Overcoming limitations of<scp>FRET</scp>measurements

Leavesley, Silas J.; Rich, Thomas C.

doi:10.1002/cyto.a.22851

Cited by 63 publications

(55 citation statements)

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“…This utility has been extended by the development of a wide range of fluorescent proteins, facilitating the use of FRET probes for live cell studies. However, FRET measurements have also been fraught with low SNR when compared with single label measurements . SNR may be further reduced when using fluorescent protein‐based FRET reporters, due to limitations in expression levels and quantum efficiencies.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Discussionmentioning

confidence: 99%

“…To overcome the inherent low SNR of FRET probes, we have implemented spectral imaging approaches . These approaches were developed by NASA to solve remote sensing/satellite imaging problems and offer tremendous potential for the study of biological systems .…”

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

et al. 2018

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“…The main drawback is the low signal-to-noise ratio of FRET signals originating from the loss of energy associated with the FRET process. The use of two or more fluorescent labels that are sensitive to local changes in pH, ionic strength, temperature, oxidation, refractive index, etc., complicates the interpretation of FRET data [ 111 , 112 ]. As a result, there are only a few reports on evaluation of molecular dynamics in PEMs using FRET.…”

Section: Analysis Of Polymer Dynamics Inside Pems: Methodsmentioning

confidence: 99%

Layer-By-Layer Assemblies of Biopolymers: Build-Up, Mechanical Stability and Molecular Dynamics

Campbell

Vikulina

2020

Polymers

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Rapid development of versatile layer-by-layer technology has resulted in important breakthroughs in the understanding of the nature of molecular interactions in multilayer assemblies made of polyelectrolytes. Nowadays, polyelectrolyte multilayers (PEM) are considered to be non-equilibrium and highly dynamic structures. High interest in biomedical applications of PEMs has attracted attention to PEMs made of biopolymers. Recent studies suggest that biopolymer dynamics determines the fate and the properties of such PEMs; however, deciphering, predicting and controlling the dynamics of polymers remains a challenge. This review brings together the up-to-date knowledge of the role of molecular dynamics in multilayers assembled from biopolymers. We discuss how molecular dynamics determines the properties of these PEMs from the nano to the macro scale, focusing on its role in PEM formation and non-enzymatic degradation. We summarize the factors allowing the control of molecular dynamics within PEMs, and therefore to tailor polymer multilayers on demand.

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“…In FRET, since the distance between the donor and the acceptor must be within the Förster radius (i.e., ≈10 nm), poor positioning of fluorescent probes in protein pair might lead to defect in detection of PPI. In addition, spectral leakage, that correspond to the direct excitation of the acceptor and causing false positive detections, donor fluorescence background, photobleaching, and trouble for measurements make real PPI detection quite difficult …”

Section: Introductionmentioning

confidence: 99%

Uncovering the In Vivo Proxisome Using Proximity‐Tagging Methods

Santin

2019

BioEssays

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The development of new approaches is critical to gain further insights into biological processes that cannot be obtained by existing methods or technologies. The detection of protein-protein interaction is often challenging, especially for weak and transient interactions or for membrane proteins. Over the last decade, several proximity-tagging methodologies have been developed to explore protein interactions in living cells. Among those, the most efficient are based on protein partner modification, such as biotinylation or pupylation. Such technologies are based on engineered variants of enzymes like peroxidases or ligases that release reactive molecules, in the presence of specific substrates, that bind surrounding proteins. Fusing a protein of interest (POI) to these enzymes allows the definition of an unbiased "proxisome," that is, all of the proteins in interaction or in close vicinity of the POI. Here, the different proximity-labeling tools available are described and comprehensive comparison to discuss advantages and limitations is provided.

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Overcoming limitations ofFRETmeasurements

Cited by 63 publications

References 11 publications

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

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

Layer-By-Layer Assemblies of Biopolymers: Build-Up, Mechanical Stability and Molecular Dynamics

Uncovering the In Vivo Proxisome Using Proximity‐Tagging Methods

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