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

Abstract

AbstractcAMP 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), decent brightness, appropriate affinity (a Kd of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 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.

A multicolor suite for deciphering population coding in calcium and cAMPin vivo

“…For dual-color imaging, we employed different excitation wavelengths: 560 nm for RCaMP3 imaging and 405 nm and 470 nm for cAMPinG1 ratiometric imaging. In line with previous studies (Massengill et al, 2022; Wang et al, 2022), we observed an increase in cAMP levels during the running period following the increase in Ca 2+ levels ( Figures 6B-6D ). Surprisingly, cAMP levels decreased after the running period and remained low for tens of seconds.…”

“…The green fluorescence intensity of cAMPinG1 increased by 1,000 % upon binding to cAMP with blue light (488 nm), while the green fluorescence intensity decreased by 61 % with violet light (405 nm), resulting in a 2,700 % ratio change with a combination of blue and violet excitation in HEK293T cell lysate. The large ratio change dependent on the decrease of fluorescence intensity with violet excitation indicates that cAMPinG1 is suitable for ratiometric imaging, in contrast to some cpGFP-type indicators such as GCaMPs and G-Flamp1, which do not show a fluorescence decrease with violet excitation (Inoue et al, 2019; Wang et al, 2022) ( Figure S2A ).…”

“…Here, we simultaneously visualized Ca 2+ and cAMP signals of more than 400 neurons in L2/3 of a mouse ( Figures 4C and 4D; Movie S2 ). During the imaging, we employed a forced running task, which was reported to increase neuromodulators, including noradrenaline, cAMP, and PKA activities in the cortex (Ma et al, 2018; Massengill et al, 2022; Wang et al, 2022). The majority of neurons showed an increase in cAMP signals during running ( Figures 4D and 4E ).…”

“…Although several cAMP sensors have been developed recently, each had some drawbacks for in vivo imaging (Massengill et al, 2022; Wang et al, 2022). Due to its low cAMP affinity, G-Flamp1 limited the number of cells that could be imaged and quantified cAMP dynamics in the mouse cortex.…”

“…Since cAMP was first visualized in 1991, more than 50 cAMP indicators have been developed (Adams et al, 1991; Massengill et al, 2021). The circularly permuted green fluorescent protein (cpGFP)-type cAMP indicators have been intensively developed more recently due to their large dynamic range (Kawata et al, 2022; Liu et al, 2022; Wang et al, 2022). However, the use of these indicators has been limited because they have low cAMP affinity (> 1 μM).…”