光操作によるマルチスケール機能イメージング法の開発

• Project/Area Number: 20034001
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Hokkaido University
• Principal Investigator: 永井 健治 Hokkaido University, 電子科学研究所, 教授 (20311350)
• Project Period (FY): 2008 – 2009
• Project Status: Completed (Fiscal Year 2009)
• Budget Amount: ¥3,800,000 (Direct Cost: ¥3,800,000); Fiscal Year 2009: ¥900,000 (Direct Cost: ¥900,000); Fiscal Year 2008: ¥2,900,000 (Direct Cost: ¥2,900,000)
• Keywords: 生体分子 / 光活性化 / 可視化 / FRET / 顕微鏡

Research Abstract

本研究は、光照射によって、蛍光活性化する生理機能センサータンパク質を開発することで、生きた細胞内や個体内の、ナノからマクロに及ぶ、様々な空間スケールにおける生理機能を高感度に観察する手法を構築することを目的とした。昨年度に作製したPA-GFPとDsRedをFRETのドナー・アクセプターとして利用した光活性化型Ca^<2+>指示薬PA-cameleonは光活性化前の状態でもDsRedによる青色励起光の吸収により蛍光が観察されたため、光活性化の利点の1つである高いコントラストを実現することができなかった。その一方で、405nmのレーザーを照射し、PA-GFPを活性化すると、緑色の蛍光が現れ、リガンド刺激に伴う細胞内Ca^<2+>濃度の変化を捉えることができた。本年度はDsRed以外の赤色蛍光タンパク質や緑色光を吸収するが蛍光性の無い色素タンパク質を用いる事で光活性化によるコントラストのさらなる増加が実現できないかを検討した。その結果、試験管内、細胞内のいずれにおいても405nmの刺激光照射によって不可逆的に無蛍光状態から蛍光性を獲得することが確認され、刺激光の照射範囲をガルバノミラーなどで制御することで、培養ディッシュ上の特定の細胞にPA-cameleonを出現させ、その細胞内のCa^<2+>動態を高いコントラストで可視化することに成功した。さらに、ゼブラフィッシュ幼魚における特定の筋細胞の自発的収縮とCa^<2+>濃度の変化を同時に観察することにも成功した。本方法はCa^<2+>指示薬以外のFRET指示薬にも原理的には応用可能であるため、光活性化指示薬開発のための汎用的指針として、その利用が期待される。

Research Products

• [Journal Article] Auto-luminescent genttically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level (2010)
  • Author(s): Saito K, Hatsugai N, Horikawa K, Kobayashi K, Matsu-ura T, Mikoshiba K, Nagai T.
  • Journal Title: PLoS ONE 5
  • Peer Reviewed
• [Journal Article] Optical recording of neuronal activity with a genetically-encoded Ca^<2+> indicator in anesthetized and freely moving mice (2010)
  • Author(s): Lutcke H, Murayama M, Hahn T, Margolis DJ, Astori S, Borgloh SMzA, Gobel W, Yang Y, Tang W, Kugler S, Sprengel R, Nagai T, Miyawaki A, Larkum ME, Helmchenl F, Hasan MT.
  • Journal Title: Frontiers in Neural Circuit (印刷中)
  • Peer Reviewed
• [Journal Article] Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes (2010)
  • Author(s): Hong JH, Min CH, Jeong B, Kojiya T, Morioka E, Nagai T, Ikeda M.Lee K.
  • Journal Title: PLoS ONE 5
  • NAID: 120003762636
  • Peer Reviewed
• [Journal Article] Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators (2010)
  • Author(s): Kotera I, Iwasaki T, Imamura H, Noji H, Nagai T.
  • Journal Title: ACS Chem Biol. 5 Pages: 215-222
  • Peer Reviewed
• [Journal Article] 1波長励起 Dual FRETによる生細胞内における複数現象の可視化 (2010)
  • Author(s): 永井健治、小林健太郎
  • Journal Title: 実験医学 28 Pages: 589-598
• [Journal Article] Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators (2009)
  • Author(s): Imamura H, Nhat KPH, Togawa H, Saito K, Iino R, Kato-Yamada Y, Nagai T, Noji H.
