Abstract
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.
Highlights
Cyclic nucleotides such as cyclic adenosine monophosphate act as second messengers, transducing extracellular stimuli into intracellular signals and leading to various effects inside the cell
We find that the cyclic adenosine monophosphate (cAMP) concentration next to a single PDE molecule is undetectable by our fluorescence resonance energy transfer (FRET)-sensor (
The live cell FRET experiments carried out here (Fig 1) show that no change in the FRET ratio was recorded with isoproterenol when the sensor was fused to an active PDE (Epac1-camps-PDE4A1)
Summary
Cyclic nucleotides such as cyclic adenosine monophosphate (cAMP) act as second messengers, transducing extracellular stimuli into intracellular signals and leading to various effects inside the cell. More recent findings suggest that second messenger signaling displays an intriguing complexity requiring a more complex intracellular distribution to achieve signaling specificity. These observations gave rise to the concept of cAMP compartmentation —intracellular compartments with different concentrations of cAMP [1]. Key roles in the formation of cAMP compartments are attributed to the cAMP production by adenylyl cyclases (AC) and the degradation by phosphodiesterases (PDE) [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]
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