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.

Modular Peroxidase-Based Reporters for Detecting Protease Activity and Protein Interactions with Temporal Gating.

Enzymatic reporters have been widely applied to study various biological processes because they can amplify signal through enzymatic reactions and provide good sensitivity. However, there is still a need for modular motifs for designing a series of enzymatic reporters. Here, we report a modular peroxidase-based motif, named CLAPon, that features acid-base coil-caged enhanced ascorbate peroxidase (APEX). We demonstrate the modularity of CLAPon by designing a series of reporters for detecting protease activity and protein-protein interactions (PPIs). CLAPon for protease activity showed a 390-fold fluorescent signal increase upon tobacco etch virus protease cleavage. CLAPon for PPI detection (PPI-CLAPon) has two variants, PPI-CLAPon1.0 and 1.1. PPI-CLAPon1.0 showed a signal-to-noise ratio (SNR) of up to 107 for high-affinity PPI pairs and enabled imaging with sub-cellular spatial resolution. However, the more sensitive PPI-CLAPon1.1 is required for detecting low-affinity PPI pairs. PPI-CLAPon1.0 was further engineered to a reporter with light-dependent temporal gating, called LiPPI-CLAPon1.0, which can detect a 3-min calcium-dependent PPI with an SNR of 17. LiPPI-CLAPon enables PPI detection within a specific time window with rapid APEX activation and diverse readout. Lastly, PPI-CLAPon1.0 was designed to have chemical gating, providing more versatility to complement the LiPPI-CLAPon. These CLAPon-based reporter designs can be broadly applied to study various signaling processes that involve protease activity and PPIs and provide a versatile platform to design various genetically encoded reporters.