できることが解った. 研究2:動物個体内のアポトーシス細胞イメージング法:これまでに,アポトーシス過程におけるミトコンドリアからのSmacタンパク質の放出を,2分割蛍光タンパク質の再構成を利用した蛍光検出するactivatableプローブを開発した.本年度は,プローブを恒常的に発現するトランスジェニックゼブラフィッシュを作製した.プローブの発現は,ゼブラフィッシュからゲノムを抽出し,PCRによりスクリーニングを行った.ポジティブなクローンについて,さらにプローブの発現をWestern blotにより確認し,各プローブを全身に発現するトランスジェニック体を作成した.現在,作成したトランスジェニック体を掛け合わせ,その初期胚にUVを照射し,実際にアポトーシス細胞選択的に蛍光回復が起きるかどうかを検証している.

Research Progress Status

24年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

24年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2012 Annual Research Report
  • 2011 Annual Research Report
  • Research Products

    (31 results)

All 2013 2012 2011

All Journal Article (5 results) (of which Peer Reviewed: 5 results) Presentation (25 results) (of which Invited: 10 results) Book (1 results)

  • [Journal Article] Advances in fluorescence and bioluminescence imaging2013

    • Author(s)
      T. Ozawa, H. Yoshimura and S. B. Kim
    • Journal Title

      Anal. Chem.

      Volume: 85 Issue: 2 Pages: 590-609

    • DOI

      10.1021/ac3031724

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Fluorescent probes for imaging endogenous β-actin mRNA in living cells using fluorescent protein-tagged Pumilio.2012

    • Author(s)
      H. Yoshimura, A. Inaguma, T. Yamada and T. Ozawa.
    • Journal Title

      ACS Chem. Biol

      Volume: 7 Issue: 6 Pages: 999-1005

    • DOI

      10.1021/cb200474a

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Intelligent Design of Nano-Scale Molecular Imaging Agents2012

    • Author(s)
      S.B. Kim, M. Hattori and T. Ozawa
    • Journal Title

      Int. J. Mol. Sci.

      Volume: 13 Issue: 12 Pages: 16986-17005

    • DOI

      10.3390/ijms131216986

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Visualization of non-engineered single mRNAs in living cells using genetically encoded fluorescent probes2011

    • Author(s)
      T. Yamada, H. Yoshimura, A. Inaguma and T. Ozawa
    • Journal Title

      Anal. Chem

      Volume: 83 Issue: 14 Pages: 5708-5714

    • DOI

      10.1021/ac2009405

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Illuminating Intracellular Signaling and Molecules for Single Cell Analysis2011

    • Author(s)
      M.Awais
    • Journal Title

      Mol.Biosyst

      Volume: 7 Issue: 5 Pages: 1376-1387

    • DOI

      10.1039/c0mb00328j

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Presentation] 分割蛍光タンパク質再構成法による細胞内在性RNAのイメージング法2013

    • Author(s)
      小澤岳昌
    • Organizer
      日本薬学会第133年会
    • Place of Presentation
      パシフィコ横浜(神奈川県)
    • Year and Date
      2013-03-29
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] タンパク質再構成法を利用した生理機能のイメージングと光制御技術2013

    • Author(s)
      小澤岳昌
    • Organizer
      蛋白研セミナー
    • Place of Presentation
      大阪大学(大阪府)
    • Year and Date
      2013-03-04
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] 生理機能を探る蛍光イメージングの高度化と新たな光分析技術2012

    • Author(s)
      小澤岳昌
    • Organizer
      第17回先端光量子科学アライアンスセミナー
    • Place of Presentation
      東京大学(東京都)
    • Year and Date
      2012-12-18
    • Related Report
      2012 Annual Research Report
  • [Presentation] Opto-bioanalysis for imaging and control of biological functions using split protein-fragment reconstitution2012

    • Author(s)
      T. Ozawa
    • Organizer
      6th International Symposium on Nanomedicine
    • Place of Presentation
      くにびきメッセ(島根県)
    • Year and Date
      2012-12-01
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] 細胞生理機能の光操作と蛍光イメージング技術2012

    • Author(s)
      小澤岳昌
    • Organizer
      第1回分光イノベーションシンポジウム
    • Place of Presentation
      東京工業大学蔵前工業会館(神奈川)
    • Year and Date
      2012-11-30
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] Imaging and Control of Biological Functions Using Protein-Fragment Reconstitution Analyses2012

    • Author(s)
      T. Ozawa
    • Organizer
      The 1st International Symposium on Biofunctional Chemistry
    • Place of Presentation
      東京工業大学(神奈川県)
    • Year and Date
      2012-11-28
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] Opto-bioanalysis for imaging and control of biological functions in live cells2012

    • Author(s)
      T. Ozawa
    • Organizer
      Seminar in the Central Drug Research Institute
    • Place of Presentation
      Lucknow, India
    • Year and Date
      2012-11-23
    • Related Report
      2012 Annual Research Report
  • [Presentation] Rational Design of Functional Proteins for Living Cell Analyses2012

    • Author(s)
      T. Ozawa
    • Organizer
      The 7th International Forum on Chemistry of Functional Organic Chemicals
    • Place of Presentation
      東京大学(東京都)
    • Year and Date
      2012-11-19
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] 生理機能の理解に向けた光プローブの開発と新たな生体分子イメージング2012

    • Author(s)
      小澤岳昌
    • Organizer
      日本分析化学会第61年会
    • Place of Presentation
      金沢大学(石川県)
    • Year and Date
      2012-09-19
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] Functional Imaging of Intracellular Signals Using Protein-Fragment Reconstitution Techniques2012

