Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging

Abstract

Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca2+ biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations.

Cytochrome b5 Reductase 3 Modulates Soluble Guanylate Cyclase Redox State and cGMP Signaling

“…We observed no direct effects of the Cyb5R3 inhibitor on the ability of sGC to generate cGMP in response to NO using purified sGC (Online Figure IV). To probe the effects of decreased Cyb5R3 expression on downstream signaling, we first transfected cells with a genetically encoded cGMP biosensor, which harbors the cGMP sensitive N -terminal domain of protein kinase G (PKG) linked to a circularly permuted eGFP 28 . Following transfection into nt shRNA and Cyb5R3 shRNA transduced RASMCs, cells were stimulated with DEA NONOate and increases in fluorescence were captured using high-speed confocal microscopy.…”

From synaptically localized to volume transmission by nitric oxide

“…With the continuing development of fluorescent biosensors responsive to physiological levels of cGMP (Gorshkov & Zhang, ) it is becoming possible to visualize NO signalling through its receptors in real time and even in small synaptic dimensions (Bhargava et al . ). Being genetically encoded, these biosensors could be expressed in specific cell types in the brain, or in specific subcellular compartments, and are likely to prove invaluable in putting theory to the test and so further advance our understanding of NO‐mediated transmission.…”

A synthetic cGMP-sensitive gene switch providing Viagra®-controlled gene expression in mammalian cells and mice

“…Indeed, various fluorescence-based cGMP sensors have been developed to visualize cGMP signaling in mammalian cells, but this technology remains limited to microscopic single-cell analysis (Bhargava et al, 2013;Nausch et al, 2008;Nikolaev et al, 2006). By contrast, the GTA/P GTA -device of the present study was able to directly evaluate the intracellular cGMP pool by converting second messenger levels into a robust, precise and specific transcriptional output that can capture dynamic Fig.…”