cGMP Trigger for Cell Signaling By Optogenetic Fungal Protein

Cyclic GMP (cGMP) signaling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. The optogenetic toolbox keeps expanding. Besides containing light-sensitive proteins that can be used to adjust membrane potential, protein-protein interactions, and gene expression, the optogenetic toolbox includes sensors that can be used to generate intracellular second messengers, triggering signaling cascades associated with vision, blood pressure, programmed cell death, and male erection.

The most successful second-messenger optogenetic tools work by generating cAMP. Optogenetic tools that work via cGMP have been less satisfactory. Thus far, these tools have been mutated versions of natural light-sensitive proteins. They lack not only elegance, but also efficiency.

Optogenetic work, however, needn’t struggle with clumsy cGMP-generating tools any longer, according to research from Goethe University Frankfurt. This research, like much prior optogenetic research, focused on a type I (microbial) rhodopsin, a light-sensitive membrane protein. The new work, however, investigated a new type I rhodopsin, one that is fused with a guanylyl cyclase domain. It was found in the fungus Blastocladiella emersonii, and it was called BeGC1. It was shown to be essential for phototaxis of zoospores in B. emersonii through green light-regulated cGMP production.

The Goethe University research, which was led by Prof. Alexander Gottschalk, went further to demonstrate directly that BEGC1’s guanylyl cyclase activity was integral to the light-regulated mechanism. These results were detailed September 8 in Nature Communications, in an article entitled, “Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.”

The article includes the suggestion that BeGC1 should be renamed guanylyl cyclase opsin from B. emersonii, or BeCyclOp: “Here we implement [BeCyclOp] as a new optogenetic tool,enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans,” wrote the authors. “Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ~17 cGMP s−1).”

“The light-activated enzyme CyclOp has outstanding molecular properties which qualify it as a valuable addition to the optogenetics toolbox for cell biologists and neurobiologists,” explained Prof. Gottschalk.

Prof Gottschalk’s research group has introduced the protein into oxygen sensing cells in order to find out what role the second messenger cGMP plays in these cells. To do so, the translucent nematode is exposed to light leading to intracellular generation of cGMP. The cells respond by acting as if they had detected an increase in the oxygen level. In this way the researchers can use CyclOp to get a better understanding of how the natural signal for these cells is turned into a cellular response.