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Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans
GENETICS (2019)
  • Sarah Woldemariam, University of California, San Francisco
  • Jatin Nagpal, The Goethe University
  • Tyler Hill, Brandeis University
  • Joy Li, San Jose State University
  • Martin W. Schneider, The Goethe University
  • Raakhee Shankar, San Jose State University
  • Mary Futey, University of California, San Francisco
  • Aruna Varshney, San Jose State University
  • Nebat Ali, San Jose State University
  • Jordan Mitchell, San Jose State University
  • Kristine Andersen, San Jose State University
  • Benjamin Barsi-Rhyne, San Jose State University
  • Alan Tran, San Jose State University
  • Wagner Steuer Costa, The Goethe University
  • Michelle C. Krzyzanowski, The State University of New York at Buffalo
  • Yanxun V. Yu, Brandeis University
  • Chantal Brueggemann, University of California, San Francisco
  • O. Scott Hamilton, University of California, Davis
  • Denise M. Ferkey, The State University of New York at Buffalo
  • Miri VanHoven, San Jose State University
  • Piali Sengupta, Brandeis University
  • Alexander Gottschalk, The Goethe University
  • Noelle L’Etoile, University of California, San Francisco
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
  • FLINCG3,
  • CGMP,
  • VISUAL REPORTER,
  • SENSORY NEURON,
  • C. ELEGANS
Publication Date
September 1, 2019
DOI
10.1534/genetics.119.302392
Publisher Statement
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Citation Information
Sarah Woldemariam, Jatin Nagpal, Tyler Hill, Joy Li, et al.. "Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans" GENETICS Vol. 213 Iss. 1 (2019) p. 59 - 77
Available at: http://works.bepress.com/miri_vanhoven/13/