多点時空間相関解析法による細胞内分子複合体研究

• Project/Area Number: 21221006
• Research Category: Grant-in-Aid for Scientific Research (S)
• Allocation Type: Single-year Grants
• Research Field: Nanomaterials/Nanobioscience
• Research Institution: Hokkaido University
• Principal Investigator: 金城 政孝 北海道大学, 先端生命科学研究科(研究院), 教授 (70177971)
• Co-Investigator(Kenkyū-buntansha): 北村 朗 北海道大学, 先端生命科学研究科(研究院), 助教 (10580152) ; 三國 新太郎 北海道大学, 医学(系)研究科(研究院), 特任助教 (40435954)
• Project Period (FY): 2009-05-11 – 2014-03-31
• Project Status: Declined (Fiscal Year 2013)
• Budget Amount: ¥176,670,000 (Direct Cost: ¥135,900,000、Indirect Cost: ¥40,770,000); Fiscal Year 2013: ¥26,000,000 (Direct Cost: ¥20,000,000、Indirect Cost: ¥6,000,000); Fiscal Year 2012: ¥26,000,000 (Direct Cost: ¥20,000,000、Indirect Cost: ¥6,000,000); Fiscal Year 2011: ¥36,400,000 (Direct Cost: ¥28,000,000、Indirect Cost: ¥8,400,000); Fiscal Year 2010: ¥41,600,000 (Direct Cost: ¥32,000,000、Indirect Cost: ¥9,600,000); Fiscal Year 2009: ¥46,670,000 (Direct Cost: ¥35,900,000、Indirect Cost: ¥10,770,000)
• Keywords: 1分子計測 / 可視化 / 生物物理 / 細胞・組織 / シグナル伝達 / 時間相関 / 空間相関 / 空間光位相変調

Research Abstract

蛍光相関分光法（Fluorescence Correlation Spectroscopy，FCS）はダイナミックな分子間相互作用を単一分子レベルで解析する手法でるが、その一方で、励起光源にレーザー光が必要であり、その焦点領域である、特定の１点でしか測定出来ない。このことは生細胞が様々な区画に分かれていて、さらにその区画の中も不均一であるため、細胞生物学的応用の際の問題となっている。その解決のために本年も引き続き多点同時測定FCSの構築と生細胞測定への応用を目指ざしている。
そこでこれまで多点測定のためのまず、多点の焦点領域を作る必要がある。そのために空間光変調素子を利用したホログラフィ技術により複数照射の励起光パターンを作りだすことを可能とした。次に検出側としてマルチ光ファイバーと各端面に光電子増倍管を接続した多点検出装置を用いて，実際に生細胞内の多点におけるタンパク質の拡散運動を検出する装置を作成した。
次に本年は細胞応用測定に重点を置き、細胞質ならび細胞核を区別して7点同時測定を行った。
生細胞内GFPを対象としてガラス面からZ軸方向（上方向）に0.5ミクロンステップの空間分解能があることを証明し、次に細胞核膜を境にして同時7点測定FCS測定が可能であることを実証した。次に機能性タンパク質としてリガンド刺激により、細胞質から核へ移行するグルココルチコイドレセプター（GR）のGFP融合タンパク質を対象とした測定を行い、15秒おきのFCS多点同時測定に成功した。さらに本年は精度を上げるために細胞運動の抑制方法を種々検討し、ノコダゾール処理と、緩衝液を調整することで、長期の観測を可能とした。
安定測定が可能となったので、さらに時間分解を上げることが可能となったので、単一タンパク質分子の動態について、検出と解析を試みている。

Current Status of Research Progress

Reason

翌年度、交付申請を辞退するため、記入しない。

Strategy for Future Research Activity

翌年度、交付申請を辞退するため、記入しない。

Research Products

• [Journal Article] Virus-like particles with removable cyclodextrins enable glutathione-triggered drug release in cells. (2013)
  • Author(s): Niikura K.
