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

Autor: Annamdevula NS; Department of Chemical & Biomolecular Engineering, University of South Alabama, Mobile, Alabama.; Center for Lung Biology, University of South Alabama, Mobile, Alabama., Sweat R; Department of Chemical & Biomolecular Engineering, University of South Alabama, Mobile, Alabama., Griswold JR; Department of Chemical & Biomolecular Engineering, University of South Alabama, Mobile, Alabama., Trinh K; Department of Chemical & Biomolecular Engineering, University of South Alabama, Mobile, Alabama., Hoffman C; Medical Sciences, University of South Alabama, Mobile, Alabama., West S; Department of Biomedical Sciences, University of South Alabama, Mobile, Alabama., Deal J; Department of Chemical & Biomolecular Engineering, University of South Alabama, Mobile, Alabama.; Center for Lung Biology, University of South Alabama, Mobile, Alabama., Britain AL; Center for Lung Biology, University of South Alabama, Mobile, Alabama.; Department of Pharmacology, University of South Alabama, Mobile, Alabama., Jalink K; The Netherlands Cancer Institute and van Leeuwenhoek Center for Advanced Microscopy, Amsterdam, the Netherlands., Rich TC; Center for Lung Biology, University of South Alabama, Mobile, Alabama.; Department of Pharmacology, University of South Alabama, Mobile, Alabama.; College of Engineering, University of South Alabama, Mobile, Alabama., Leavesley SJ; Department of Chemical & Biomolecular Engineering, University of South Alabama, Mobile, Alabama.; Center for Lung Biology, University of South Alabama, Mobile, Alabama.; Department of Pharmacology, University of South Alabama, Mobile, Alabama.
Jazyk: angličtina
Zdroj: Cytometry. Part A : the journal of the International Society for Analytical Cytology [Cytometry A] 2018 Oct; Vol. 93 (10), pp. 1029-1038. Date of Electronic Publication: 2018 Sep 03.
DOI: 10.1002/cyto.a.23572
Abstrakt: 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.
(© 2018 International Society for Advancement of Cytometry.)
Databáze: MEDLINE
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