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Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes

Author:
Colombo, Sonia, Broggi, Serena, Collini, Maddalena, D'Alfonso, Laura, Chirico, Giuseppe, Martegani, Enzo
Source:
Biochemical and biophysical research communications 2017 v.487 pp. 594-599
ISSN:
0006-291X
Subject:
Saccharomyces cerevisiae, biosensors, cAMP-dependent protein kinase, cell cycle, cyclic AMP, energy transfer, fluorescence, glucose, mammals, metabolism, second messengers, stress tolerance, yeasts
Abstract:
In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell biosensors for cAMP levels and PKA activity in mammalian cells. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce cAMP. In addition, we had evidence for damped oscillations in cAMP levels at least in SP1 strain. Regarding detection of PKA activity, we show that in wild type strains the FRET increased after glucose addition to derepressed cells, while no changes were observed when glucose was added to either a strain that is not able to produce cAMP or to a strain with absent PKA activity. Taken together these probes are useful to follow activation of the cAMP/PKA pathway in single yeast cells and for long times (up to one hour).
Agid:
5675799