  • Journal Title: Proc.Natl, Acad, Sci.USA 106 Pages: 15651-15656
  • Peer Reviewed
• [Journal Article] Fine-tuning of the cytoplasmic Ca2+ concentration is essential for pollen tube growth (2009)
  • Author(s): Iwano M, Entani T, Shiba H, Kakita H, Nagai T, Mizuno H, Miyawaki A, Shoji T, Kubo K, Isogai A, Takayama S.
  • Journal Title: Plant Physiology 150 Pages: 1322-1334
  • Peer Reviewed
• [Journal Article] An ultramarine fluorescent protein with increased photostability and pH insensitivity (2009)
  • Author(s): Tomosugi W, Matsuda T, Tani T, Nemoto T, Kotera I, Saito K, Horikawa K, Nagai T.
  • Journal Title: Nature Methods 6 Pages: 351-353
  • NAID: 120001648573
  • Peer Reviewed
• [Journal Article] 生命システムの階層間をまたぐイメージング技術 (2009)
  • Author(s): 野地博行、永井健治
  • Journal Title: 蛋白質核酸酵素 54 Pages: 1913-1917
• [Journal Article] 細胞結合振動しシステムを対象にした超高感度大規模イメージング (2009)
  • Author(s): 堀川一樹、永井健治
  • Journal Title: 蛋白質核酸酵素 54 Pages: 1945-1951
• [Journal Article] 生細胞内でのタンパク質動態測定とその意義 (2009)
  • Author(s): 永井健治、松田知己
  • Journal Title: 生物物理 49 Pages: 181-196
  • NAID: 10025975726
  • Peer Reviewed
• [Journal Article] 光スイッチング蛍光タンパク質を用いた生細胞内でのタンパク質動態定量法 (2009)
  • Author(s): 永井健治、松田知己
  • Journal Title: 組織細胞生物化学2009 Pages: 99-105
• [Journal Article] 酸性の環境でも光る蛍光タンパク質 (2009)
  • Author(s): 永井健治
  • Journal Title: 化学 64 Pages: 72-73
• [Journal Article] ワンステップ全自動プラスミド構築方法 (2009)
  • Author(s): 小寺一平、永井健治
  • Journal Title: 細胞工学 28 Pages: 402-407
• [Journal Article] 光スイッチング蛍光タンパク質を用いた超解像観察法 (2009)
  • Author(s): 永井健治
  • Journal Title: 分光研究 58 Pages: 7-9
  • NAID: 10024730583
  • Peer Reviewed
• [Journal Article] An ultramarine fluorescent protein with increased photostability and pH insensitivity (2009)
  • Author(s): Tomosugi W, Matsuda T, Tani T, Nemoto T, Kotera I, Saito K, Horikawa K, Nagai T
  • Journal Title: Nature Methods (印刷中)
  • NAID: 120001648573
  • Peer Reviewed
• [Journal Article] Two-photon excitation behavior of thiophene-based oligomeres and apolymer. n press, 2008 (2009)
  • Author(s): Yamamoto T, Kumagai A, Saito K & Nagai T.
  • Journal Title: Journal of Nanoscience and Nanotechnology 9 Pages: 670-672
  • Peer Reviewed
• [Journal Article] 蛍光プローブ開発秘話-Pericam- (2009)
  • Author(s): 永井健治
  • Journal Title: 蛋白質核酸酵素 54 Pages: 86-92
• [Journal Article] A mercury arc lamp-based multi-color confocal real time imaging system for cellular structure and function (2008)
  • Author(s): Saito K, Kobayashi K, Tani T, Nagai T
  • Journal Title: Cell Structure Function 33 Pages: 133-141
  • NAID: 130004137533
  • Peer Reviewed
• [Journal Article] A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme. 137 : 1-7, 2008 (2008)
  • Author(s): Kotera I, Nagai T
  • Journal Title: Journal of Biotechnology 137 Pages: 1-7
  • Peer Reviewed
• [Journal Article] Cell-cycle-specific nestin expression coordinates with morphological changes in embryonic cortical neural progenitors. 121 : 1204-1212, 2008 (2008)
  • Author(s): Sunabori T, Tokunaga A, Nagai T, Sawamoto K, Okabe M, Miyawaki A, Matsuzaki Y, Miyata T & Okano H.
