RRC ID 59621
Author Woldemariam S, Nagpal J, Hill T, Li J, Schneider MW, Shankar R, Futey M, Varshney A, Ali N, Mitchell J, Andersen K, Barsi-Rhyne B, Tran A, Costa WS, Krzyzanowski MC, Yu YV, Brueggemann C, Hamilton OS, Ferkey DM, VanHoven M, Sengupta P, Gottschalk A, L'Etoile N.
Title Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans.
Journal Genetics
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.
Volume 213(1)
Pages 59-77
Published 2019-9-1
DOI 10.1534/genetics.119.302392
PII genetics.119.302392
PMID 31331946
PMC PMC6727795
MeSH Animals Caenorhabditis elegans Cells, Cultured Cyclic GMP / metabolism* Fluorescence Resonance Energy Transfer / methods* Green Fluorescent Proteins / genetics Green Fluorescent Proteins / metabolism* Guanylate Cyclase / genetics Guanylate Cyclase / metabolism Opsins / genetics Opsins / metabolism Optical Imaging / methods Optogenetics / methods* Sensory Receptor Cells / cytology Sensory Receptor Cells / metabolism
IF 3.564
Times Cited 2
C.elegans tm2364