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Imaging of Synamics of Cellular Functions using GFP-based probes

Research Project

Project/Area Number 11480186
Research Category

Grant-in-Aid for Scientific Research (B).

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionThe Institute of Physical and Chemical Research, RIKEN

Principal Investigator

MIYAWAKI Atsushi  Lab for Cell Function Dynamics, Laboratory Head, The Institute of Physical and Chemical Research, RIKEN, 細胞機能探索技術開発チーム, チームリーダー(研究職) (80251445)

Co-Investigator(Kenkyū-buntansha) MIZUNO Hideaki  Lab for Cell Function Dynamics, Laboratory Staff Scientist, The Institute of Physical and Chemical Research, RIKEN, 細胞機能探索技術開発チーム, 研究員 (80301779)
HAMA Hiroshi  Lab for Cell Function Dynamics, Laboratory Staff Scientist, The Institute of Physical and Chemical Research, RIKEN, 細胞機能探索技術開発チーム, 研究員 (30261796)
Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥15,700,000 (Direct Cost: ¥15,700,000)
Fiscal Year 2000: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1999: ¥11,200,000 (Direct Cost: ¥11,200,000)
KeywordsGFP / RFP / circularly-permuted GFP / Ca^<2+> indicator / calmodulin / random mutagenesis / in vitro mutagenesis / pericam / カルシウムイメージング / cameleon / レーザー走査顕微鏡 / プレートイメージアナライザー / 蛍光偏光度測定
Research Abstract

Our primary goal is to gain a better understanding of how the molecules for life behave in space and time. Signal transduction cascades involve multiple enzymes and are orchestrated by specific protein-protein interactions. Such dynamics is revealed by optical means such as fluorescence readout. The green fluorescent protein (GFP)of the jellyfish Arequioreare victoria is a spontaneously fluorescent protein that can be ncorporated into other proteins by genetic fusion. One of our approaches is to use two GFPs of different colors to permit fluorescence resonance energy transfer(FRET), which is highly sensitive to the relative orientation and distance between the two fluorophores and alters the ratio of their emission intensities, and ideal readout for fast imaging and confocal microscopy. Cameleons are genetically-encoded fluorescent indicators for Ca^<2+>-based on FRET using GFPs. Another approach is to use circularly permuted GFP(cpGFP), in which the aminoand carboxy-portions have been interchanged and reconnected by a short spacer between the original termini. We have created Ca^<2+> -sensitive cpGFP, "pericam". Because cameleons and pericams can be targeted genetically and imaged by one- or two-photon excitation microscopy, they allowed monitoring Ca^<2+> in whole organisms, tissues, organelles, and submicroscopic environments where measurements were previously impossible. Dynamic changes in intracellular Ca^<2+> concentrations control many important celluar events. We have extended optical methods to develop some fluorescent indicators for visualization of the Ca^<2+> -related events, which are currently assayed by grinding millions of cells. Furthermore, we have developed optical hardwares for multi-color fluorescence imaging : the simultaneous observation of the Ca^<2+>-rerated events in conjunction with ca^<2+> dynamics in cells loaded with two or more fluorescent indicators.

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] Miyawaki A. , and Tsien R. Y.: "Monitoring Protein Conformations and Interactions by Fluorescent Resonance Energy Transfer between Mutants of Green Fluorescent Protein"Methods in Enzymology. 327. 472-500 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Sawano A. and Miyawaki A.: "Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis"Nucleic Acids Research . 28, No. 16. e78 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Miyawaki A. , Mizuno H. , Llopis J. , Tsien R. Y. and Jalink K.: "Cameleons as Cytosolic and intra-organellar calcium probes"Calcium Signalling . second Edition. 3-16 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nagai T. , Sawano A. , Park E. , and Miyawaki A.: "Circularly permuted green fluorescent proteins engineered to sense to Ca^<2+>"Proceeding National Academy Science USA. 98, No. 6. 3197-3203 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Mizuno H. , Sawano A. , Eli P. , Hama H. and Miyawaki A.: "Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer "Biochemistry. 40(8). 2502-2510 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Miyawaki A.and Tsien R.Y.: "Monitoring Protein Conformations and Interactions by Fluorescent Resonance Energy Transfer between Mutants of Green Fluorescent Protein"Methods in Enzymology. 327. 472-500 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Sawano A.and Miyawaki A.: "Directed evolution of green fluorescent protein by a new Versatile PCR strategy for site-directed and semi-random Mutagenesis"Nucleic Acids Research. 28, No. 16. e78 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Miyawaki A.Mizuno H., Llopis J., Tsien R.Y.and Jalink K.: "Cameleon as Cytosolic and intra-organellar calcium probes"Calcium Signalling second EditionMethods in Enzymology. 3-16 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nagai t., Sawano A., Park E.and Miyawaki A.: "Circularly permuted green fluorescent proteins engineered to sense to Ca^<2+>"Proceeding National Academy Science USA 98. No. 6. 3197-3202 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Mizuno H., Sawano A.Eli P., Hama H.and Miyawaki A.: "Red fluorescent protein from Discosoma as a fusion tag and a parnter for fluorescence resonance energy transfer"Biochemistry. 40(8). 2502-2510 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Matsushita F.,Miyawaki A.,and Mikoshiba K.: "Vomeroglandin/CRP-ductin is strongly expressed in the glands associated with the mouse vomeronasal organ : Identification and characterization of mouse vomeroglandin"Biochemical and Biophysical Research Communications. 268. 275-281 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Sawano A.and Miyawaki A.: "Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis"Nucleic Acids Research. 28,No.16. e78 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Mizuno H.,Sawano A.,Eli P.,Hama H.and Miyawaki A.: "Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer"Biochemistry. 40(8). 2502-2510 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nagai T.,Sawano A.,Park E.,and Miyawaki A.: "Circularly permuted green fluorescent proteins engineered to sense to Ca^<2+>"Proceeding National Academy Science USA. (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Miyawaki A.,Tsien R.Y.: "Monitoring Protein Conformations and Interactions by Fluorescence Resonance Energy Transfer Between Mutants of Green Fluorescent Protein"Methods in Enzymology, Chimeric Proteins. (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Miyawaki A.,Llopis J.,Mizuno H.,Jelink K.,Tsien R.Y.: "Cameleons as cytosolic and intra-organellar calcium probes"Oxford University Press, Calcium Signaling : A Practical Approach. (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Mikoshiba K.,Furuichi T.,Miyawaki A.et al.: "Role of Inositol 1,4,5-Trisphosphate(IP_3) Receptor/Ca^<2+> Signaling in Cell Function"Japan Scientific Societies Press, Challenges for Neuroscience in the 21^<st> Century. 121-146 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Matsushita F.,Miyawaki A.,Mikoshiba K.: "Vomeroglandin/CRP-ductin is strongly expressed in the gland associated with the mouse vomeronasal organ : identification and characterization of mouse vomeroglandin"Biochemical and Biophysical Research Communications. (2000)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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