TY - JOUR A1 - Lohse, Christian A1 - Bock, Andreas A1 - Maiellaro, Isabella A1 - Hannawacker, Annette A1 - Schad, Lothar R. A1 - Lohse, Martin J. A1 - Bauer, Wolfgang R. T1 - Experimental and mathematical analysis of cAMP nanodomains JF - PLoS ONE N2 - In their role as second messengers, cyclic nucleotides such as cAMP have a variety of intracellular effects. These complex tasks demand a highly organized orchestration of spatially and temporally confined cAMP action which should be best achieved by compartmentalization of the latter. A great body of evidence suggests that cAMP compartments may be established and maintained by cAMP degrading enzymes, e.g. phosphodiesterases (PDEs). However, the molecular and biophysical details of how PDEs can orchestrate cAMP gradients are entirely unclear. In this paper, using fusion proteins of cAMP FRET-sensors and PDEs in living cells, we provide direct experimental evidence that the cAMP concentration in the vicinity of an individual PDE molecule is below the detection limit of our FRET sensors (<100nM). This cAMP gradient persists in crude cytosol preparations. We developed mathematical models based on diffusion-reaction equations which describe the creation of nanocompartments around a single PDE molecule and more complex spatial PDE arrangements. The analytically solvable equations derived here explicitly determine how the capability of a single PDE, or PDE complexes, to create a nanocompartment depend on the cAMP degradation rate, the diffusive mobility of cAMP, and geometrical and topological parameters. We apply these generic models to our experimental data and determine the diffusive mobility and degradation rate of cAMP. The results obtained for these parameters differ by far from data in literature for free soluble cAMP interacting with PDE. Hence, restricted cAMP diffusion in the vincinity of PDE is necessary to create cAMP nanocompartments in cells. KW - fluorescence resonance energy transfer KW - yellow fluorescent protein KW - radii KW - adenylyl cyclase signaling cascade KW - cell fusion KW - cytosol KW - isoproterenol KW - absorption KW - cyclic nucleotides such as cyclic adenosine monophosphate Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170972 VL - 12 IS - 4 ER - TY - JOUR A1 - Calebiro, Davide A1 - Maiellaro, Isabella T1 - cAMP signaling microdomains and their observation by optical methods JF - Frontiers in Cellular Neuroscience N2 - The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains. KW - G protein-coupled receptor KW - cyclic AMP KW - signaling microdomain KW - fluorescence resonance energy transfer KW - neurons Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118252 SN - 1662-5102 VL - 8 ER -