Förster Resonance Energy Transfer Biosensors Research Articles

Automated screening of AURKA activity based on a genetically encoded FRET biosensor using fluorescence lifetime imaging microscopy

Fluorescence Lifetime Imaging Microscopy (FLIM) is a robust tool to measure Förster Resonance Energy Transfer (FRET) between two fluorescent proteins, mainly when using genetically-encoded FRET biosensors. It is then possible to monitor biological processes such as kinase activity with a good spatiotemporal resolution and accuracy. Therefore, it is of interest to improve this methodology for future high content screening purposes. We here implement a time-gated FLIM microscope that can image and quantify fluorescence lifetime with a higher speed than conventional techniques such as Time-Correlated Single Photon Counting (TCSPC). We then improve our system to perform automatic screen analysis in a 96-well plate format. Moreover, we use a FRET biosensor of AURKA activity, a mitotic kinase involved in several epithelial cancers. Our results show that our system is suitable to measure FRET within our biosensor paving the way to the screening of novel compounds, potentially allowing to find new inhibitors of AURKA activity.

An Antiviral Drug Screening Platform with a FRET Biosensor for Measurement of Arenavirus Z Assembly.

The smallest arenavirus gene product, Z protein, plays critical roles in the virus life cycle. Z is the major driving force of budding and particle production because of a unique property that defines self-assembly. In addition to the roles in budding, Z also participates in the suppression of type I interferon production to evade host antiviral immunity. Therefore, Z and its assembled form are an attractive drug target for arenaviral hemorrhagic fever, such as Lassa fever. Here, we developed a biosensor that enabled the evaluation of the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), Z assembly using the principle of Förster resonance energy transfer (FRET). This FRET biosensor consisted of three tandem Z that were sandwiched between super-enhanced cyan-emitting fluorescent protein and variant of a yellow-emitting mutant of green fluorescent protein so that Z-Z intermolecular binding via the really interesting new gene finger domain increased the emission ratio. To identify novel anti-arenavirus compounds, the FRET biosensor was employed to screen the PathogenBox400 for inhibitors of Z assembly in a 96-well plate format. The assay performed well, with a Z'-factor of 0.89, and identified two compounds that decreased the emission ratio of the FRET biosensor in a dose-dependent manner. Of them, the compound, 5,6,7,8-tetrahydro-7-(benzyl)-pyrido[4',3':4,5]thieno[2,3-d]pyrimidin-2,4-diamine, was found to significantly inhibit LCMV propagation in infected cells. Thereby, the present study demonstrated that a novel FRET biosensor incorporating Z assembly built on FRET and named Zabton, was a valuable screening tool to identify anti-arenavirus compounds in the context of inhibition of Z assembly.Key words: Arenavirus, Förster resonance energy transfer, anti-viral drugs, Z protein.

FRET Microscopy in Yeast.

Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts.

A FRET biosensor reveals free zinc deficiency in diabetic beta-cell vesicles

The concentration of free zinc within insulin-storing vesicles is important for vesicle maturity and therefore requires accurate measurement. However, common small-molecule intensity-based probes and most available genetically encoded Förster resonance energy transfer (FRET)-based sensors for zinc are unsuitable for estimating the free zinc concentration in insulin-storing vesicles. Therefore, we have developed a novel FRET-based zinc sensor based on the RING motif of TRIM72, referred to as ZnT72R, which has an approximate Kd that varies from 6.07 ± 0.28 μmol/L to 7.84 ± 0.42 μmol/L in vitro and a cytosol-calibrated Kd of approximately 55.56 ± 4.59 μmol/L in HEK293 T cells. To pinpoint the free zinc concentration of insulin-storing vesicles, we initially targeted ZnT72R to beta-cell vesicles by fusing them to NPY (neuropeptide Y). Following NPY-ZnT72R labeling, the FRET intensity ratios of vesicles were quantified. We found that the free zinc concentration in insulin-storing vesicles of diabetic db/db mice (28.30 ± 1.33 μmol/L) was significantly lower than that of control mice (41.46 ± 3.53 μmol/L).

