Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators

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 ε subunit of the bacterial FoF1-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 μM 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 FoF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

[1]  W. Gan,et al.  ATP mediates rapid microglial response to local brain injury in vivo , 2005, Nature Neuroscience.

[2]  Rodrigue Rossignol,et al.  Energy Substrate Modulates Mitochondrial Structure and Oxidative Capacity in Cancer Cells , 2004, Cancer Research.

[3]  D. Burk,et al.  On respiratory impairment in cancer cells. , 1956, Science.

[4]  F. Ashcroft,et al.  A Novel Method for Measurement of Submembrane ATP Concentration* , 2000, The Journal of Biological Chemistry.

[5]  T. Pozzan,et al.  Improved strategies for the delivery of GFP-based Ca2+ sensors into the mitochondrial matrix. , 2005, Cell calcium.

[6]  Masasuke Yoshida,et al.  Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase* , 2005, Journal of Biological Chemistry.

[7]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[8]  F. Ashcroft,et al.  ATP-dependent interaction of the cytosolic domains of the inwardly rectifying K+ channel Kir6.2 revealed by fluorescence resonance energy transfer , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Hiroyuki Fujita,et al.  Highly coupled ATP synthesis by F1-ATPase single molecules , 2005, Nature.

[10]  H. Kennedy,et al.  Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria. , 1999, The Journal of biological chemistry.

[11]  Y. Kato-Yamada Isolated ε subunit of Bacillus subtilis F1‐ATPase binds ATP , 2005, FEBS letters.

[12]  Jim Berg,et al.  A genetically encoded fluorescent reporter of ATP/ADP ratio , 2008, Nature Methods.

[13]  A Miyawaki,et al.  Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  N. Dale,et al.  Purine-mediated signalling triggers eye development , 2007, Nature.

[15]  Masasuke Yoshida,et al.  Isolated ϵ Subunit of Thermophilic F1-ATPase Binds ATP* , 2003, Journal of Biological Chemistry.

[16]  T. Finger,et al.  ATP Signaling Is Crucial for Communication from Taste Buds to Gustatory Nerves , 2005, Science.

[17]  F. Ashcroft ATP-sensitive potassium channelopathies: focus on insulin secretion. , 2005, The Journal of clinical investigation.

[18]  Takeharu Nagai,et al.  Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein , 2008, Nature Methods.

[19]  Masasuke Yoshida,et al.  ε Subunit, an Endogenous Inhibitor of Bacterial F1-ATPase, Also Inhibits F0F1-ATPase* , 1999, The Journal of Biological Chemistry.

[20]  Masasuke Yoshida,et al.  Role of the ϵ Subunit of Thermophilic F1-ATPase as a Sensor for ATP* , 2007, Journal of Biological Chemistry.

[21]  Takeharu Nagai,et al.  Shift anticipated in DNA microarray market , 2002, Nature Biotechnology.

[22]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[23]  Masasuke Yoshida,et al.  F0F1-ATPase/Synthase Is Geared to the Synthesis Mode by Conformational Rearrangement of ϵ Subunit in Response to Proton Motive Force and ADP/ATP Balance* , 2003, Journal of Biological Chemistry.

[24]  A. Miyawaki,et al.  Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Masasuke Yoshida,et al.  Structures of the thermophilic F1-ATPase ε subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1 , 2007, Proceedings of the National Academy of Sciences.

[26]  L. Reitzer,et al.  Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells. , 1979, The Journal of biological chemistry.