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Protein Phosphorylation || FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells
摘要： Fluorescence microscopy is widely used in biology to localize, to track, or to quantify proteins in single cells. However, following particular events in living cells with good spatio-temporal resolution is much more complex. In this context, Forster resonance energy transfer (FRET) biosensors are tools that have been developed to monitor various events such as dimerization, cleavage, elasticity, or the activation state of a protein. In particular, genetically encoded FRET biosensors are strong tools to study mechanisms of activation and activity of a large panel of kinases in living cells. Their principles are based on a conformational change of a genetically encoded probe that modulates the distance between a pair of fluorescent proteins leading to FRET variations. Recent advances in fluorescence microscopy such as fluorescence lifetime imaging microscopy (FLIM) have made the quantification of FRET efficiency easier. This review aims to address the different kinase biosensors that have been developed, how they allow specific tracking of the activity or activation of a kinase, and to give an overview of the future challenging methods to simultaneously track several biosensors in the same system.
关键词： multiplex,biosensor,fluorescence microscopy,FRET,protein conformation,kinase
更新于2025-09-09 09:28:46
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A multifunctional sensor for selective and sensitive detection of vitamin B12 and tartrazine by F?rster resonance energy transfer
摘要： We used thiamine nitrate (TN) as single material to fabricate nitrogen and sulfur co-doped carbon quantum dots (N,S-CQDs) with a quantum yield of 10.4% through one-pot hydrothermal method, and its properties were characterized by TEM, XPS, FTIR, fluorescence (FL) and UV-vis spectrophotometer, respectively. The fluorescence of N,S-CQDs was effectively quenched in the presence of vitamin B12 (VB12)/tartrazine due to F?rster resonance energy transfer (FRET). Moreover, the rate (KT) and efficiency (E%) of energy transfer from N,S-CQDs (as a donor) to VB12/tartrazine (as an acceptor) enhanced with increasing the concentrations of acceptor, and the KT and E% were also varied with the change of excitation wavelengths (from 338 to 408 nm). Based on this principle, a multifunctional fluorescence probe was designed for selective and sensitive detection of VB12/tartrazine with a detection limit (3σ/slope) of 15.6/18.0 nmol/L. Meanwhile, the proposed method was successfully employed to detect VB12/tartrazine in milk and several beverages with a recovery range of 97.5-104.2% /91.0-110.6%.
关键词： Tartrazine,Multifunctional sensor,FRET,N,S-CQDs,Vitamin B12
更新于2025-09-09 09:28:46
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Improving Quality, Reproducibility, and Usability of FRET-Based Tension Sensors
摘要： Mechanobiology, the study of how mechanical forces affect cellular behavior, is an emerging field of study that has garnered broad and significant interest. Researchers are currently seeking to better understand how mechanical signals are transmitted, detected, and integrated at a subcellular level. One tool for addressing these questions is a F?rster resonance energy transfer (FRET)-based tension sensor, which enables the measurement of molecular-scale forces across proteins based on changes in emitted light. However, the reliability and reproducibility of measurements made with these sensors has not been thoroughly examined. To address these concerns, we developed numerical methods that improve the accuracy of measurements made using sensitized emission-based imaging. To establish that FRET-based tension sensors are versatile tools that provide consistent measurements, we used these methods, and demonstrated that a vinculin tension sensor is unperturbed by cell fixation, permeabilization, and immunolabeling. This suggests FRET-based tension sensors could be coupled with a variety of immuno-fluorescent labeling techniques. Additionally, as tension sensors are frequently employed in complex biological samples where large experimental repeats may be challenging, we examined how sample size affects the uncertainty of FRET measurements. In total, this work establishes guidelines to improve FRET-based tension sensor measurements, validate novel implementations of these sensors, and ensure that results are precise and reproducible.
关键词： FRET efficiency,mechanotransduction,FRET-based biosensor,sensitized emission
更新于2025-09-04 15:30:14
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Two Orders of Magnitude Variation of Diffusion-Enhanced F?rster Resonance Energy Transfer in Polypeptide Chains
摘要： A ?exible peptide chain displays structural and dynamic properties that correspond to its folding and biological activity. These properties are mirrored in intrachain site-to-site distances and diffusion coef?cients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of F?rster resonance energy transfer (FRET) between two sites labeled with a FRET donor and acceptor. The relatively large F?rster radii of traditional FRET methods (R0 > 20 ?) lead to a fairly low contribution of diffusion. We introduced short-distance FRET (sdFRET) where Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, acts as acceptor paired with donors, such as naphtylalanine (NAla), tryptophan, 5-L-?uorotryptophan, or tyrosine. The F?rster radii are always close to 10 ?, which makes sdFRET highly sensitive to diffusional motion. We recently found indications that the FRET enhancement caused by diffusion depends symmetrically on the product of the radiative ?uorescence lifetime of the donor and the diffusion coef?cient. In this study, we varied this product by two orders of magnitude, using both donors of different lifetime, NAla and FTrp, as well as a varying viscogen concentration, to corroborate this statement. We demonstrate the consequences of this relationship in evaluating the impact of viscogenic coadditives on peptide dimensions.
关键词： chain dynamics,viscosity,radiative ?uorescence lifetime,diffusion,short-distance FRET,peptide structure
更新于2025-09-04 15:30:14
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Novel FRET-Based Src Biosensor Reveals Mechanisms of Src Activation and Its Dynamics in Focal Adhesions
摘要： Koudelkova′ et al. developed and functionally verified a Src kinase biosensor. The biosensor represents a unique tool to monitor Src structure, activity, and localization both in vitro and in cells. Using the biosensor, the action of Src inhibitors and Src dynamics in focal adhesions was described.
关键词： Src activation,Src inhibitors,focal adhesions,FRET-based Src biosensor,Src dynamics
更新于2025-09-04 15:30:14
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[Methods in Molecular Biology] BCL-2 Family Proteins Volume 1877 (Methods and Protocols) || Rapid Imaging of BCL-2 Family Interactions in Live Cells Using FLIM-FRET
摘要： The Bcl-2 proteins control cell death via interchanging interactions within the Bcl-2 family. Fluorescence lifetime imaging microscopy (FLIM) is used to detect F?rster resonance energy transfer (FRET) between two fluorescent-fusion proteins in live cells. FLIM-FRET has been used to detect specific interactions and their disruption, for Bcl-2 family proteins. To date, this has been possible only in low throughput, due to the time required for serial data acquisition. We developed an automated optical system with eight parallel detectors for rapid and efficient data collection. Here we describe how to use this system for FLIM-FRET imaging of Bcl-2 family protein interactions in a 384-well plate format.
关键词： BH3 mimetic,FLIM Hyperspectral,FLIM-FRET,Bcl-2 family,mCerulean3,High throughput,Fluorescence lifetime imaging microscopy
更新于2025-09-04 15:30:14