A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

Abstract

cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F0 of 1100% in HEK293T cells), relatively high brightness, appropriate affinity (a Kd of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 s and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted β-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening

“…However, other studies such as Di Benedetto et al and Lefkimmiatis et al demonstrated that cAMP generated in the cytosol did not enter the mitochondrial matrix, except during mitochondrial permeability transition ( Di Benedetto et al, 2013 ; Lefkimmiatis et al, 2013 ). These different observations might be due to different sensitivities of the indicators used ( Wang et al, 2022 ). For those biological systems in which cytosolic cAMP can enter mitochondrial matrix, further studies are required to dissect the underlying mechanism.…”

“…Quantum yields were determined using mEGFP as a standard (QY = 0.60). pH titrations were performed by mixing 10 μL concentrated protein solution with 110 μL buffers of different pH ranging from 2 to 11 and the pKa was determined as described previously ( Wang et al, 2022 ). cAMP titrations were performed by mixing 1 μM of purified protein in HEPES buffer with varying concentrations of cAMP (0.001, 0.01, 0.1, 0.5, 1, 2, 5, 10, 25, 100 and 500 μM) or cGMP (0.01, 0.1, 0.5, 1, 2, 5, 10, 25, 100, 500, 1,000 and 2000 μM) and K d and Hill coefficient were determined as described previously ( Wang et al, 2022 ).…”

“…To quantitatively compare the brightness of selected variants, bacterial patches on the agar plates cultured overnight at 34°C were imaged in the home-made system mentioned above and analyzed by ImageJ software (National Institutes of Health). The fluorescence change (ΔF/F 0 ) of the cAMP sensor mutants were tested as previously described ( Wang et al, 2022 ). Briefly, selected bacterial colonies were patched on LB agar plate and grew at 25°C for 3 days.…”

“…However, current cAMP single-FP indicators (Flamindo2, cAMPr, Pink Flamindo and R-FlincA) exhibit small fluorescence change (|ΔF/F 0 | < 150%) and most are dim in mammalian cells at 37°C ( Odaka et al, 2014 ; Harada et al, 2017 ; Hackley et al, 2018 ; Ohta et al, 2018 ). To address these problems, we recently developed a high-performance single-FP cAMP indicator named G-Flamp1 ( Wang et al, 2022 ), which is created by inserting a circularly permuted GFP (cpGFP) into a bacterial cAMP-binding domain of the bacterial Mloti K1 channel (mlCNBD). G-Flamp1 exhibits a large fluorescence increase (a maximum ΔF/F 0 of 1,100% in HEK293T cells), appropriate affinity (a K d of 2.17 μM) and sub-second response kinetics.…”

“…G-Flamp1 exhibits a large fluorescence increase (a maximum ΔF/F 0 of 1,100% in HEK293T cells), appropriate affinity (a K d of 2.17 μM) and sub-second response kinetics. However, compared to the well-known single-FP calcium indicator GCaMP6s, the brightness of G-Flamp1 is still relatively low, which could hinder the cAMP imaging of subcellular structures such as mitochondria ( Wang et al, 2022 ).…”