  • Journal Title: Mol Biosyst. Volume: 9(3) Issue: 3 Pages: 501-507
  • DOI: 10.1039/c2mb25420d
  • Peer Reviewed
• [Journal Article] Nonmuscle myosin II folds into a 10S form via two portions of tail for dynamic subcellular loacalization. (2013)
  • Author(s): Kiboku T.
  • Journal Title: Genes to Cells. Volume: 18(2) Issue: 2 Pages: 90-109
  • DOI: 10.1111/gtc.12021
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• [Journal Article] pH Dependence of the Fluorescence Lifetime of FAD in Solution and in Cells. (2013)
  • Author(s): Islam MS.
  • Journal Title: Int J Mol Sce. Volume: 14(1) Issue: 1 Pages: 1952-1963
  • DOI: 10.3390/ijms14011952
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• [Journal Article] Local nucleosome dynamics facilitate chromatin accessibility in living Mammalian cells. (2012)
  • Author(s): Hihara S.
  • Journal Title: Cell Rep. Volume: 2(6) Issue: 6 Pages: 1645-1656
  • DOI: 10.1016/j.celrep.2012.11.008
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• [Journal Article] Direct association of unfolded proteins with mammalian ER stress sensor, IRE1 beta. (2012)
  • Author(s): Oikawa D.
  • Journal Title: PLoS One. Volume: 7(12) Issue: 12 Pages: e51290-e51290
  • DOI: 10.1371/journal.pone.0051290
  • Peer Reviewed
• [Journal Article] Atg9 vesicles are an important membrane source during early steps of autophagosome formation. (2012)
  • Author(s): Yamamoto H.
  • Journal Title: J. Cell Biol. Volume: 198(2) Issue: 2 Pages: 219-233
  • DOI: 10.1083/jcb.201202061
  • Peer Reviewed
• [Journal Article] A small peptide sequence is sufficient for initiating Kinesin-1 activation through part of TPR region of KLC1 (2012)
  • Author(s): Kawano T, Araseki M, Araki Y, Kinjo M, Yamamoto T, Suzuki T
  • Journal Title: Traffic Volume: (掲載確定) Issue: 6 Pages: 834-848
  • DOI: 10.1111/j.1600-0854.2012.01350.x
  • Peer Reviewed
• [Journal Article] Fluorescence Imaging of Mitochondria in Living Cells Using a Novel Fluorene Derivative with a Large Two-Photon Absorption Cross-Section. (2012)
  • Author(s): Tani S.
  • Journal Title: Curr Pharm Biotechnol. Volume: 13(14) Pages: 2649-2654
  • Peer Reviewed
• [Journal Article] Use of Carbohydrate-Conjugated Nanoparticles for an Integrated Approach to Functional Imaging of Glycans and Understanding of Their Molecular Mechanisms. (2012)
  • Author(s): Nagahori N.
  • Journal Title: Curr Pharm Biotechnol. Volume: 13(14) Pages: 2612-2616
  • Peer Reviewed
• [Journal Article] Current Research on Protein-Protein Interactions Among Auxin-Singnaling Factors in Regulation of Plant Grouwth and Development. (2012)
  • Author(s): Muto H.
  • Journal Title: Curr Pharm Biotechnol. Volume: 13(14) Pages: 2604-2611
  • Peer Reviewed
• [Journal Article] Bovine serum albumin-coated quantum dots as a cytoplasmic viscosity probe in a single living cell (2012)
  • Author(s): Nakane Y.
  • Journal Title: Anal. Methods Volume: 4 Issue: 7 Pages: 1903-1905
  • DOI: 10.1039/c2ay25318f
  • Peer Reviewed
• [Journal Article] 蛍光相互相関分光法を駆使した生細胞内の分子状態解析 (2012)
  • Author(s): 佐々木 章
  • Journal Title: 生化学 Volume: 84（12） Pages: 1024-1027
  • Peer Reviewed
• [Journal Article] Fluorescence Imageing of Mitochondria in Living Cells Using a Novel Fluorene Derivative with a Large Two-Photon Absorption Cross-Section (2012)
  • Author(s): Tani S.