A FRET biosensor based on MnO2 nanosphere/copper nanocluster complex: From photoluminescence quenching to recovery and magnification

A Förster resonance energy transfer (FRET)-based biosensor using Cu nanoclusters (NCs) as energy donors and signal magnifiers and MnO2 nanospheres as both energy acceptors and target recognizers for selective detection of glutathione (GSH) was developed. MnO2 nanospheres, synthesized through an ultrasonication assisted top down method, can quench the photoluminescence (PL) of Cu NCs through FRET, after forming complex through electrostatic interactions. GSH can recover PL of Cu NCs by triggering the decomposition of complex through digesting MnO2 nanospheres into Mn2+. Additionally, the PL can be further magnified through the host-guest interactions between GSH and Cu NCs, with around 25 times higher than the original intensity and 3 times higher than the initial PL intensity of Cu NCs. Under the optimum conditions, the FRET based sensor can detect GSH in the concentration range from 0 to 140 μM, with a detection limit of 17 μM. Moreover, the proposed method shows a strong anti-interference ability, achieving the sensitive directly detection of GSH in human whole blood samples. Our results provide insights in designing FRET based sensors. Moreover, all the components are nontoxic and biodegradable, demonstrating their potential applications in other fields such as nanomedicine and environmental science.

Development of novel methods that monitor necroptosis and the release of DAMPs at the single cell resolution.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). While danger-associated molecular pattern (DAMP)s are released from dead cells and involved in various pathological conditions, the mechanisms underlying regulation of the release of DAMPs are not fully understood. Apoptosis and pyroptosis can be detected by several types of sensors such as Forster resonance energy transfer (FRET) biosensors, termed SCAT1 (a sensor for caspase 1 activation based on FRET) and SCAT3, respectively. These sensors have provided better understanding of pyroptosis and apoptosis in vitro and in vivo. However, there have been no biosensors to monitor necroptosis. Development of a FRET biosensor that monitors necroptosis and generation of transgenic mice expressing such FRET biosensor might be useful to understand the mechanisms underlying the execution of necroptosis and also the consequences of necroptosis in vivo. In our recent study (Nat Commun, 9(1):4457), we developed a FRET biosensor for necroptosis, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Moreover, we recently developed a platform called Live-Cell Imaging for Secretion activity (LCI-S) to monitor protein secretion at the single cell level. This platform has enabled us to monitor the release of HMGB1 (High Mobility Group Box 1), one of the DAMPs, at the single cell level and reveals two different modes of the release of HMGB1 from necroptotic cells.

Genetically Encoded FRET Biosensor for Visualizing EphA4 Activity in Different Compartments of the Plasma Membrane

The EphA4 receptor tyrosine kinase is well-known for its pivotal role in development, cancer progression, and neurological disorders. However, how EphA4 kinase activity is regulated in time and space still remains unclear. To visualize EphA4 activity in different membrane microdomains, we developed a sensitive EphA4 biosensor based on Förster resonance energy transfer (FRET), and targeted it in or outside raft-like microdomains in the plasma membrane. We showed that our biosensor can produce a robust and specific FRET response upon EphA4 activation, both in vitro and in live cells. Interestingly, we observed stronger FRET responses for the non-raft targeting biosensor than for the raft targeting biosensor, suggesting that stronger EphA4 activation may occur in non-raft regions. Further investigations revealed the importance of the actin cytoskeleton in suppressing EphA4 activity in raft-like microdomains. Therefore, our FRET-based EphA4 biosensor could serve as a powerful tool to visualize and investigate EphA4 activation and signaling in specific subcellular compartments of single live cells.

Two Decades of Genetically Encoded Biosensors Based on Förster Resonance Energy Transfer.

Two decades have passed since the development of the first calcium indicator based on the green fluorescent protein (GFP) and the principle of Förster resonance energy transfer (FRET). During this period, researchers have advanced many novel ideas for the improvement of such genetically encoded FRET biosensors, which have allowed them to expand their targets from small molecules to signaling proteins and physicochemical properties. Although the merits of "genetically encoded" FRET biosensors became clear once various cell lines were established and several transgenic organisms were generated, the road to these developments was not necessarily a smooth one. Moreover, even today the development of new FRET biosensors remains a very labor-intensive, trial-and-error process. Therefore, at this junction, it may be worthwhile to summarize the progress of the FRET biosensor and discuss the future direction of its development and application.Key words: FRET, biosensor, fluorescent protein.

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET.

Direct visualization and light control of several cellular processes is a challenge due to spectral overlap of available genetically-encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein miRFP720 and the fully NIR Förster Resonance Energy Transfer (FRET) pair miRFP670-miRFP720 that enables design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use for multiplexed imaging and light control of RhoGTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and then concurrently used LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified the antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK, showed that Rac1 activity and GDI binding depend exquisitely on the spatiotemporal coordination between these two molecules, and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.

Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors.

Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.

