Use of single-cell imaging techniques to assess the regulation of cAMP dynamics

cAMP is a ubiquitous intracellular signalling molecule that can regulate a wide array of cellular processes. The diversity of action of this second messenger owes much to the localized generation, action and hydrolysis of cAMP within discrete subcellular regions. Further signalling specificity can be achieved by the ability of cells to modulate the frequency or incidence of such cAMP signals. Here, we discuss the use of two cAMP biosensors that measure real-time cAMP changes in the single cell, to address the mechanisms underlying the generation of dynamic cAMP signals. The first method monitors sub-plasmalemmal cAMP changes using mutant cyclic nucleotide-gated channels and identifies an AKAP (A-kinase-anchoring protein)–protein kinase A–PDE4 (phosphodiesterase-4) signalling complex that is central to the generation of dynamic cAMP transients in this region of the cell. The second study uses a fluorescence resonance energy transfer-based cAMP probe, based on Epac1 (exchange protein directly activated by cAMP 1), to examine interplay between Ca²⁺ and cAMP signals. This study demonstrates real-time oscillations in cAMP driven by a Ca²⁺-stimulated AC (adenylate cyclase) (AC8) and subsequent PDE4 activity. These studies, using two very different single-cell cAMP probes, broaden our understanding of the specific spatiotemporal characteristics of agonist-evoked cAMP signals in a model cell system.