Simultaneous assessment of cAMP signaling events in different cellular compartments using FRET-based reporters

Methods in Molecular Biology
Alex Burdyga, Konstantinos Lefkimmiatis

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.

Citations

Dec 15, 2016·Scientific Reports·Hege E LarsenDavid J Paterson
Jun 27, 2018·Proceedings of the National Academy of Sciences of the United States of America·Alex BurdygaKonstantinos Lefkimmiatis
Jul 4, 2019·Progress in Biophysics and Molecular Biology·Francesca GrisanKonstantinos Lefkimmiatis
Mar 21, 2021·Cell Death and Differentiation·Francesca GrisanKonstantinos Lefkimmiatis

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