2000 Fiscal Year Final Research Report Summary
Imaging of Synamics of Cellular Functions using GFP-based probes
| Project/Area Number |
11480186
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | The Institute of Physical and Chemical Research, RIKEN |
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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)
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| 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)
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| Project Period (FY) |
1999 – 2000
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| Keywords | GFP / RFP / circularly-permuted GFP / Ca^<2+> indicator / calmodulin / random mutagenesis / in vitro mutagenesis / pericam |
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| 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.
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