  • Journal Title: Curr Pharm Biotechnol Volume: (in press)
  • Peer Reviewed
• [Journal Article] Use of Carbohydrate-Conjugated Nanoparticles for an Integrated Approach to Functional Imaging of Glycans and Understanding of Their Mnlerular Mechanisms (2012)
  • Author(s): Nagahori N.
  • Journal Title: Curr Pharm Biotechnol Volume: (in press)
  • Peer Reviewed
• [Journal Article] 蛍光相関分光法を用いた分子間相互作用解析の応用 (2012)
  • Author(s): 長堀紀子
  • Journal Title: 化学工業 Volume: 63(1) Pages: 18-23
  • Peer Reviewed
• [Journal Article] Current Research on Protein-Protein Interactions Among Auxin-Singnaling Factors in Regulation of Plant Grouwth and Development (2011)
  • Author(s): Muto H.
  • Journal Title: Curr Pharm Biotechnol Volume: (in press)
  • Peer Reviewed
• [Journal Article] A modified FCCS procedure applied to Ly49A-MHC class I cis-interaction sudies in cell membranes (2011)
  • Author(s): Stromqvist J.
  • Journal Title: Biophys J Volume: 101(5) Pages: 1257-69
  • Peer Reviewed
• [Journal Article] Intracellular pH sensing using autofluorescence lifetime microscopy (2011)
  • Author(s): Ogikubo S, Nakabayashi T, Adachi T, Islam MS, Yoshizawa T, Kinjo M, Ohta N.
  • Journal Title: J Phys Chem B. Volume: 115(34) Issue: 34 Pages: 10385-10390
  • DOI: 10.1021/jp2058904
  • Peer Reviewed
• [Journal Article] Importance of sialic acid residues illuminated by live animal imaging using phosphorylcholine self-assembled monolayer-coated quantum dots (2011)
  • Author(s): Ohyanagi T, Nagahori N, Shimawaki K, Hinou H, Yamashita T, Sasaki A, Jin T, Iwanaga T, Kinjo M, Nishimura S.
  • Journal Title: J Am Chem Soc. Volume: 133(32) Issue: 32 Pages: 12507-12517
  • DOI: 10.1021/ja111201c
  • Peer Reviewed
• [Journal Article] Direct observation of dimerization between different CREB1 isoforms in a living cell (2011)
  • Author(s): Sadamoto H.
  • Journal Title: PLoS One Volume: 6(6)
  • Peer Reviewed
• [Journal Article] Two distinct amyloid {beta}-protein(A{betal} assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology and toxicity analyses (2011)
  • Author(s): Matsumura S.
  • Journal Title: J Biol Chem Volume: 286(13) Pages: 11555-62
  • Peer Reviewed
• [Journal Article] 生細胞における蛍光相関分光分析 (2011)
  • Author(s): 金城政孝
  • Journal Title: ぶんせき Volume: 436 Pages: 221-228
  • NAID: 10028118190
  • Peer Reviewed
• [Journal Article] Single-particle trackillg of quantum dot-conjugated prion proteins inside yeast cells. (2011)
  • Author(s): Tsuji T
  • Journal Title: Biochem Biophys Res Commun Volume: 405(4) Pages: 638-643
  • Peer Reviewed
• [Journal Article] Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate. (2011)
  • Author(s): Sun F
  • Journal Title: Biochem Biophys Res Commun Volume: 404(2) Pages: 706-710
  • Peer Reviewed
• [Journal Article] Dynamic Assembly Properties of Nonmuscle Myosin II Isoforms Revealed by Combination of Fluorescence Correlation Spectroscopy and Fluorescence Cross-Correlation Spectroscopy. (2010)
  • Author(s): Mitsuhashi M
  • Journal Title: J Biochem Volume: 149(3) Pages: 253-263
