Robust and sensitive GFP-based cGMP sensor for real time imaging in intact Caenorhabditis elegans
Martin W. Schneider,
Wagner Steuer Costa,
O. Scott Hamilton,
Denise M. Ferkey,
Posted 02 Oct 2018
bioRxiv DOI: 10.1101/433425
Posted 02 Oct 2018
cGMP is a ubiquitous second messenger that plays a role in sensory signaling and plasticity through its regulation of ion channels and kinases. Previous studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities, including light, heat, gases, salt and odor. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other sensors and fluorescent markers. Here, we demonstrate that WincG2, a codon-optimized version of the cpEGFP-based cGMP sensor FlincG3, can be used in C. elegans to visualize rapidly changing cGMP levels in living, behaving animals using a single fluorophore. We coexpressed the sensor with the blue light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles and found that the rate of WincG2 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that WincG2 responds linearly upon NaCl concentration changes and SDS presentation in the cell bodies of the gustatory neuron ASER and the nociceptive phasmid neuron PHB, respectively. Intriguingly, WincG2 fluorescence in the ASER cell body decreased in response to a NaCl concentration downstep and either stopped decreasing or increased in response to a NaCl concentration upstep, which is opposite in sign to previously published calcium recordings. These results illustrate that WincG2 can be used to report rapidly changing cGMP levels in an intact animal and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.
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