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

Abstract

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 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 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 oF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment. fluorescent indicator ͉ FRET ͉ live imaging ͉ oxidative phosphorylation A denosine 5Ј-triphosphate (ATP) is the ubiquitous energy currency of all living organisms. The high phosphatetransfer potential of ATP is used for many biological processes, including muscle contraction, synthesis and degradation of biological molecules, and membrane transport. In addition, it has been suggested that ATP acts as an intracellular or extracellular signaling molecule in cellular processes, such as insulin secretion (1), neurotransmission (2), cell motility (3), and organ development (4). However, it has been difficult to precisely understand how ATP controls cellular processes and how the intracellular ATP level is regulated at the single cell level, because the conventional ATP quantification methods can only provide the averaged ATP level of an ensemble of cells based on cell extract analysis. Moreover, the distribution pattern of ATP between different intracellular compartments is unclear. Several attempts have been made to monitor ATP levels real-time in individual cells; however, these methods present several problems. For example, in chemiluminescence imaging from cells expressing firefly luciferase (5), chemiluminescence by luciferase depends not only on the intracellular ATP level but also on the luciferase concentration, as well as the other substrates, oxygen, and luciferin. Moreover, pH also affects luciferase activity. Another drawback of this method is that the intracellular ATP level could be perturbed because of ATP consumption. Furthermore, the dim luminescence of luciferase requires longer exposure time for image acquisition, making real-time observation cumbersome. Other approaches include measurement of the ion channel activity (6) or conformational change (7) of the ATP-s...

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration

“…We first asked whether the ATP level was altered in the axons of Jsap1:Jlp dKO primary neurons, using a lentiviral vector for ATeam, a fluorescence resonance energy transfer (FRET)-based ATP indicator. 29 We detected a significantly lower [ATP]i (intra-axonal ATP concentration) at 5 d.p.i. in the axons of AxCANCre-infected Jsap1:Jlp dKO neurons than in those of AxCANCre-uninfected (control) Jsap1 f/f :Jlp f/f neurons (Supplementary Figure S7).…”

“…The expression plasmid pCL20c-CMV-G-CaMP7 was generated by inserting the entire coding sequence of G-CaMP7 (Ohkura et al 2012) into pCL20c-CMV. To generate expression plasmids for the FRET-based ATP indicator AT1.03-YEMK (Imamura et al 2009) and its negative mutant AT1.03-R122K/R126K, 29 pCL20c-CMV-AT1.03-YEMK and -R122K/R126K, the respective entire coding regions were inserted into pCL20c-CMV. Substitutions and deletions in cDNAs were introduced by overlapping PCR, as described previously.…”

P2X receptors regulate adenosine diphosphate release from hepatic cells

“…Intracellular ATP is equally distributed between the nucleus, mitochondria, and cytosol in hepatic cells [35], while ADP levels are normally very low. This suggests that ADP release from hepatic cells is likely to involve a regulated pathway.…”

Low‐dose suramin in autism spectrum disorder: a small, phase I/II, randomized clinical trial

“…Extracellular purines like ATP, ADP, and adenosine, and pyrimidines like UTP are ligands for 19 different purinergic (P2X, P2Y, and P1) receptors 23. The intracellular concentration of ATP (iATP) in mammalian cells is typically 1–5 mmol/L,24 but drops when ATP is released through membrane channels under stress. Typical concentrations of extracellular adenine nucleotides in the unstirred water layer at the cell surface where receptors and ligands meet are about 1–10 μ mol/L, near the effective concentration for most purinergic receptors,25 but can increase when ATP is released during cell stress.…”