For more details, please, refer to corresponding sections of the text
For more details, please, refer to corresponding sections of the text. over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight controlsomething, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs. of cAMP levels in Risedronate sodium pooled cellular populations, thus leaving us to guess what is going on with cAMP molecules in any given single cell. Apart from the limited spatial resolution, biochemical assays typically require cAMP liberation Risedronate sodium from specimens under study, which is usually accomplished by cell lysis (Williams, 2004; Hill et al., 2010). This way, biochemical assays in essence provide a single time point measurement, reflecting the total cAMP levels present in a specimen at the time of cell disruption. Though it is possible to deduce the overall kinetic trend of total cAMP over a time period by preparing a set of biological replicates and lysing them at certain intervals, the resulting kinetic curve is usually only a faint reflection of the actual cAMP oscillations in a given biological sample. Experimental data on cAMP, obtained with biochemical methods with limited temporal and spatial resolution, formed the basis for a widely accepted model of cAMP signaling. This model implies cAMP generation by membrane-bound adenylyl cyclases (ACs) in response to GPCRs activation and its subsequent free diffusion into the cytoplasm. The ensuing activation of immediate cytoplasmic effectors of cAMP, such as protein kinase A (PKA), convey the signal further to the level of cell nucleus, eventually translating extracellular stimuli into transcriptional response (Beavo and Brunton, 2002). However, cAMP network and governing principles of its functional and structural organization happen to be far more complex. Indeed, the conceptualization of cAMP signaling as of a highly compartmentalized process, occurring in separated subcellular domains, shaped by anchoring proteins and phosphodiesterases (PDEs), with organization of the key players of cAMP-mediated signal relay machinery into supramolecular complexes or signalosomes, has just started to evolve (Willoughby and Cooper, 2007; Lefkimmiatis and Zaccolo, 2014). Apart from the intricate laws of spatial organization of cAMP generation, trafficking and degradation, this burgeoning model recognizes the multifaceted nature of signal Risedronate sodium encoding by cAMP (strength vs. duration vs. frequency) and pays due regards to the crosstalk between cAMP and other intracellular regulators (Rich et al., 2014). It wound Risedronate sodium not be an overstatement to say, that the major insights into the complexity of cAMP signaling, served to fuel the above conceptual framework, were gained by studies exploiting next generation of tools for cAMP probing and modulation. Most of these tools are genetically encoded proteins, tailored for sensing and modulation of cAMP in living systems. These engineered proteins provide excellent spatial resolution down to desired subcellular domains, can respond to genuine oscillations of cAMP levels in real time and are designed to uncover cAMP signaling partners, and as such have enabled a paradigm-shift in cyclic nucleotide research. Evidently, in order to scrutinize a complex phenomenon, a set of diverse probing tools is required. Align with this and thanks to the intricate nature of cAMP signaling relay and never-ceasing attempts to gain insights into the of biosensors for cAMP have been developed (reviwed in Willoughby and Cooper, 2008; Hill et al., 2010; Sprenger and Nikolaev, 2013). However, besides being genetically-encoded proteins and hence applicable to studies in living cells, the modern biosensors do not have much in common, as they strive to probe different aspects of cAMP signaling, are governed by unique biological phenomena and rely on varied biophysical techniques. Considering this heterogeneity and in order to make this review more sound and cohesive, we decided to categorize the biosensors into two major groups: tools for direct measurement of cAMP and tools for indirect cAMP probing. As the name indicates, direct probes provide cAMP measurements, having a readout typically becoming generated immediately after binding between cAMP and the sensor molecules. Additionally, the readout intensity from direct biosensors is usually proportional to the intensity of activation, which allows the direct probes to convey valid Rabbit polyclonal to c-Myc (FITC) data on actual oscillations of intracellular cAMP.