  • Journal Title: Journal of Cell Sciince 121 Pages: 1204-1212
  • Peer Reviewed
• [Journal Article] 光変換蛍光タンパク質を用いた生体分子の動態解析法 (2008)
  • Author(s): 永井健治、松田知己
  • Journal Title: 実験医学 26 Pages: 156-162
• [Journal Article] 新規の光変換蛍光蛋白質プローブを用いた生体イメージングと動態解析 (2008)
  • Author(s): 松田知己、永井健治
  • Journal Title: 蛋白質核酸酵素 53 Pages: 1858-1864
  • NAID: 40016306372
• [Journal Article] 光活性化・光変換蛍光タンパク質を用いたタンパク質動態解析 (2008)
  • Author(s): 松田知己、永井健治
  • Journal Title: バイオインダストリー 25 Pages: 21-26
• [Presentation] 蛍光タンパク質技術が拓いたもの (2010)
  • Author(s): 永井健治
  • Organizer: 第115回日本解剖学会学術集会
  • Place of Presentation: 岩手医科大学、盛岡
  • Year and Date: 2010-03-29
• [Presentation] Approaches to in vivo functional imaging by engineering bacterial luminescent protein (2010)
  • Author(s): 永井健治
  • Organizer: 第83回日本細菌学会
  • Place of Presentation: パシフィコ横浜、横浜
  • Year and Date: 2010-03-27
• [Presentation] 結晶構造情報に基づく蛍光タンパク質の改変とその生物学的応用 (2010)
  • Author(s): 永井健治
  • Organizer: ソフトナノ材料研究ステーションシンポジウム
  • Place of Presentation: 東北大、仙台
  • Year and Date: 2010-03-11
• [Presentation] Toward elucidation of biological enigma by genetically-encoded molecular spies (2009)
  • Author(s): 永井健治
  • Organizer: International Symposium on Waching Biomolecules in Action
  • Place of Presentation: Senri Life Science Center, Osaka
  • Year and Date: 2009-12-16
• [Presentation] 蛍光タンパク質テクノロジーの展望 -観て触って探る生命の不思議- (2009)
  • Author(s): 永井健治
  • Organizer: シナプス研究会「シナプス機能と病態」
  • Place of Presentation: 生理研、岡崎
  • Year and Date: 2009-12-14
• [Presentation] 蛍光タンパク質性機能プローブの精密設計とバイオイメージングへの応用 (2009)
  • Author(s): 永井健治
  • Organizer: 第7回脳科学研究教育センターシンポジウム
  • Place of Presentation: 北海道大学、札幌
  • Year and Date: 2009-12-09
• [Presentation] Toward understanding biological phenomena by genetically-encoded molecular spies (2009)
  • Author(s): 永井健治
  • Organizer: Molecular Imaging for Syetems Biology
  • Place of Presentation: Okazaki Conference Center, Okazaki
  • Year and Date: 2009-12-06
• [Presentation] Toward understanding biological phenomena by genetically-encoded molecular spies (2009)
  • Author(s): 永井健治
  • Organizer: BIT's 3rd Annual Eorld Congress of Gene-2009
  • Place of Presentation: Foshan Sansui Golden Sun Hotel, China
  • Year and Date: 2009-12-04
• [Presentation] Toward understanding biological phenomena by genetically-encoded molecular spies (2009)
  • Author(s): 永井健治
  • Organizer: 2009年生物物理・生化学・分子生物学会北海道支部会合同年会
  • Place of Presentation: 北海道大学、札幌
  • Year and Date: 2009-11-13
• [Presentation] 蛍光タンパク質を巧妙に用いた生理機能動態の可視化 (2009)
  • Author(s): 永井健治
  • Organizer: 第47回日本生物物理学会年会
  • Place of Presentation: アスティ徳島、徳島
  • Year and Date: 2009-10-30
• [Presentation] Toward elucidation of dynamic living system by hierarchical molecular imaging (2009)
  • Author(s): 永井健治
  • Organizer: 82th Annual meeting of JBS
  • Place of Presentation: 神戸国際会議場、神戸
  • Year and Date: 2009-10-21
• [Presentation] Toward understanding biological enigma by genetically-encoded molecular spies (2009)
  • Author(s): 永井健治
  • Organizer: Hokkaido University-Academia Sinica Joint Symposium
  • Place of Presentation: Hokkaido University, Sapporo
  • Year and Date: 2009-10-07
• [Presentation] 新規蛍光タンパク質の開発とバイオイメージングへの応用 (2009)
  • Author(s): 永井健治
  • Organizer: BioOpto Japan 2009
  • Place of Presentation: パシフィコ横浜、横浜
  • Year and Date: 2009-09-17
