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相互相関分光法を利用した細胞内分子間相互作用の解析

Research Project

Project/Area Number 19021001
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Science and Engineering
Research InstitutionHokkaido University

Principal Investigator

金城 政孝  Hokkaido University, 大学院・先端生命科学研究院, 教授 (70177971)

Co-Investigator(Kenkyū-buntansha) 野村 保友  山形大学, 医学研究科, 准教授 (80237883)
藤井 文彦  大阪大学, 免疫学フロンティア研究センター, 助教 (40374657)
Project Period (FY) 2007 – 2008
Project Status Completed (Fiscal Year 2008)
Budget Amount *help
¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 2008: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2007: ¥2,300,000 (Direct Cost: ¥2,300,000)
Keywords生物物理 / 分子認識 / 分子機械 / 蛍光測定 / ゆらぎ / 単1細胞 / 拡散 / 拡散定数 / 全反射顕微鏡 / 空間相関 / 空間相互相関
Research Abstract

単1細胞機能の時間的・空間的な情報を得るためにはイメージングの手法が重要であるが、細胞内を早い速度で「離合集散」しながら複合体形成を行い細胞機能を支えている分子を捉えるために申請者は蛍光相関分光法(Fluorescence Correlation Spectroscopy ; FCS)に着目した。なぜならFCSは動きの速い分子の相互作用検出に適し、しかも1分子レベルまでも検出が可能だからである。しかし一方で単1細胞内の分子間相互作用や、分子数の変化は細胞内の測定場所での違いが大きく反映されるために、正しい評価を行うためには多数の点で同時にFCS測定を行い、空間情報として分子の「動き」や「結合」の情報を得る必要がある。これらの点を克服するために、多点同時測定のFCSが有効であると考えた。まず全反射光学系と斜光光学系を検討し,励起光強度が安定に維持できることなどから本研究においては,最終的に全反射光学系を採用した。次に,細胞中の多点として4点でFCS測定を同時に行うことができる測定システムの開発を行ない、細胞内分子間相互作用の統合的理解を目的とした。当初計画していた単1細胞内4点ではなく, 検出部としての光ファイバーの設置の安定性から7点測定とし, 装置を完成させた。さらに, 膜結合性GFPの構築と細胞内発現を行い, 実際に単1生細胞中におけるタンパク質の7点同時に蛍光強度変化,ならびそこからのFCS測定が可能であることを実証した。7点からの情報のすべての組み合わせである21通りが可能であるが, その中で数点の空間相互相関解析を試みた。また, 膜結合タンパク質としてNaポンプタンパク質等の測定を行った。その結果,複合体を形成することで確実に拡散速度が低下することを明らかにした。今後の研究の方向性として, 直接相互作用の検出,細胞内の任意の空間での検出などの必要性が明らかになった。現在, 各点の時間相互相関解析が可能とするために, 各測定点にダイクロイックミラーの設置を行い, 直接相互作用の解析を試みている。

Report

(2 results)
  • 2008 Annual Research Report
  • 2007 Annual Research Report
  • Research Products

    (42 results)

All 2009 2008 2007

All Journal Article (25 results) (of which Peer Reviewed: 25 results) Presentation (15 results) Book (2 results)

  • [Journal Article] Multipoint fluorescence correlation spectroscopy with total internal reflection fluorescence microscope2009

    • Author(s)
      Ohsugi Y
    • Journal Title

      J Biomed Opt 14(1)

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Fluorescence cross-correlation spectroscopy of plant proteins2009

    • Author(s)
      Muto H
    • Journal Title

      Methods Mol Biol. 479

      Pages: 203-215

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] The A- and B-type nuclear lamin networks : microdomains involved in chromatin organization and transcription2008

    • Author(s)
      Shimi T
    • Journal Title

      Genes Dev. 22(24)

      Pages: 3409-3421

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cross-talk-free fluorescence cross-correlation spectroscopy by the switching method2008

    • Author(s)
      Takahashi Y
    • Journal Title

      Cell Struct Funct 33(1)

      Pages: 143-150

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking2008

    • Author(s)
      Noda Y
    • Journal Title

      J Cell Biol. 182(3)

      Pages: 587-601

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy2008

    • Author(s)
      Park H
    • Journal Title

      Mol Cells. 26(1)

      Pages: 87-92

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Stress-induced environmental changes in a single cell as revealed by fluorescence lifetime imaging2008

    • Author(s)
      Nakabayashi T
    • Journal Title

      Photochem Photobiol Sci. 7(6)

