Mechanisms Restricting Diffusion of Intracellular cAMP.

Shailesh R Agarwal,Colleen E Clancy,Robert D Harvey

doi:10.1038/srep19577

Shailesh R Agarwal, Colleen E Clancy + Show 1 more

Open Access

https://doi.org/10.1038/srep19577

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Abstract

Although numerous receptors stimulate cAMP production in a wide array of cells, many elicit distinct, highly localized responses, implying that the subcellular distribution of cAMP is not uniform. One often used explanation is that phosphodiesterases, which breakdown cAMP, act as functional barriers limiting diffusion. However, several studies refute the notion that this is sufficient, suggesting that phosphodiesterase-independent movement of cAMP must occur at rates slower than free diffusion. But, until now this has never been demonstrated. Using Raster Image Correlation Spectroscopy (RICS), we measured the diffusion coefficient of a fluorescently-labeled cAMP derivative (φ450-cAMP) as well as other fluorescent molecules in order to investigate the role that molecular size, cell morphology, and buffering by protein kinase A (PKA) play in restricting cAMP mobility in different cell types. Our results demonstrate that cytosolic movement of cAMP is indeed much slower than the rate of free diffusion and that interactions with PKA, especially type II PKA associated with mitochondria, play a significant role. These findings have important implications with respect to cAMP signaling in all cells.

Highlights

Numerous receptors stimulate cAMP production in a wide array of cells, many elicit distinct, highly localized responses, implying that the subcellular distribution of cAMP is not uniform
Our results demonstrate that cytosolic movement of cAMP is much slower than the rate of free diffusion and that interactions with protein kinase A (PKA), especially type II PKA associated with mitochondria, play a significant role
Intracellular diffusion is affected by three independent factors: the fluid-phase viscosity of the cytoplasm, collisional interactions caused by molecular crowding, and binding interactions with mobile and fixed molecules in cells[32]

Summary

Introduction

Numerous receptors stimulate cAMP production in a wide array of cells, many elicit distinct, highly localized responses, implying that the subcellular distribution of cAMP is not uniform. Our results demonstrate that cytosolic movement of cAMP is much slower than the rate of free diffusion and that interactions with PKA, especially type II PKA associated with mitochondria, play a significant role These findings have important implications with respect to cAMP signaling in all cells. A number of modeling studies have concluded that while PDE activity is necessary for compartmentalized behavior, it alone is not sufficient to establish cAMP gradients[13,14,15,16,17,18,19] These simulations suggest that for compartmentalized responses to occur, diffusion of cAMP in the cytoplasm must be much slower than the rate of free diffusion measured in dilute aqueous solutions. The idea that cAMP movement occurs at rates slower than free diffusion is still a theoretical prediction that has yet to be demonstrated experimentally

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