  • Peer Reviewed
• [Journal Article] STPR, a 23-amino acid tandem repeat domain, found in the human function-unknown protein ZNF821. (2010)
  • Author(s): Nonaka Y
  • Journal Title: Biochemistry Volume: 49(38) Pages: 8367-8375
  • Peer Reviewed
• [Journal Article] In vivo evidence for the fibrillar structures of Sup35 prions in yeast cells. (2010)
  • Author(s): Kawai-Noma S
  • Journal Title: J Cell Biol Volume: 190(2) Pages: 223-231
  • Peer Reviewed
• [Journal Article] A quantum dot-based ratiometric pH sensor. (2010)
  • Author(s): Jin T
  • Journal Title: Chem Commun (Camb) Volume: 46(14) Pages: 2408-2410
  • Peer Reviewed
• [Journal Article] Kinesin-1/Hsc70-dependent mechanism of slow axonal transport and its relation to fast axonal transport. (2010)
  • Author(s): Terada S
  • Journal Title: EMBO J Volume: 29(4) Pages: 843-854
  • Peer Reviewed
• [Journal Article] 蛍光相関法によるタンパク質の機能解析 (2010)
  • Author(s): 金城政孝
  • Journal Title: 生化学 Volume: 82(12) Pages: 1103-1116
  • NAID: 10027880119
  • Peer Reviewed
• [Presentation] 蛍光相関分光法によるタンパク質機能解析-生細胞への応用- (2012)
  • Author(s): 金城政孝
  • Organizer: 細胞生物学セミナー
  • Place of Presentation: 秋田大学(秋田市)(招待講演)
  • Year and Date: 2012-02-22
• [Presentation] 蛍光相関分光法とイメージングを用いたタンパク質凝集過程の研究 (2012)
  • Author(s): 金城政孝
  • Organizer: 神戸大学先端融合科学シンポジウム「タンパク質アセンブリ～会合、超分子化、凝集～」
  • Place of Presentation: 神戸大学(神戸市)(招待講演)
  • Year and Date: 2012-02-01
• [Presentation] 多点蛍光相関分光測定による生細胞観察 (2012)
  • Author(s): 金城政孝
  • Organizer: レーザー学会学術講演会第32回年次大会
  • Place of Presentation: TKP仙台加フルンスセンター(宮城県)(招待講演)
  • Year and Date: 2012-01-30
• [Presentation] 多点蛍光相関分光法の開発と生細胞測定 (2012)
  • Author(s): 金城 政孝
  • Organizer: 第9回バイオオプティクス研究会
  • Place of Presentation: 山口大学（山口市）
  • Invited
• [Presentation] Study of transport dynamics in living cell using fluorescence correlation spectroscopy (2012)
  • Author(s): Masataka Kinjo
  • Organizer: The 25th Annual and International Meeting of the Japanese Association for Animal Cell Technology (JAACT2012)
  • Place of Presentation: Nagoya Congress Center（Nagoya)
  • Invited
• [Presentation] Unraveling nuclear pore transport of glucocorticoid receptor using multipoint fluorescence correlation spectroscopy (2012)
  • Author(s): Masataka Kinjo
  • Organizer: The Second Japan-Korea Workshop on Digital Holography and Information Photonics (DHIP2012)
  • Place of Presentation: The University of Tokushima(Tokushima)
  • Invited
• [Presentation] 多点蛍光相関分光法を用いた核内受容体の機能解明 (2012)
  • Author(s): 金城 政孝
  • Organizer: 日本顕微鏡学会第56回シンポジウム
  • Place of Presentation: 北海道大学（札幌市）
  • Invited
• [Presentation] First step for fluorescence correlation spectroscopy and fluorescence cross correlation spectroscopy of living cell (2012)
  • Author(s): Masataka Kinjo
  • Organizer: Fluorescence Microscopy: from Basics to Advanced
  • Place of Presentation: Okinawa Institute of Science and Technology Graduate University(Okinawa)
  • Invited
• [Presentation] ALS関連TDP43タンパク質のカルボキシル末端断片の凝集体形成と毒性はRNAによって防御されている (2012)
  • Author(s): 北村 朗
