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Genetics. 2019 Sep;213(1):59-77. doi: 10.1534/genetics.119.302392. Epub 2019 Jul 22.

Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans.

Author information

1
Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, California 94158.
2
Department of Cell and Tissue Biology, University of California, San Francisco, California 94143.
3
Department of Molecular Membrane Biology and Neurobiology, The Goethe University, 60323 Frankfurt, Germany.
4
Neuroscience Graduate Program, Brandeis University, Waltham, Massachusetts 02453.
5
Department of Biology, Brandeis University, Waltham, Massachusetts 02454.
6
Department of Biological Sciences, San Jose State University, California 95192.
7
Department of Biological Sciences, University at Buffalo, The State University of New York, New York 14260.
8
Center for Neuroscience, University of California, Davis, California 95618.
9
Department of Cell and Tissue Biology, University of California, San Francisco, California 94143 noelle.letoile@ucsf.edu.

Abstract

cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. 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 fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 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.

KEYWORDS:

C. elegans; FlincG3; cGMP; sensory neuron; visual reporter

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