      Pages: 671-674

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements2008

    • Author(s)
      Nakabayashi T
    • Journal Title

      Photochem Photobiol Sci. 7(6)

      Pages: 668-670

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Regulated motion of glycoproteins revealed by direct visualization of a single car go in the endoplasmic reticulum.2008

    • Author(s)
      Nagaya H
    • Journal Title

      J Cell Biol 180

      Pages: 129-143

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Analysis of the molecular dynamics of medaka nuage proteins by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching.2008

    • Author(s)
      Nagao I
    • Journal Title

      FEBS J 275

      Pages: 341-349

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Temporal and spatial localization of three germline-specific proteins in medaka.2008

    • Author(s)
      Aoki Y
    • Journal Title

      Dev Dyn 237

      Pages: 800-807

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] 蛍光相関分光法によるダイナミックバイオイメージング2008

    • Author(s)
      金城政孝
    • Journal Title

      電気学会誌 128

      Pages: 11-15

    • NAID

      10020026175

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Diffusion analysis of glucocorticoid receptor and antagonist effect in living cell nucleus.2007

    • Author(s)
      Shintaro Mikuni
    • Journal Title

      Experimental and Molecular Pathology 82

      Pages: 163-168

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Triplet Fraction Buildup Effect of the DNA-YOYO Complex Studied with Fluorescence Correlation Spectroscopy.2007

    • Author(s)
      Masafumi Shimizu
    • Journal Title

      Analytical Biochem 366

      Pages: 87-92

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance.2007

    • Author(s)
      Kabayama K
    • Journal Title

      Proc Natl Acad Sci USA 104

      Pages: 13678-13683

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Detection of polyglutamine protein oligomers in cells by fluorescence correlation spectroscopy.2007

    • Author(s)
      Takahashi Y
    • Journal Title

      J Biol Chem 282

      Pages: 24039-24048

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy.2007

    • Author(s)
      Fujii F
    • Journal Title

      Anal Biochem 370

      Pages: 131-141

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Preparation and characterization of thiacalix[4]arene coated water-soluble CdSe/ZnS quantum dots as a fluorescent probe for Cu2+ ions.2007

    • Author(s)
      Jin T
    • Journal Title

      Comb Chem High Throughput Screen 10

      Pages: 473-479

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Effect of cholesterol on diffusion in surfactant bilayers.2007

    • Author(s)
      Pieper T
    • Journal Title

      J Chem Phys 127

      Pages: 165102-165102

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Detection of antigen protein by using fluorescence cross-correlation spectroscopy and quantum-dot-labeled antibodies.2007

    • Author(s)
      Fujii F
    • Journal Title

      Chembiochem 8

      Pages: 199-203

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] 蛍光相関分光法と分子ものさしを用いた細胞内微環境の解析2007

    • Author(s)
      金城政孝
    • Journal Title

      北海道大学出版会

      Pages: 73-85

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] 蛍光相関分光法による生体1分子解析2007

    • Author(s)
      金城政孝
    • Journal Title

      化学 別冊「分子イメージング」

      Pages: 72-76

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] FCSによる解析法2007

    • Author(s)
      三國新太郎
    • Journal Title

      別冊「分子間相互相互作用解析ハンドブック」

      Pages: 85-89

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] FCCSによるタンパク質相互作用解析2007

    • Author(s)
      三國新太郎
    • Journal Title

      別冊「分子間相互相互作用解析ハンドブック」

      Pages: 95-95

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] 相互作用の定量化:イメージングと蛍光相関分光法2007

    • Author(s)
      金城政孝
    • Journal Title

      化学と生物 45

      Pages: 750-756

    • NAID

      10019578227

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Presentation] 多点全反射蛍光相関装置による細胞測定2008

    • Author(s)
      金城政孝
    • Organizer
      第5回バイオオプティクス研究会
    • Place of Presentation
      浜松市
    • Year and Date
      2008-12-19
    • Related Report
      2008 Annual Research Report
  • [Presentation] Study of Mombrane-Binding Protein Dynamics with Fluorescence Correlation Spectroscopy2008

    • Author(s)
      Masataka Kinjo
    • Organizer
      The Annual Meeting of the Spectroscopical Society of Japan
    • Place of Presentation
      Sendai JAPAN
    • Year and Date
      2008-11-20
    • Related Report
      2008 Annual Research Report
  • [Presentation] Dybamic Aspects of Protein in Living Cell Investigated by Total Internal Reflection Fluourescence Correlation Spectroscopy2008

