Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

GPCRs transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors to determine GPCR agonist localization in the whole brain. We previously engineered integrator sensors for the mu and kappa opioid receptor agonists called M- and K-SPOTIT, respectively. Here, we show a new integrator sensor design platform called SPOTall that we used to engineer sensors for the beta-2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. For multiplexed imaging of SPOTIT and SPOTall, we engineered a red version of the SPOTIT sensors. Finally, we used M-SPOTIT and B2AR-SPOTall to detect morphine, isoproterenol, and epinephrine in the mouse brain. The SPOTIT and SPOTall sensor design platform can be used to design a variety of GPCR integrator sensors for unbiased agonist detection of many synthetic and endogenous neuromodulators across the whole brain.

A Modular Fluorescent Sensor Motif Used to Detect Opioids, Protein-Protein Interactions, and Protease Activity.

Modular fluorescent sensor motifs are needed to design fluorescent sensors for detecting various cellular processes and functional molecules. Here, we took advantage of the versatility of a new sensor motif to design a series of sensors called SPOTon. SPOTon sensors integrate the signal from either opioids, protein-protein interactions, or protease activities to generate persistent green fluorescence. We demonstrate that SPOTon can be engineered with temporal gating to allow detection of these cellular events during a user-defined time window, providing temporal information about cellular processes and functional molecule release. These SPOTon sensors all show a high signal-to-noise ratio, up to 38 for chemical gated opioid detection, 147 for chemical gated protein-protein interaction detection, and 85 for protease activity detection.