title: Simultaneous assessment of cAMP signaling events in different cellular compartments using FRET-based reporters.
abstract: Several aspects of the cAMP signaling cascade, including the levels of the messenger itself and the activity of its main effector protein kinase A (PKA), can be measured in living cells, thanks to genetically encoded probes based on fluorescence resonance energy transfer (FRET). While these biosensors enable the assessment of cAMP or PKA activity with great spatial and temporal resolution, concomitant events triggered by the same stimuli at the same or other cellular compartments are not easily assessed. In this chapter we present a simple approach that allows the simultaneous measurement of cAMP and its actions in subcellular compartments of neighboring cells. As proof of principle, we compare cAMP signals and PKA activity in the cytosol of neighboring HEK cells. We propose that this flexible and powerful method can significantly improve the direct comparison of cAMP signals and their action in specific cellular domains.
authors.affiliation: Department of Physiology, Anatomy and Genetics, Burdon Sanderson Cardiac Science Centre, BHF Centre of Research Excellence, University of Oxford, Sherrington Building, Parks Road, Oxford, OX1 3PT, UK.
authors.forename: Alex
authors.initials: A
authors.lastname: Burdyga
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Lefkimmiatis
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D015398:Signal Transduction; D012984:Software
chemical_list: D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases