2009年9月
Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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- 巻
- 106
- 号
- 37
- 開始ページ
- 15651
- 終了ページ
- 15656
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1073/pnas.0904764106
- 出版者・発行元
- NATL ACAD SCIENCES
Adenosine 5'-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the epsilon subunit of the bacterial F(o)F(1)-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 mu M to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of F(o)F(1)-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.
- リンク情報
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- DOI
- https://doi.org/10.1073/pnas.0904764106
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/19720993
- PubMed Central
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735558
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000269806600028&DestApp=WOS_CPL
- URL
- http://europepmc.org/abstract/med/19720993
- URL
- http://orcid.org/0000-0002-1896-0443
- ID情報
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- DOI : 10.1073/pnas.0904764106
- ISSN : 0027-8424
- ORCIDのPut Code : 33757958
- PubMed ID : 19720993
- PubMed Central 記事ID : PMC2735558
- Web of Science ID : WOS:000269806600028