Spectral imaging of FRET‐based sensors reveals sustained cAMP gradients in three spatial dimensions

Abstract

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.

cAMP Compartmentalization in Cerebrovascular Endothelial Cells: New Therapeutic Opportunities in Alzheimer’s Disease

“…Subsequently, other highly useful sensors were developed for the performance of cAMP signal sublocation experiments, such as the one that uses mutants of the green fluorescent protein (GFP) fused to the different subunits of PKA [76] or those based on the cAMP-binding domains of the exchange proteins directly activated by cAMP (Epac) [77]. More recently, visualization of three-dimensional spatial gradients of cAMP concentration in individual cells was achieved by implementing spectral imaging approaches to estimate FRET efficiency when using multiple fluorescent markers [78,79].…”

Milestones in the development and implementation of FRET-based sensors of intracellular signals: A biological perspective of the history of FRET

“…A recent study by Annamdevula and colleagues demonstrates that agonist-induced cAMP gradients exist in three spatial dimensions. [99] Thus, standard FRET microscopy approaches may not provide sufficient information to evaluate the distributions of intracellular FRET or the underlying biological signal.…”

I Am the Alpha and the …Gamma, and the G. Calibration of Intensity‐Based FRET Measurements

“…Its flexibility and versatility are enhanced by the generation of sensors, that is, donor-acceptor fluorescence protein constructs connected by a linker, whose conformation responds to certain properties of the environment. The FRET efficiency of such sensors can be used as a readout parameter for a multitude of biological processes (3,4). Although a large number of approaches are available for the determination of FRET, intensity-based (ratiometric) measurements remain the cornerstone in its biological applications since these experiments are the easiest to implement and they usually provide the answer to the questions asked about protein clustering by ordinary biologists (1).…”