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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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FRET signal as a function of cAMP concentration.(A) Steady state dose-response simulation (black) and experimental (gray) curves show excellent agreement. Ordinates are background-subtracted FRET emission ratio changes, ΔR, measured relative to the prestimulus ratio R(0). (B) Time course of simulated and experimental FRET signal shows excellent agreement. FRET ratio trace obtained by delivery of 30 µM cAMP to cell under whole-cell recording conditions. All experiments were performed in HeLa cells transfected with H30 (acquisition conditions: 1 frame/5 s). The microscope was equipped with a CCD camera (Sensicam QE; PCO), a software-controlled monochromator (Polychrome IV; TILL Photonics), and a beam-splitter optical device (Multispec Microimager; Optical Insights).
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pone-0011725-g002: FRET signal as a function of cAMP concentration.(A) Steady state dose-response simulation (black) and experimental (gray) curves show excellent agreement. Ordinates are background-subtracted FRET emission ratio changes, ΔR, measured relative to the prestimulus ratio R(0). (B) Time course of simulated and experimental FRET signal shows excellent agreement. FRET ratio trace obtained by delivery of 30 µM cAMP to cell under whole-cell recording conditions. All experiments were performed in HeLa cells transfected with H30 (acquisition conditions: 1 frame/5 s). The microscope was equipped with a CCD camera (Sensicam QE; PCO), a software-controlled monochromator (Polychrome IV; TILL Photonics), and a beam-splitter optical device (Multispec Microimager; Optical Insights).

Mentions: In order to precisely compare simulated results with FRET imaging data, a theoretical FRET signal was calculated from simulated concentrations of cAMP-bound-H30 and free H30 sensor, and included a FRET efficiency term and contamination terms due to overlap of the sensor emission and excitation spectra. Thus, the simulated FRET ratio, R, was the same as the experimental FRET ratio:(6)with(7)(8)and β = 0.35, γ = 0.12, δ = 0.67. When H30 was bound to cAMP there was no transfer of energy and all of the donor emission was detected as the cyan signal. β, γ, δ and H30 affinity for cAMP were obtained from experimental measurements and were adjusted slightly to yield better agreement with experimental calibration data (Fig. 2). R/R0 was calculated by dividing FRET ratio, R(t), by the initial FRET ratio, R(0), measured before stimulation was applied, as in original experiments. Simulation of cAMP dose-FRET response curves were constructed and compared to experiments (Fig. 2A). In addition, the time course of simulated H30 binding to cAMP, as measured by the FRET signal, also was compared to experimental results. Both FRET calibration curves showed good agreement (Fig. 2B), providing precise quantitative comparison with experimental results.


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)

FRET signal as a function of cAMP concentration.(A) Steady state dose-response simulation (black) and experimental (gray) curves show excellent agreement. Ordinates are background-subtracted FRET emission ratio changes, ΔR, measured relative to the prestimulus ratio R(0). (B) Time course of simulated and experimental FRET signal shows excellent agreement. FRET ratio trace obtained by delivery of 30 µM cAMP to cell under whole-cell recording conditions. All experiments were performed in HeLa cells transfected with H30 (acquisition conditions: 1 frame/5 s). The microscope was equipped with a CCD camera (Sensicam QE; PCO), a software-controlled monochromator (Polychrome IV; TILL Photonics), and a beam-splitter optical device (Multispec Microimager; Optical Insights).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908681&req=5

pone-0011725-g002: FRET signal as a function of cAMP concentration.(A) Steady state dose-response simulation (black) and experimental (gray) curves show excellent agreement. Ordinates are background-subtracted FRET emission ratio changes, ΔR, measured relative to the prestimulus ratio R(0). (B) Time course of simulated and experimental FRET signal shows excellent agreement. FRET ratio trace obtained by delivery of 30 µM cAMP to cell under whole-cell recording conditions. All experiments were performed in HeLa cells transfected with H30 (acquisition conditions: 1 frame/5 s). The microscope was equipped with a CCD camera (Sensicam QE; PCO), a software-controlled monochromator (Polychrome IV; TILL Photonics), and a beam-splitter optical device (Multispec Microimager; Optical Insights).
Mentions: In order to precisely compare simulated results with FRET imaging data, a theoretical FRET signal was calculated from simulated concentrations of cAMP-bound-H30 and free H30 sensor, and included a FRET efficiency term and contamination terms due to overlap of the sensor emission and excitation spectra. Thus, the simulated FRET ratio, R, was the same as the experimental FRET ratio:(6)with(7)(8)and β = 0.35, γ = 0.12, δ = 0.67. When H30 was bound to cAMP there was no transfer of energy and all of the donor emission was detected as the cyan signal. β, γ, δ and H30 affinity for cAMP were obtained from experimental measurements and were adjusted slightly to yield better agreement with experimental calibration data (Fig. 2). R/R0 was calculated by dividing FRET ratio, R(t), by the initial FRET ratio, R(0), measured before stimulation was applied, as in original experiments. Simulation of cAMP dose-FRET response curves were constructed and compared to experiments (Fig. 2A). In addition, the time course of simulated H30 binding to cAMP, as measured by the FRET signal, also was compared to experimental results. Both FRET calibration curves showed good agreement (Fig. 2B), providing precise quantitative comparison with experimental results.

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