title: Imaging changes in the cytosolic ATP-to-ADP ratio.
abstract: Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside the cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We, therefore, developed a genetically encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here, we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Mathew
authors.initials: M
authors.lastname: Tantama
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. Electronic address: gary_yellen@hms.harvard.edu.
authors.forename: Gary
authors.initials: G
authors.lastname: Yellen
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002138:Calibration; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D004734:Energy Metabolism; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate
keywords: ADP; ATP; Energy; Fluorescence; Genetically encoded; Imaging; Metabolism; Microscopy; Ratiometric; Sensor