    • Author(s)
      T. Ozawa
    • Organizer
      14th International Congress of Histochemistry and Cytochemistry
    • Place of Presentation
      京都国際会館(京都府)
    • Year and Date
      2012-08-27
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] タンパク質再構成法を利用する生理機能のイメージングと制御法2012

    • Author(s)
      小澤岳昌
    • Organizer
      ナノ学会第10回大会
    • Place of Presentation
      大阪大学(大阪府)
    • Year and Date
      2012-06-15
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] 光プローブが拓く生体分子イメージングと細胞制御技術2012

    • Author(s)
      小澤岳昌
    • Organizer
      第1回国際先端生物学・医学・工学会議
    • Place of Presentation
      名古屋大学(愛知県)
    • Year and Date
      2012-04-21
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] Imaging and Control of Biological Functions Using Split-reporter Reconstitution Analyses2012

    • Author(s)
      T.Ozawa
    • Organizer
      Molecular Imaging and Systems Biology
    • Place of Presentation
      東京(招待講演)
    • Year and Date
      2012-01-30
    • Related Report
      2011 Annual Research Report
  • [Presentation] Fluorescent and Bioluminescent Proteins Engineered for Visualizing Biological Function in Live Cells2011

    • Author(s)
      T.Ozawa
    • Organizer
      Seminar in the Central Drug Research Institute
    • Place of Presentation
      Lucknow, (India)(招待講演)
    • Year and Date
      2011-12-19
    • Related Report
      2011 Annual Research Report
  • [Presentation] Fluorescent and Bioluminescent Proteins Engineered for Visualizing Biological Function in Live Cells2011

    • Author(s)
      T.Ozawa
    • Organizer
      Seminar in the Sun Yat-sen University
    • Place of Presentation
      Guangzhou, (China)(招待講演)
    • Year and Date
      2011-12-15
    • Related Report
      2011 Annual Research Report
  • [Presentation] 生理機能の解析を目指したタンパク質再構成法の基礎と応用研究-再構成法の原理と使い方-2011

    • Author(s)
      小澤岳昌
    • Organizer
      第17回細胞生物学ワークショップ
    • Place of Presentation
      札幌(北海道)(招待講演)
    • Year and Date
      2011-11-22
    • Related Report
      2011 Annual Research Report
  • [Presentation] Imaging Analysis of Biological functions in Living Cells and Animals2011

    • Author(s)
      T.Ozawa
    • Organizer
      Seminar in the Center Hospital Shantou University Medical College
    • Place of Presentation
      Shantou (China)(招待講演)
    • Year and Date
      2011-10-31
    • Related Report
      2011 Annual Research Report
  • [Presentation] Imaging Analysis of Biological functions in Living Cells and Animals Using Split-Reporter Reconstitution Techniques2011

    • Author(s)
      T.Ozawa
    • Organizer
      Seminar in Chonnam National University Medical School
    • Place of Presentation
      Jeonnam (Korea)(招待講演)
    • Year and Date
      2011-10-07
    • Related Report
      2011 Annual Research Report
  • [Presentation] Imaging and Control of Biological functions Using Split-Reporter Reconstitution Analyses2011

    • Author(s)
      T.Ozawa
    • Organizer
      Korean Society of Molecular and Cellular Biology
    • Place of Presentation
      Seoul (Korea)(招待講演)
    • Year and Date
      2011-10-06
    • Related Report
      2011 Annual Research Report
  • [Presentation] タンパク質再構成技術を用いた生体分子イメージングと光分析法2011

    • Author(s)
      小澤岳昌
    • Organizer
      第85回日本生化学会大会
    • Place of Presentation
      京都(招待講演)
    • Year and Date
      2011-09-24
    • Related Report
      2011 Annual Research Report
  • [Presentation] Real-time analysis of intracellular signaling using the protein fragment reconstitution methods2011

    • Author(s)
      T.Ozawa
    • Organizer
      14^<th> Asian Chemical Congress
    • Place of Presentation
      Bangkok (Thailand)(招待講演)
    • Year and Date
      2011-09-05
    • Related Report
      2011 Annual Research Report
  • [Presentation] Imaging Biological functions Using Split-Reporter Reconstitution Analyses2011

    • Author(s)
      T.Ozawa
    • Organizer
      1^<st> International Symposium of Molecular Imaging and Nanomedicine
    • Place of Presentation
      Lijiang (China)(招待講演)
    • Year and Date
      2011-08-21
    • Related Report
      2011 Annual Research Report
  • [Presentation] Real-time analysis of intracellular signaling using the protein fragment reconstitution techniques2011

    • Author(s)
      T.Ozawa
    • Organizer
      Seminar in Department of Surgery & Oncology
    • Place of Presentation
      Liverpool (UK)(招待講演)
    • Year and Date
      2011-06-17
    • Related Report
      2011 Annual Research Report
  • [Presentation] Fluorescent and Bioluminescent Proteins Engineered for Visualizing Biological Function in Live Cells2011

    • Author(s)
      T.Ozawa
    • Organizer
      IUPAC Internation Congress on Analytical Sciences (ICAS) 2011
    • Place of Presentation
      Kyoto (Japan)(招待講演)
    • Year and Date
      2011-05-23
    • Related Report
      2011 Annual Research Report
  • [Presentation] Fluorescent and Bioluminescent Proteins Engineered for Visualizing Biological Function in Live Cells2011

    • Author(s)
      T.Ozawa
    • Organizer
      NCCR Chemical Biology Mini-Symposium
    • Place of Presentation
      Lausanne (Swiss)
    • Year and Date
      2011-04-20
    • Related Report
      2011 Annual Research Report
  • [Book] 学術の動向5月号(寄稿)2011

    • Author(s)
      小澤岳昌
    • Publisher
      日本学術会議
    • Related Report
      2011 Annual Research Report

URL: 

Published: 2011-04-06   Modified: 2018-03-28  

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