• [Presentation] Toward elcidation of biological enigma by genetically-encoded molecular spies and snipers (2009)
  • Author(s): 永井健治
  • Organizer: 2^<nd> Swiss-Japan Chemical Biology Symposium
  • Place of Presentation: Tokyo, Japan
  • Year and Date: 2009-09-12
• [Presentation] Perspective of bioimaging and biomanipulation techniques (2009)
  • Author(s): 永井健治
  • Organizer: International Symposium of post-silicon materials and devices research alliance projecInternational Symposium "Innovative Nanoscience of Supermolecular Motor Proteins"
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2009-09-08
• [Presentation] Toward elcidation of biological enigma by genetically-encoded functional indicator (2009)
  • Author(s): 永井健治
  • Organizer: International Symposium of post-silicon materials and devices research alliance project
  • Place of Presentation: Osaka, Japan
  • Year and Date: 2009-09-06
• [Presentation] Rational design of photoswitchable fluorescent probes and its use for bioimaging (2009)
  • Author(s): 永井健治
  • Organizer: 238th American Chemical Society Meeting
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2009-08-18
• [Presentation] Deciphering Enigma of Biological Function by Genetically-encoded Molecular Spies (2009)
  • Author(s): 永井健治
  • Organizer: International Symposium on Photonic Bioimaging
  • Place of Presentation: Sapporo, Japan
  • Year and Date: 2009-08-01
• [Presentation] 蛍光タンパク質テクノロジーの最前線 (2009)
  • Author(s): 永井健治
  • Organizer: 第34回組織細胞化学会講習会
  • Place of Presentation: 徳島大学、徳島
  • Year and Date: 2009-07-29
• [Presentation] Tune-up of FRET-based functional indicators for high sensitive bioimaging (2009)
  • Author(s): 永井健治
  • Organizer: Application Trens of Live Cell Imaging
  • Place of Presentation: Soul, Korea
  • Year and Date: 2009-07-17
• [Presentation] Deciphering enigma of biological function by a genetically-encoded fluorescent indicator (2009)
  • Author(s): 永井健治
  • Organizer: Gordon Research Conferences-Fertilization & Activation of Development-
  • Place of Presentation: NH, USA
  • Year and Date: 2009-07-13
• [Presentation] 生理機能・動態を計測するための蛍光タンパク質技術 (2009)
  • Author(s): 永井健治
  • Organizer: 第9回日本蛋白質科学会年会
  • Place of Presentation: 熊本全日空ホテルニュースカイ、熊本市
  • Year and Date: 2009-05-22
• [Presentation] Measurement of diffusion coefficient of biomolecules by fluorescence decay after photostimulation of photoswichable fluorescent proteins in living cells (2009)
  • Author(s): 永井健治
  • Organizer: 9^<th> NIBB-EMBL Symposium
  • Place of Presentation: Okazaki, Japan
  • Year and Date: 2009-04-21
• [Presentation] Multifunctional imaging by genetically-encoded homo-FRET-based indicator (2009)
  • Author(s): 永井健治
  • Organizer: Focus On Microscopy 2009
  • Place of Presentation: Krakow, Poland
  • Year and Date: 2009-04-21
• [Presentation] 改変蛍光タンパク質を利用した生理機能の可視化 (2009)
  • Author(s): 永井健治
  • Organizer: 日本学術振興会「未踏・ナノデバイステクノロジー」第151委員会研究会「ナノ計測の先端共用施設利用とナノ時空研究の一側面」
  • Place of Presentation: 物質・材料研究機構、つくば
  • Year and Date: 2009-01-31
• [Presentation] GFPを巧妙に用いた分子機能・動態の可視化 (2009)
  • Author(s): 永井健治
  • Organizer: 京都府立医科大学研究開発センター第8回学術講演会
  • Place of Presentation: 京都府立医科大学、京都
  • Year and Date: 2009-01-21
• [Presentation] Deciphering enigma of biological function by a genetically-encoded molecular spy (2009)
  • Author(s): Takeharu Nagai
