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Simultaneous Assessment of cAMP Signaling Events in Different Cellular Compartments Using FRET-Based Reporters

  • Alex Burdyga
  • Konstantinos LefkimmiatisEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1294)

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.

Keywords

cAMP PKA FRET Imaging Fluorescence Microdomains Compartmentalization 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Physiology, Anatomy and Genetics, Burdon Sanderson Cardiac Science Centre, BHF Centre of Research ExcellenceUniversity of OxfordOxfordUK

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