Characterization of Genetically Encoded FRET Biosensors for Rho-Family GTPases.

Genetically encoded FRET-based biosensors are increasingly popular and useful tools for examining signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic techniques used to characterize and to validate single-chain, genetically encoded Förster resonance energy transfer (FRET) biosensors of the Rho GTPase-family proteins. Methods described here are generally applicable to other genetically encoded FRET-based biosensors by modifying the tested conditions to include additional/different regulators and inhibitors, as appropriate for the specific protein of interest.

Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells.

Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS’s capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.

Live imaging of extracellular signal‐regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer

Background The dynamic features of thrombus formation have been visualized by conventional video widefield microscopy or confocal microscopy in live mice. However, owing to technical limitations, the precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives To visualize, by the use of two-photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles. Results When a core of densely packed platelets had developed, ERK activity was increased from the basal region close to the injured arterioles. PKA was activated at the downstream side of an unstable shell overlaying the core of platelets. Intravenous administration of a MEK inhibitor, PD0325901, suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog, dbcAMP, inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase. Conclusion In vivo imaging of transgenic mice expressing FRET biosensors will open a new opportunity to visualize the spatiotemporal changes in signaling molecule activities not only during thrombus formation but also in other hematologic disorders.

Development of FRET biosensor based on aptamer/functionalized graphene for ultrasensitive detection of bisphenol A and discrimination from analogs

We report an ultrasensitive, effective and efficient platform for small molecule bisphenol A (BPA) detection based on Förster resonance energy transfer (FRET) between functionalized graphene (graphene oxide; GO and reduced GO; rGO) as acceptor and fluorescently tagged BPA specific aptamer (5’-FAM-tagged single-stranded DNA; FAM-ssDNA) as donor design. Due to noncovalent assembly and specific adsorption, fluorescent quenching of FAM probe takes place due to good overlap between FAM emission and the GO (and rGO) absorption spectrum. On addition of BPA analyte, the aptamer preferentially bounds to BPA, forming FAM-ssDNA–aptamer–BPA complexes, the FRET is disrupted possibly by switching its configuration or re-hybridization preventing aptamer from molecular adsorption on or away from GO or rGO surface and the fluorescence signal is restored (turn-on’ bioassay). This method relies upon competitive interaction between two hydroxyphenyl groups of BPA and “cytosine–cytosine” mismatches in ssDNA. The intensity of optical fluorescence signal was changed in linear proportion to BPA concentration ranging 50 pg/mL − 100 ng/mL, with low limit of detection <10 pg/mL. The sensitivity and specificity of biosensor toward BPA was demonstrated viaseveral BP, BPB, BPC, DES analogs. The developed aptasensor assay is successfully implemented for real water (tap, bottled and river) samples and recovery rate is between 96.0% and 104% indicative of higher accuracy. These remarkable findings are attributed to interplay of large surface area and topologically interconnectedness provided by functional moieties in graphene scaffolds thus favorable interaction with FAM-ssDNA. This strategy can be extended to detection of other molecules by simply substituting the aptamer.

Triple FRET Imaging of Non-Muscle Myosin II Localization in Migrating Cells

Non-muscle myosin II (NMII) is critical for cell migration because myosin motors drive cell contraction by regulating pulling forces on actin filaments. Myosin regulatory light chain phosphorylation leads to stress fiber assembly and fiber contraction. It is undetermined if NMII phosphorylation increases or decreases during cell migration due to the inherent complexity of myosin regulation. Single metric approaches for illuminating the roles of these proteins have been unsuccessful in determining where the proteins are active simultaneously in the same cell. Our lab has developed two Forster resonance energy transfer (FRET) biosensors to make dynamic measurements of myosin activity. Our NMII phosphorylation sensor together with our myosin light chain phosphatase (MLCP) sensor are able to measure myosin activity in a single cell. Mutations were introduced into both sensors to change the homotransfer and reflect conformational changes that indicate activation. In order to optimize the myosin light chain kinase (MLCK) sensor, KCl was added to determine the range of min/max. During the migration process, changes in NMII phosphorylation, MLCK, and MLCP activity were observed through the use of three separate FRET biosensors transfected together into living cells. We are able to track MLCP, MLCK, and NMII phosphorylation in the same pathway with high accuracy of spatial and temporal resolution. Our data suggests differential regulation of MLCK and MLCP in along a moving membrane. This allows for a more sophisticated understanding of the intermediate and long-term action of myosin phosphorylation during cell migration.

