Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans
| Autor: | Nebat Ali, Jordan Mitchell, Joy Li, Yanxun V. Yu, Tyler Hill, Martin W. Schneider, Noelle D. L'Etoile, Wagner Steuer Costa, Benjamin Barsi-Rhyne, Chantal Brueggemann, Kristine Andersen, O. Scott Hamilton, Mary Futey, Raakhee Shankar, Aruna Varshney, Jatin Nagpal, Sarah Woldemariam, Miri K. VanHoven, Alan Tran, Denise M. Ferkey, Alexander Gottschalk, Michelle C. Krzyzanowski, Piali Sengupta |
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| Rok vydání: | 2019 |
| Předmět: |
Sensory Receptor Cells
Cells sensory neuron Green Fluorescent Proteins chemistry.chemical_element Investigations Calcium Biology visual reporter Cyclase Green fluorescent protein 03 medical and health sciences 0302 clinical medicine C . elegans medicine Fluorescence Resonance Energy Transfer FlincG3 Genetics Animals Caenorhabditis elegans Cyclic GMP Cells Cultured Ion channel 030304 developmental biology 0303 health sciences Cultured Opsins Optical Imaging elegans Methods Technology and Resources Phosphodiesterase biology.organism_classification Sensory neuron Cell biology Optogenetics cGMP Förster resonance energy transfer medicine.anatomical_structure chemistry Guanylate Cyclase C. elegans 030217 neurology & neurosurgery Developmental Biology |
| Zdroj: | Genetics, vol 213, iss 1 Genetics |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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