    • Author(s)
      Masataka Kinjo
    • Organizer
      International Congress on Cell Biology
    • Place of Presentation
      Seoul KOREA
    • Year and Date
      2008-10-09
    • Related Report
      2008 Annual Research Report
  • [Presentation] FCSとFCCS2008

    • Author(s)
      金城政孝
    • Organizer
      第4回ライブセルイメージング講習会
    • Place of Presentation
      つくば市
    • Year and Date
      2008-10-07
    • Related Report
      2008 Annual Research Report
  • [Presentation] 蛍光相関分光法を用いた生命システムの解析2008

    • Author(s)
      金城政孝
    • Organizer
      日本物理学会2008年秋季大会
    • Place of Presentation
      盛岡市
    • Year and Date
      2008-09-22
    • Related Report
      2008 Annual Research Report
  • [Presentation] Dynamics and Interactions of Protein in Living Cells Analyzed by Fluorescence Correlation Spectroscopy2008

    • Author(s)
      Masataka Kinjo
    • Organizer
      10th Seoul National University & Hokkaido University Joint Symposium
    • Place of Presentation
      Seoul
    • Year and Date
      2008-01-25
    • Related Report
      2007 Annual Research Report
  • [Presentation] 蛍光相関分光法を利用したin vivo分子間相互作用解析2007

    • Author(s)
      金城政孝
    • Organizer
      第30回日本分子生物学会年会・第80回日本生化学会大会 合同大会
    • Place of Presentation
      横浜
    • Year and Date
      2007-12-13
    • Related Report
      2007 Annual Research Report
  • [Presentation] 蛍光相関分光法による生細胞内微環境の解析2007

    • Author(s)
      金城政孝
    • Organizer
      第3回ライブセルイメージング講習会
    • Place of Presentation
      つくば
    • Year and Date
      2007-10-12
    • Related Report
      2007 Annual Research Report
  • [Presentation] 蛍光相関分光法によるin vitro 並びにin vivo分子間相互作用解析2007

    • Author(s)
      金城政孝
    • Organizer
      第48回日本組織細胞化学会総会
    • Place of Presentation
      甲府
    • Year and Date
      2007-09-29
    • Related Report
      2007 Annual Research Report
  • [Presentation] 蛍光相関分光法を用いた細胞内分子間相互作用解析と創薬への応用2007

    • Author(s)
      金城政孝
    • Organizer
      第56回高分子討論会
    • Place of Presentation
      名古屋
    • Year and Date
      2007-09-19
    • Related Report
      2007 Annual Research Report
  • [Presentation] 蛍光相関分光法2007

    • Author(s)
      金城政孝
    • Organizer
      日本分光学会第43回夏期セミナー
    • Place of Presentation
      千葉
    • Year and Date
      2007-08-30
    • Related Report
      2007 Annual Research Report
  • [Presentation] 次世代バイオイメージングとしての蛍光相関分光法:分子間相互作用の解析による新たな医学研究の発展2007

    • Author(s)
      金城政孝
    • Organizer
      産学官連携を指向した最前線セミナー
    • Place of Presentation
      東京
    • Year and Date
      2007-07-27
    • Related Report
      2007 Annual Research Report
  • [Presentation] 光相関分光法を用いた細胞内微環境の解析2007

    • Author(s)
      金城政孝
    • Organizer
      第53回高分子夏季大学
    • Place of Presentation
      北海道
    • Year and Date
      2007-07-19
    • Related Report
      2007 Annual Research Report
  • [Presentation] Dynamics and interaction of protein in living cell analysis by FCS2007

    • Author(s)
      Masataka Kinjo
    • Organizer
      EMBO Practical Course: Imaging in 3D and the F-techniques: FRET, FCS, FLIM and FRAP
    • Place of Presentation
      Singapore
    • Year and Date
      2007-06-18
    • Related Report
      2007 Annual Research Report
  • [Presentation] 蛍光相関分光法によるタンパク質の機能解析2007

    • Author(s)
      金城政孝
    • Organizer
      第59回日本細胞生物学会
    • Place of Presentation
      福岡
    • Year and Date
      2007-05-29
    • Related Report
      2007 Annual Research Report
  • [Book] 動物実験代替のためのバイオマテリアル・デバイス2007

    • Author(s)
      齋藤有香
    • Total Pages
      13
    • Publisher
      シーエムシー出版
    • Related Report
      2007 Annual Research Report
  • [Book] 生細胞蛍光イメージング2007

    • Author(s)
      金城政孝
    • Total Pages
      40
    • Publisher
      共立出版
    • Related Report
      2007 Annual Research Report

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Published: 2007-04-01   Modified: 2018-03-28  

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