  • Organizer: 第5回タンパク質の異常凝集とその防御・修復機構に関する研究会
  • Place of Presentation: 京都大学原子炉実験所（大阪府泉南郡）
  • Invited
• [Presentation] 蛍光相関分光法による生体高分子機能解析 (2012)
  • Author(s): 金城 政孝
  • Organizer: 蛍光相関分光法を用いた解析に関する講習会
  • Place of Presentation: 富士フィルム足柄サイト（南足柄市）
  • Invited
• [Presentation] Inside view of molecular dynamics of Glucocorticoid Receptor by using Fluorescence Cross Correlation Spectroscopy in living cell (2012)
  • Author(s): 金城 政孝
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学（名古屋市）
  • Invited
• [Presentation] 蛍光相関分光法と超解像顕微鏡法によるタンパク質凝集の研究 (2011)
  • Author(s): 金城政孝
  • Organizer: 第8回バイオオプティクス研究会・理研シンポジウム「蛍光相関分光と票法伝達(8)」
  • Place of Presentation: 北里大学(相模原市)(招待講演)
  • Year and Date: 2011-12-16
• [Presentation] Detection of Biomolecular Dynamics in Living Cell Using Multipoint Fluorescence Correlation Spectroscopy (2011)
  • Author(s): Masataka Kinjo
  • Organizer: The 13th Takayanagi Kenjiro memorial Symposium
  • Place of Presentation: 静岡大学(浜松市)(招待講演)
  • Year and Date: 2011-11-18
• [Presentation] 蛍光相関分光法による細胞機能解析 (2011)
  • Author(s): 金城政孝
  • Organizer: 光サイエンス特別講義
  • Place of Presentation: 奈良先端科学技術大学院大学(生駒市)(招待講演)
  • Year and Date: 2011-11-16
• [Presentation] 蛍光相関法の原理と細胞生物学における利用 (2011)
  • Author(s): 金城政孝
  • Organizer: 第23回散乱研究会
  • Place of Presentation: 日本薬学会館(東京都)(招待講演)
  • Year and Date: 2011-11-11
• [Presentation] 蛍光相関分光法と超解像顕微鏡法を用いたタンパク質凝集過程の研究 (2011)
  • Author(s): 金城政孝
  • Organizer: 京都大学原子炉実験所専門研究会第4回タンパク質の異常凝集とその防御・修復機構に関する研究会
  • Place of Presentation: 京都大学原子炉実験所(大阪府泉南郡)(招待講演)
  • Year and Date: 2011-11-10
• [Presentation] Analysis for the relationship of intranuclrear diffusional landsdcape and transctiptional activity of glucocorticoid receptor (2011)
  • Author(s): Shintaro Mikuni
  • Organizer: International Symposium on Photonic Bioimaging
  • Place of Presentation: 京王プラザホテル札幌(北海道)
  • Year and Date: 2011-10-22
• [Presentation] 蛍光相関分光イメージングによる生体分子間相互作用の解析 (2011)
  • Author(s): 金城政孝
  • Organizer: 第20回日本バイオイメージング学会学術集会
  • Place of Presentation: 千歳科学技術大学(千歳市)(招待講演)
  • Year and Date: 2011-09-02
• [Presentation] Study of protein dynamics in live cell using multipoint temporal and spatial correlation spectroscopy analysis (2011)
  • Author(s): Masataka Kinjo
  • Organizer: STELLA(School for Training in Experiments with Lasers and Laser Applications)
  • Place of Presentation: インスブリア大学(イタリア)(招待講演)
  • Year and Date: 2011-06-20
• [Presentation] Aggregation and disaggregation of aggregate-prone proteins : Which is toxic? (2011)
  • Author(s): 北村朗
  • Organizer: 奈良先端未来開拓コロキウム
  • Place of Presentation: 奈良先端科学技術大学院大学(生駒市)(招待講演)
  • Year and Date: 2011-05-30
• [Presentation] Fluorescence correlation analysis of intranuclear transcriptional activity of glucocorticoid receptor. (2011)
  • Author(s): Shintaro Mikuni
  • Organizer: 2^<nd> International Symposium on Photonic Bioimaging
  • Place of Presentation: Hilton Niseko Village (Niseko, Japan) 招待講演
  • Year and Date: 2011-02-10
• [Presentation] Study of protein dynamics in live cell using multipoint temporal and spatial correlation spectroscopy analysis. (2011)
