Document Detail


Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.
MedLine Citation:
PMID:  19720993     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 muM 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.
Authors:
Hiromi Imamura; Kim P Huynh Nhat; Hiroko Togawa; Kenta Saito; Ryota Iino; Yasuyuki Kato-Yamada; Takeharu Nagai; Hiroyuki Noji
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-08-31
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  106     ISSN:  1091-6490     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2009 Sep 
Date Detail:
Created Date:  2009-10-06     Completed Date:  2009-11-13     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  15651-6     Citation Subset:  IM    
Affiliation:
Precursory Research for Embryonic Science, Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. imamura@sanken.osaka-u.ac.jp
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MeSH Terms
Descriptor/Qualifier:
Adenosine Triphosphate / metabolism*
Bacterial Proteins / chemistry,  genetics,  metabolism
Bacterial Proton-Translocating ATPases / chemistry,  genetics,  metabolism
Cell Compartmentation
Fluorescence Resonance Energy Transfer / methods*
Fluorescent Dyes / chemistry,  metabolism
Green Fluorescent Proteins / chemistry,  genetics,  metabolism
Hela Cells
Humans
Luminescent Proteins / chemistry,  genetics,  metabolism
Microscopy, Fluorescence
Models, Molecular
Oxidative Phosphorylation
Recombinant Fusion Proteins / chemistry,  genetics,  metabolism
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/Cyan Fluorescent Protein; 0/Fluorescent Dyes; 0/Luminescent Proteins; 0/Recombinant Fusion Proteins; 0/yellow fluorescent protein, Bacteria; 147336-22-9/Green Fluorescent Proteins; 56-65-5/Adenosine Triphosphate; EC 3.6.1.-/Bacterial Proton-Translocating ATPases
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