5 Dimensional Hyperspectral FRET Imaging and Analysis Approaches for Measuring cAMP Gradients

cAMP is a second messenger that regulates numerous cellular and physiological functions. A growing body of evidence suggests that cAMP signals are localized resulting in the formation of spatial cAMP gradients. Recently, Forster Resonance energy transfer (FRET) imaging has become a standard method for assessing changes in cAMP concentration. However, many FRET probes have low signal-to-noise ratios and limited dynamic range, making measurements of cAMP difficult. Our previous work suggested that hyperspectral imaging and analysis allows quantitative measurement of FRET efficiencies in 3D (x, y, and t) within single cells. However, traditional (x, y, and t) approaches of measuring FRET often lead to misinterpretation of data. Here, we present data demonstrating that hyperspectral imaging approaches can be used for 5D (x, y, z, λ and t) assessment of FRET efficiency in living cells allowing the study of spatially-localized cAMP signals. We utilized H188-2 (cAMP reporter) and AKAR4 (PKA activity reporter) FRET biosensors that were transfected into PMVECs and HEK 293 cells, respectively. Time lapse hyperspectral Z-stack images were acquired using a Nikon A1R confocal microscope. We used custom MATLAB scripts to calculate FRET efficiencies from image data. These approaches allowed the measurement of spatial cAMP gradients and PKA activity induced by agonist treatment. We found that cAMP gradients developed from the apical to the basolateral side of PMVECs when treated with isoproterenol, while gradients developed from both the basolateral and apical side when treated with PGE1. In addition, we observed an increase in localized phosphorylation upon treatment with isoproterenol and PGE1 in HEK293 cells. Thus, these studies demonstrate the feasibility for simultaneously studying cAMP gradients and PKA activity in cells.This work was supported by AHA 16PRE27130004, NIH P01HL066299, NIH S10RR027535, and the Abraham Mitchell Cancer Research Fund.

Intravital Förster resonance energy transfer imaging reveals osteopontin-mediated polymorphonuclear leukocyte activation by tumor cell emboli.

Myeloid‐derived suppressor cells (MDSCs) cause paraneoplastic leukemoid reactions and facilitate tumor cell metastasis. However, the interaction of MDSCs with tumor cells in live tissue has not been adequately visualized. To accomplish this task, we developed an intravital imaging protocol to observe metastasized tumor cells in mouse lungs. For visualization of the activation of MDSCs, bone marrow cells derived from transgenic mice expressing a Förster resonance energy transfer biosensor for ERK were implanted into host mice. Under a two‐photon excitation microscope, numerous polymorphonuclear cells (PMNs) were found to infiltrate the lungs of tumor‐bearing mice in which 4T1 mammary tumor cells were implanted into the footpads. By Förster resonance energy transfer imaging, we found ERK activation in PMNs around the 4T1 tumor emboli in the lungs. Because antibody array analysis implied the involvement of osteopontin (OPN) in the metastasis of 4T1 cells, we further analyzed the effect of OPN knockdown. The OPN knockdown in 4T1 cells did not affect the cell growth, but markedly suppressed lung metastasis of 4T1 cells and ERK activation in PMNs in the lung. Intravenous injection of recombinant OPN restored the lung metastasis of OPN‐deficient 4T1 cells, suggesting that OPN functioned in a paracrine manner. It has been reported that ERK activation of neutrophils causes NETosis and that PMNs promote metastasis of tumor cells by NETosis. In agreement with previous reports, the NETosis inhibitor DNase I inhibited lung metastasis of 4T1 cells. These observations suggest that OPN promotes metastasis of 4T1 cells by activating PMNs and inducing NETosis.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

High-Content Imaging Platform for Profiling Intracellular Signaling Network Activity in Living Cells

SummaryEssential characteristics of cellular signaling networks include a complex interconnected architecture and temporal dynamics of protein activity. The latter can be monitored by Förster resonance energy transfer (FRET) biosensors at a single-live-cell level with high temporal resolution. However, these experiments are typically limited to the use of a couple of FRET biosensors. Here, we describe a FRET-based multi-parameter imaging platform (FMIP) that allows simultaneous high-throughput monitoring of multiple signaling pathways. We apply FMIP to monitor the crosstalk between epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor signaling, signaling perturbations caused by pathophysiologically relevant EGFR mutations, and the effects of a clinically important MEK inhibitor (selumetinib) on the EGFR network. We expect that in the future the platform will be applied to develop comprehensive models of signaling networks and will help to investigate the mechanism of action as well as side effects of therapeutic treatments.