title: Use of genetically encoded sensors to monitor cytosolic ATP/ADP ratio in living cells.
abstract: ATP is not only recognized as the universal energy "currency" in most cells but also plays a less well-known role as an intracellular and extracellular messenger. Here, we review novel approaches for measuring free ATP (or ATP/ADP ratios) in living mammalian cells by using genetically encoded sensors. We also discuss the key technical aspects of routine real-time ATP/ADP monitoring using as a model one of the last-generation fluorescent probes, a fusion protein commonly known as "Perceval." Finally, we present detailed guidelines for the simultaneous measurement of cytosolic ATP/ADP ratios and Ca(2+) concentrations alongside electrical parameters in individual pancreatic β cells, in which energy metabolism is tightly linked to plasma membrane excitability to control the secretion of insulin. With appropriate variations, this approach can be adapted to the study of cytosolic ATP/ADP ratios and Ca(2+) concentrations in malignant cells, two important aspects of oncometabolism.
authors.affiliation: Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, United Kingdom.
authors.forename: Andrei I
authors.initials: AI
authors.lastname: Tarasov
authors.affiliation: Section of Cell Biology, Department of Medicine, Imperial College London, London, United Kingdom. Electronic address: g.rutter@imperial.ac.uk.
authors.forename: Guy A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004734:Energy Metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation; D050417:Insulin-Secreting Cells / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D018408:Patch-Clamp Techniques
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002118:Calcium
keywords: Cytosolic ATP; Glycolysis; Live-cell imaging; Pancreatic β-cells; Patch clamp; Wide-field microscopy