  • Organizer: The 4^<th> LSW symposium
  • Place of Presentation: 北海道大学
  • Year and Date: 2009-01-09
• [Presentation] Engineering fluorescent proteins to visualize and manipulate biological functions (2008)
  • Author(s): Takeharu Nagai
  • Organizer: The 11^<th> Japan and America Frontier of Science
  • Place of Presentation: Beckman center, Irvine, USA
  • Year and Date: 2008-12-05
• [Presentation] Engineering fluorescent proteins to visualize biological functions (2008)
  • Author(s): Takeharu Nagai
  • Organizer: The 8^<th> Japan and America Frontier of Engineering
  • Place of Presentation: Kobe convention center, Kobe
  • Year and Date: 2008-11-17
• [Presentation] 蛍光タンパク質を巧妙に用いた分子機能・動態の可視化 (2008)
  • Author(s): 永井健治
  • Organizer: 日本薬学会関東支部第33回学術講演会
  • Place of Presentation: 長井記念館、東京
  • Year and Date: 2008-11-01
• [Presentation] Imagaing biological functions by using FRET-based sensor proteins (2008)
  • Author(s): Takeharu Nagai
  • Organizer: ICCB2008
  • Place of Presentation: COEX, Seoul, Korea
  • Year and Date: 2008-10-10
• [Presentation] Engineering fluorescent and bioluminescent proteins to visualize biological functions (2008)
  • Author(s): Takeharu Nagai
  • Organizer: NEWroscience2008
  • Place of Presentation: de Sao Paulo university, Brazil
  • Year and Date: 2008-09-08
• [Presentation] Engineering fluorescent and bioluminescent proteins to visualize biological functions (2008)
  • Author(s): Takeharu Nagai
  • Organizer: IBRO (I Neurolatam)
  • Place of Presentation: Buzios, Brazil
  • Year and Date: 2008-09-03
• [Presentation] 改変蛍光タンパク質によるタンパク質機能・動態のリアルタイム可視化 (2008)
  • Author(s): 永井健治
  • Organizer: 岩手医科大学先端医療研究センター公開シンポジウム「バイオイメージングと分子生物学による脳・血管解明一健康増進に向けた最先端研究」
  • Place of Presentation: 岩手県医師会館ホール、盛岡
  • Year and Date: 2008-07-19
• [Presentation] Direct measurement of protein dynamics in single living cells using a rationally designed photoconvertible fluorescent protein (2008)
  • Author(s): 永井健治
  • Organizer: 第60回日本細胞生物学会大会
  • Place of Presentation: パシフィコ横浜、横浜
  • Year and Date: 2008-06-29
• [Presentation] Direct measurement of protein dynamics in living cells using a rationally designed photoconvertible fluorescent protein (2008)
  • Author(s): Takeharu Nagai
  • Organizer: NIPS-JST international workshop
  • Place of Presentation: Okazaki conference center, Okazaki
  • Year and Date: 2008-04-18
• [Presentation] Development of a fluorescent protein with deep blue color (2008)
  • Author(s): Takeharu Nagai
  • Organizer: FOM2008
  • Place of Presentation: Awaji Yume Butai, Osaka
  • Year and Date: 2008-04-14
• [Remarks]
  • URL: http://nano.es.hokudai.ac.jp/
• [Remarks]
  • URL: http://nano.es.hokudai.ac.jp/
• [Patent(Industrial Property Rights)] 光増感性蛍光タンパク質 (2010)
  • Inventor(s): 永井健治、松田知己、竹本研、高橋里佳
  • Industrial Property Rights Holder: 北海道大学
  • Industrial Property Number: 2010-022603
  • Filing Date: 2010-02-03
• [Patent(Industrial Property Rights)] 光活性化生理機能センサータンパク質 (2009)
  • Inventor(s): 永井健治、松田知己
  • Industrial Property Rights Holder: 北海道大学
  • Industrial Property Number: 2009-233826
  • Filing Date: 2009-10-07
• [Patent(Industrial Property Rights)] 蛍光温度プローブおよびそれを用いた温度測定装置 (2009)
  • Inventor(s): 永井健治、小寺一平、岩崎卓也
  • Industrial Property Rights Holder: 北海道大学
  • Industrial Property Number: 2009-149338
  • Filing Date: 2009-06-24