Dataset Information

Dual-Mode FRET and BRET Sensors for Detecting cAMP Dynamics.

ABSTRACT: Genetically encoded fluorescent and luminescent indicators have revolutionized our ability to monitor physiology in real time, but the separate development of new sensors for each of these imaging modalities involves substantial effort and resources. Methods to rapidly engineer multimodal sensors would, therefore, significantly accelerate the diversification of sensors for simultaneous use in different systems and applications. We hypothesized that the enhanced Nano-lanterns could be incorporated into modular ratiometric sensors as an efficient approach to creating dual-mode fluorescent-luminescent sensors. As a proof-of-concept, we engineered an Epac1-based sensor that responds to cyclic adenosine monophosphate binding with a greater than 80% change in both Förster Resonance Energy Transfer and bioluminescent resonance energy transfer (BRET) modes. We also demonstrate that our new sensor reports cellular changes in G-protein-coupled signaling, and that the ratiometric BRET mode is bright enough for subcutaneous measurements in mice.

SUBMITTER: French AR

PROVIDER: S-EPMC6761615 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Dual-Mode FRET and BRET Sensors for Detecting cAMP Dynamics.

French Alexander R AR Tesmer Alexander L AL Tantama Mathew M

ACS omega 20190912 13

Genetically encoded fluorescent and luminescent indicators have revolutionized our ability to monitor physiology in real time, but the separate development of new sensors for each of these imaging modalities involves substantial effort and resources. Methods to rapidly engineer multimodal sensors would, therefore, significantly accelerate the diversification of sensors for simultaneous use in different systems and applications. We hypothesized that the enhanced Nano-lanterns could be incorporate ...[more]

PMID: 31572851

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Project description:Cyclic AMP is a ubiquitous second messenger that orchestrates a variety of cellular functions over different timescales. The mechanisms underlying specificity within this signaling pathway are still not well understood. Several lines of evidence suggest the existence of spatial cAMP gradients within cells, and that compartmentalization underlies specificity within the cAMP signaling pathway. However, to date, no studies have visualized cAMP gradients in three spatial dimensions (3D: x, y, z).This is in part due to the limitations of FRET-based cAMP sensors, specifically the low signal-to-noise ratio intrinsic to all intracellular FRET probes. Here, we overcome this limitation, at least in part, by implementing spectral imaging approaches to estimate FRET efficiency when multiple fluorescent labels are used and when signals are measured from weakly expressed fluorescent proteins in the presence of background autofluorescence and stray light. Analysis of spectral image stacks in two spatial dimensions (2D) from single confocal slices indicates little or no cAMP gradients formed within pulmonary microvascular endothelial cells (PMVECs) under baseline conditions or following 10 min treatment with the adenylyl cyclase activator forskolin. However, analysis of spectral image stacks in 3D demonstrates marked cAMP gradients from the apical to basolateral face of PMVECs. Results demonstrate that spectral imaging approaches can be used to assess cAMP gradients-and in general gradients in fluorescence and FRET-within intact cells. Results also demonstrate that 2D imaging studies of localized fluorescence signals and, in particular, cAMP signals, whether using epifluorescence or confocal microscopy, may lead to erroneous conclusions about the existence and/or magnitude of gradients in either FRET or the underlying cAMP signals. Thus, with the exception of cellular structures that can be considered in one spatial dimension, such as neuronal processes, 3D measurements are required to assess mechanisms underlying compartmentalization and specificity within intracellular signaling pathways.

Dataset Information

Dual-Mode FRET and BRET Sensors for Detecting cAMP Dynamics.

Publications

Dual-Mode FRET and BRET Sensors for Detecting cAMP Dynamics.

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