Assessment of cAMP-PKA Signaling in Candida glabrata by FRET-Based Biosensors.

Fluorescence or Förster resonance energy transfer (FRET)-based biosensors are used to monitor activity through molecular pathways inside the cell. Binding of secondary metabolites or enzyme-guided modification of protein targets can be assessed by quantifying the rate of energy transfer between two adequate fluorophores. The AKAR3 sensor contains a protein kinase A (PKA) phosphorylation site, which upon phosphorylation interacts with a ligand domain, bringing together FRET donor and acceptor fluorophores and allowing FRET. The EPAC2 sensor contains a cyclic adenosine monophosphate (cAMP)-binding domain. Upon binding of cAMP, donor and acceptor molecules are separated, thereby lowering energy transfer. Since the cAMP-PKA pathway is of great importance for regulation of growth, survival, and virulence in Candida species, monitoring the activity of this pathway in a time- and space-resolved manner allows for detailed investigation of potential drug targets. In this chapter, we describe how these FRET-based biosensors can be used in a practical setup.