Local PDE activity creates a cAMP gradient in cytosolic fractions.

<p>Concentration-effect curves of cAMP-induced changes of the FRET ratios of the cAMP sensors Epac1-camps (grey curve) and Epac1-camps-PDE4A1 (red curve) in soluble cytosolic preparations of transiently transfected HEK-TsA cells. The presence of PDE activity in the fusion protein leads to a loss of apparent affinity of the FRET-sensor for cAMP (rightward shift of the concentration-effect curve). (b)Separate expression of equal amounts of Epac1-camps and PDE4A1 (blue curve) leads to a right-shift of the concentration-effect-curve, albeit to a lesser extent than the fusion protein. The curves for Epac1-camps and Epac1-camps-PDE4A1 are shown for comparison as grey and red dashed lines, respectively. (c) Apparent affinities (pEC₅₀) of Epac1-camps, Epac1-camps-PDE4A1, and Epac1-camps + PDE4A1 for cAMP are 5.60 ± 0.03(= 2.5<i>μ</i>M), 4.60 ± 0.02(= 25<i>μ</i>M), and 5.14 ± 0.02(= 7.2<i>μ</i>M), respectively. This indicates that the cAMP concentration in close proximity to the PDE is less than the concentration of the surrounding solution. Experiments were carried out in 10mM TRIS, 10mM MgCl₂, pH7.4 and FRET changes were recorded upon addition of increasing concentrations of cAMP. Data are normalized to the maximum change of the FRET ratio at saturating concentrations of cAMP (= 100%) and the basal FRET ratio in the absence of cAMP (= 0%), respectively. The slope of all curves is not significantly different from n = 1 (P = 0.53, P = 0.37, P = 0.80 for Epac1-camps, Epac1-camps-PDE4A1, and Epac1-camps + PDE4A1, respectively). Data are means ± s.e.m. of at least three independent experiments carried out with 3-5 repetitions. ****, ####, (P < 0.0001) according to one-way ANOVA with Tukey’s multiple comparison test.</p

Schematic description of the experimental design used to measure cAMP nanocompartments on a single molecule resolution.

<p>The PDE molecule is modeled as an absorbing sphere of radius <i>R</i>_PDE and the sensor protein attached adjacent (distance <i>d</i>) is used to measure the cAMP concentration.</p

Depth <i>γ</i> of the nanocompartments formed by a single PDE molecule as a function of the cAMP concentration <i>c</i> for different values of absorptive action <i>η</i>.

<p>The concentration <i>c</i> is given in multiples of the Michaelis-Menten constant of the PDE , the absorptive action <i>η</i> is defined as . The deepest compartments are found in the limit <i>c</i> → 0, where . When the cAMP concentration is increased (<i>c</i> → ∞), the compartments get flooded and disappear due to saturation of the PDE. Dots indicate the concentration at which the compartment is half way flooded and are found at .</p

Concentration of cAMP inside a spherical cluster of PDE for different numbers of degrading molecules <i>N</i>_PDE.

<p>For both plots the absorptive action of a single PDE was set to <i>η</i>₂ = 6.1 ± 1.4. Left: cluster with radius <i>R</i>_• = 100nm; to form a microcompartment of this size there required number of PDEs is about <i>N</i>_PDE = 10. Right: cluster with radius <i>R</i>_• = 200nm. The depth of the microcompartment is smaller when the number of degrading molecules is kept constant.</p

Models of clusters of a degrading enzyme.

<p>Left: the degrading enzyme (here PDE) has a homogeneous distribution within a spherical region of diameter <i>R</i>_•. Right: the degrading enzyme forms a thin layer acting as a protective border for the inner region.</p

Fusion of PDE4A1 to Epac1-camps generates cAMP nanodomains in living cells.

<p>(a) and (c) Representative traces of the normalized FRET (YFP/CFP) ratio of the indicated constructs. Increases of cAMP were obtained by activation of endogenous <i>β</i>-receptors by isoproterenol (a) or upon stimulation with the direct activators of adenylyl cyclase forskolin (c) and (d) Amplitude of the cAMP response elicited by isoproterenol (b) or forskolin (d) expressed as a percentage of maximal stimulation induced by rolipram. Data are shown as means ± s.e.m. of at least 6 independent experiments. Differences vs. Epac1-camps were statistically significant by one-way ANOVA followed by Bonferroni post hoc-test **, P < 0, 001 ****, P < 0,00001.</p

Binding curves of the free sensor protein Epac1-camps + PDE (blue) and the fusion protein Epac1-camps-PDE4A1 (red).

<p>The overall PDE activity is equal in both experiments, indicating that the right shift of the binding curve is solely caused by local PDE degradation. The curves were determined by fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174856#pone.0174856.e028" target="_blank">Eq (18)</a> to the corresponding experimental data. The distance between sensor and absorbing enzyme was set to <i>d</i> = 0nm as for an ideal sensor-enzyme construct. The fit of the free sensor data (blue) yields the dissociation constant of the sensor <i>K</i>_<i>D</i> = 7.3 ± 0.66<i>μ</i>M (goodness of fit: <i>χ</i>² = 3.2, <i>p</i> = 0.66). From the sensor-enzyme construct (red) we obtain the absorptive action of a PDE <i>η</i>₂ = 6.1 ± 1.4, (<i>χ</i>² = 9.6, <i>p</i> = 0.08). The flooding of the nanocompartment as predicted by our model could not be observed in the experiments.</p