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

Sarah Woldemariam,Kristine Andersen,Nebat Ali,Jordan Mitchell,Miri Vanhoven,Jatin Nagpal,Joy Li,Piali Sengupta,Chantal Brueggemann,Alan Tran,Wagner Steuer Costa,Aruna Varshney,Raakhee Shankar,Mary Futey,Tyler Hill,Alexander Gottschalk,Martin Schneider ,Denise M Ferkey ,O Scott Hamilton ,Yanxun V Yu ,Benjamin Barsi‐Rhyne ,Michelle Krzyzanowski ,Noëlle D L’étoile

doi:10.1534/genetics.119.302392

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.

Highlights

ABSTRACT cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases
While evidence suggests that these receptor guanylyl cyclases (rGCs) and TAX-2/TAX-4 are localized to the dendrite tip of AFD, it was unknown whether cGMP levels were spatially regulated in this neuron (Inada et al 2006; Nguyen et al 2014)
Like FlincG, FlincG3 contains the N-terminal region of protein kinase G (PKG) I a, which is comprised of two cGMP-binding domains that bind cGMP cooperatively

Summary

Introduction

ABSTRACT cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Because so many neurobiological processes revolve around cGMP, having a robust, easy to use, visual reporter for cGMP with precise temporal and spatial fidelity is critical to complement the primarily pharmacological, biochemical, and genetic approaches used to study this second messenger’s role in these processes Such a reporter can be used to illuminate how producers (guanylyl cyclases) and degraders (phosphodiesterases) of cGMP shape the landscape of this cyclic nucleotide in neurons. Couto et al (2013) suggested that compartmentalization of cGMP levels by the action of a PDE might allow cGMP to increase in one compartment while decreasing in another compartment of the cell While these studies demonstrate that the FRET-based sensor can be used to visualize the cGMP landscape in neurons, the complexity of a multiple emission system raises a barrier to its use. Such a tool would be maximally efficient for probing the interplay between cGMP and calcium dynamics within sensory compartments in this transparent organism

Methods

Results

Conclusion