  • Author(s): Masataka Kinjo
  • Organizer: 2^<nd> International Symposium on Photonic Bioimaging
  • Place of Presentation: Hilton Niseko Village (Niseko, Japan) 招待講演
  • Year and Date: 2011-02-08
• [Presentation] The relationship between the landscape of the diffusion property and transcriptional activity of glucocorticoid recetor. (2011)
  • Author(s): Shintaro Mikuni
  • Organizer: International Symposium Physicochemical Field for Genetic Activities
  • Place of Presentation: Awaji Yumebutai International Conference Center (Awaji, Japan)
  • Year and Date: 2011-01-24
• [Presentation] Dynamic properties of cell structure analyzed by fluorescence correlation spectroscopy. (2010)
  • Author(s): Masataka Kinjo
  • Organizer: The International Chemical Congress of Pacific Basin Societies
  • Place of Presentation: Hilton Hawaiian Village (Honolulu, Hawaii, U.S.A). 招待講演
  • Year and Date: 2010-12-19
• [Presentation] 多焦点蛍光相関分光法:蛍光相関イメージングへ向けて (2010)
  • Author(s): 金城政孝
  • Organizer: 平成22年度日本分光学会ナノ分光部会・生細胞分光部会合同シンポジウム
  • Place of Presentation: 東京大学小柴ホール(東京都) 招待講演
  • Year and Date: 2010-12-10
• [Presentation] 多点蛍光相関分光装置の構築と細胞内測定 (2010)
  • Author(s): 金城政孝
  • Organizer: 第3回タンパク質の異常凝集とその防御・修復機能に関する研究会
  • Place of Presentation: 京都大学原子炉実験所(大阪府泉南郡) 招待講演
  • Year and Date: 2010-11-12
• [Presentation] Study of molecular complexes in live cell using multipoint temporal and spatial correlation spectroscopy analysis. (2010)
  • Author(s): Masataka Kinjo
  • Organizer: 13^<th> Carl Zeiss Sponsored International Workshop on FCS and Related Methods
  • Place of Presentation: The National University of Singapore (Singapore) 招待講演
  • Year and Date: 2010-10-26
• [Presentation] Imaging correlation analysis of intranuclear transcriptional activity of glucocorticoid receptor (2010)
  • Author(s): Shintaro Mikuni
  • Organizer: 日本生物物理学会第48回年会
  • Place of Presentation: 東北大学川内北キャンパス(仙台市)
  • Year and Date: 2010-09-22
• [Presentation] Study of molecular interaction in live cell using multipoint temporal and spatial correlation spectroscopy analysis. (2010)
  • Author(s): Masataka Kinjo
  • Organizer: The 3^<rd> International Symposium on Protein Community
  • Place of Presentation: Hotel Nikko Nara (Nara, Japan) 招待講演
  • Year and Date: 2010-09-16
• [Presentation] Cell membrane-binding proteins analyzed by multi-point total internal reflection fluorescence correlation spectroscopy. (2010)
  • Author(s): Masataka Kinjo
  • Organizer: Japan-Korea Workshop of FCS and FCCS
  • Place of Presentation: Korea Advanced Institute of Science and Technology(Daejon, Korea) 招待講演
  • Year and Date: 2010-08-27
• [Presentation] 生細胞におけるタンパク質社会のイメージングに向けて (2010)
  • Author(s): 金城政孝
  • Organizer: 第10回日本蛋白質学会年会
  • Place of Presentation: 札幌コンベンションセンター(札幌市) 招待講演
  • Year and Date: 2010-06-16
• [Presentation] 蛍光相関分光法を用いた細胞内導入DNAの解析 (2010)
  • Author(s): 金城政孝
  • Organizer: 遺伝子・デリバリー研究会第10回シンポジウム
  • Place of Presentation: 北海道大学学術交流会館(札幌市) 招待講演
  • Year and Date: 2010-06-03