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The role of type 4 phosphodiesterases in generating microdomains of cAMP: large scale stochastic simulations.

Oliveira RF, Terrin A, Di Benedetto G, Cannon RC, Koh W, Kim M, Zaccolo M, Blackwell KT - PLoS ONE (2010)

Bottom Line: Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity.Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity.The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains.

View Article: PubMed Central - PubMed

Affiliation: The Krasnow Institute for Advanced Study, George Mason University, Fairfax, Virginia, United States of America.

ABSTRACT
Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.

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The theoretical FRET signal and cAMP concentration show microdomains without diffusional barriers.(A) The FRET signal for the submembrane region is 6.8% higher than the cytosol. Mean (black traces) and ±SD (gray traces, n = 5). (B) Difference between submembrane and cytosolic cAMP concentration is similar to that observed for the FRET signal, and is independent of overexpression of the H30 sensor. The model cell with H30 is shown in black; the model cell without H30 is shown in gray. SD traces are not illustrated because they overlap with the mean. No diffusional barriers are present for these simulations. The expression of the sensor does not disturb the cAMP microdomain, therefore the difference between submembrane FRET and cytosolic FRET is not an artifact of the method. (C) Representative kinetics of FRET changes recorded in cells expressing either the membrane targeted sensor mpH30 or the cytosolic sensor H30 [16], [56] upon stimulation with 1µM PGE1. FRET experiments were performed as described previously in [16].
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pone-0011725-g004: The theoretical FRET signal and cAMP concentration show microdomains without diffusional barriers.(A) The FRET signal for the submembrane region is 6.8% higher than the cytosol. Mean (black traces) and ±SD (gray traces, n = 5). (B) Difference between submembrane and cytosolic cAMP concentration is similar to that observed for the FRET signal, and is independent of overexpression of the H30 sensor. The model cell with H30 is shown in black; the model cell without H30 is shown in gray. SD traces are not illustrated because they overlap with the mean. No diffusional barriers are present for these simulations. The expression of the sensor does not disturb the cAMP microdomain, therefore the difference between submembrane FRET and cytosolic FRET is not an artifact of the method. (C) Representative kinetics of FRET changes recorded in cells expressing either the membrane targeted sensor mpH30 or the cytosolic sensor H30 [16], [56] upon stimulation with 1µM PGE1. FRET experiments were performed as described previously in [16].

Mentions: The first set of simulations evaluates whether diffusional barriers are required for the cAMP microdomain as measured by FRET. This first step aims at reproducing the results previously reported [16]. Fig. 4A shows that the model successfully reproduces the experimental FRET signal, including the difference in cAMP concentration between submembrane and cytosolic compartments. Comparison between the experimentally calculated FRET signal (Fig. 4C) and the theoretically derived FRET signal shows that both traces have a sharp increase (rising phase) right after stimulation is delivered at 100 s. Likewise, FRET peak value is reached ∼100 s after stimulation has started in both experiment and simulation. Because this simulation uses experimentally constrained values for diffusion, this result confirms the experimental result that the cAMP microdomain does not require diffusional barriers.


The role of type 4 phosphodiesterases in generating microdomains of cAMP: large scale stochastic simulations.

Oliveira RF, Terrin A, Di Benedetto G, Cannon RC, Koh W, Kim M, Zaccolo M, Blackwell KT - PLoS ONE (2010)

The theoretical FRET signal and cAMP concentration show microdomains without diffusional barriers.(A) The FRET signal for the submembrane region is 6.8% higher than the cytosol. Mean (black traces) and ±SD (gray traces, n = 5). (B) Difference between submembrane and cytosolic cAMP concentration is similar to that observed for the FRET signal, and is independent of overexpression of the H30 sensor. The model cell with H30 is shown in black; the model cell without H30 is shown in gray. SD traces are not illustrated because they overlap with the mean. No diffusional barriers are present for these simulations. The expression of the sensor does not disturb the cAMP microdomain, therefore the difference between submembrane FRET and cytosolic FRET is not an artifact of the method. (C) Representative kinetics of FRET changes recorded in cells expressing either the membrane targeted sensor mpH30 or the cytosolic sensor H30 [16], [56] upon stimulation with 1µM PGE1. FRET experiments were performed as described previously in [16].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908681&req=5

pone-0011725-g004: The theoretical FRET signal and cAMP concentration show microdomains without diffusional barriers.(A) The FRET signal for the submembrane region is 6.8% higher than the cytosol. Mean (black traces) and ±SD (gray traces, n = 5). (B) Difference between submembrane and cytosolic cAMP concentration is similar to that observed for the FRET signal, and is independent of overexpression of the H30 sensor. The model cell with H30 is shown in black; the model cell without H30 is shown in gray. SD traces are not illustrated because they overlap with the mean. No diffusional barriers are present for these simulations. The expression of the sensor does not disturb the cAMP microdomain, therefore the difference between submembrane FRET and cytosolic FRET is not an artifact of the method. (C) Representative kinetics of FRET changes recorded in cells expressing either the membrane targeted sensor mpH30 or the cytosolic sensor H30 [16], [56] upon stimulation with 1µM PGE1. FRET experiments were performed as described previously in [16].
Mentions: The first set of simulations evaluates whether diffusional barriers are required for the cAMP microdomain as measured by FRET. This first step aims at reproducing the results previously reported [16]. Fig. 4A shows that the model successfully reproduces the experimental FRET signal, including the difference in cAMP concentration between submembrane and cytosolic compartments. Comparison between the experimentally calculated FRET signal (Fig. 4C) and the theoretically derived FRET signal shows that both traces have a sharp increase (rising phase) right after stimulation is delivered at 100 s. Likewise, FRET peak value is reached ∼100 s after stimulation has started in both experiment and simulation. Because this simulation uses experimentally constrained values for diffusion, this result confirms the experimental result that the cAMP microdomain does not require diffusional barriers.

Bottom Line: Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity.Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity.The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains.

View Article: PubMed Central - PubMed

Affiliation: The Krasnow Institute for Advanced Study, George Mason University, Fairfax, Virginia, United States of America.

ABSTRACT
Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.

Show MeSH