Extracellular signals are relayed to the cell’s interior by second messengers such as cyclic AMP (cAMP), which was once thought to diffuse freely throughout the cytoplasm. Yet cAMP has also become known for its ability to bring about distinct, highly localized downstream effects. So, cAMP concentrations are now thought to vary from place to place within the cell, and to do so in ways that cannot be explained by simple diffusion.

To account for cAMP levels throughout the cell, scientists have suggested various refinements to simple diffusion. These include favorable distributions of cAMP pathway constituents, such as protein kinases and their substrates, and the presence of cAMP-degrading phosphodiesterases (PDEs). Neither of these explanations has proven to be entirely satisfactory.

Another wrinkle has been introduced by scientists working at the Max Delbrück Center for Molecular Medicine. These scientists, led by Andreas Bock and Paolo Annibale, say that under basal conditions, most cAMP in cells is bound, not free. Under stimulated conditions, that is, when extracellular receptors are engaged, some cAMP molecules are released, enough to propagate a signal, but not so much as to swamp the cell’s PDEs.

This model of cAMP signal propagation, which the scientists call “buffered diffusion,” was detailed August 25 in Cell, in an article titled, “Optical Mapping of cAMP Signaling at the Nanometer Scale.”

The article reports how the scientists used fluorescence spectroscopy to show that, contrary to earlier data, cAMP at physiological concentrations is “predominantly bound to cAMP binding sites and, thus, immobile.” The article also explains how restraining fast-moving cAMP gives slow-acting PDEs a chance to create, in their vicinities, domains that are practically free of cAMP.

“With a large fraction of cAMP being buffered, PDEs can create nanometer-size domains of low cAMP concentrations,” the article’s authors wrote. “Using FRET-cAMP nanorulers, we directly map cAMP gradients at the nanoscale around PDE molecules and the areas of resulting downstream activation of cAMP-dependent protein kinase (PKA).”

The authors concluded that their study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-size domains. Moreover, these domains, or “compartments,” are shaped by PDEs that gate activation of downstream effectors.

“You can imagine these cleared-out compartments rather like the holes in a Swiss cheese—or like tiny prisons in which the actually rather slow-working PDE keeps watch over the much faster cAMP to make sure it does not break out and trigger unintended effects in the cell,” explained Annibale. “Once the perpetrator is locked up, the police no longer have to chase after it.”

To measure the size of the holes, the researchers used their FRET-cAMP nanorulers, which are elongated proteins. The researchers’ measurements showed that most compartments are actually smaller than 10 nanometers, that is, 10 millionths of a millimeter. This way, the cell is able to create thousands of distinct cellular domains in which it can regulate cAMP separately and thus protect itself from the signaling molecule’s unintended effects.

Graphical abstract: Optical mapping of cAMP signaling at the nanometer scale. [Cell]
“We were able to show that a specific signaling pathway was initially interrupted in a hole that was virtually cAMP-free,” asserted Annibale. “But when we inhibited the PDEs that create these holes, the pathway continued on unobstructed.”

“This means the cell does not act like a single on/off switch, but rather like an entire chip containing thousands of such switches,” explained Martin Lohse, MD, scientific director, Max Delbrück Center for Molecular Medicine. “The mistake made in past experiments was to use cAMP concentrations that were far too high, thus enabling a large amount of the signaling molecule to diffuse freely in the cell because all binding sites were occupied.”

As a next step, the researchers want to further investigate the architecture of the cAMP “prisons” and find out which PDEs protect which signaling proteins. In the future, medical research could also benefit from their findings. “Many drugs work by altering signaling pathways within the cell,” noted Lohse. “Thanks to the discovery of this cell compartmentalization, we now know there are a great many more potential targets that can be searched for.”

“A study from San Diego, which was published at the same time as our article in Cell, shows that cells begin to proliferate when their individual signaling pathways are no longer regulated by spatial separation,” Bock pointed out. He added that it is already known, for example, that during heart failure, the distribution of cAMP concentration levels in heart cells is changed.

Such cell signaling subtleties in cancer and heart disease may be better understood now that the current work has detailed the phenomenon of buffered diffusion. In fact, this work may open new avenues for research into cancer, cardiovascular disease, and other diseases.

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