Encoded Fluorescence Resonance Energy Transfer Research Articles

Genetically Encoded Fluorescence Resonance Energy Transfer Biosensor for Live-Cell Visualization of Lamin A Phosphorylation at Serine 22.

Extensive phosphorylation at serine 22 (pSer22) on lamin A is the hallmark of cell mitosis, which contributes to the breakdown of nuclear envelope. In the interphase, pSer22 lamin A exists in low abundance and is involved in mechanotransduction, virus infection, and gene expression. Numerous evidences emerge to support lamin A regulation on cell function and fate by phosphorylation. However, live-cell imaging tools for visualizing the dynamics of pSer22 lamin A are yet to be established. Herein, we developed a novel lamin A phosphorylation sensor (LAPS) based on fluorescence resonance energy transfer (FRET) with high sensitivity and specificity. We observed the dynamic lamin A phosphorylation during the cell cycle progression in single living cells: the increase of pSer22 modification when cells entered the mitosis and recovered upon the mitosis exit. Our biosensor also showed the gradual reduction of pSer22 modification during cell adhesion and in response to hypotonic environment. By applying LAPS, we captured the propagation of pSer22 modification from inside to outside of the inner nuclear membrane, which further led to the breakdown of nuclear envelope. Meanwhile, we found the synchronous phosphorylation of pSer22 lamin A and H3S10ph at mitosis entry. Inhibition of Aurora B, the responsible kinase for H3S10ph, could shorten the mitotic period without obvious effect on the pSer22 modification level of lamin A. Thus, LAPS allows the spatiotemporal visualization of the lamin A pSer22, which will be useful for elucidating the molecular mechanisms underlying cell mitosis and mechanoresponsive processes.

Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors.

Selenium is a component of selenoproteins, which plays a crucial role in cellular redox homeostasis, thyroid metabolism, and DNA synthesis. Selenium has pleiotropic effects like antioxidant and anti-inflammatory activities; however, excess intake of selenium can imbalance such processes. The effects of selenium on human health are numerous and complex, demanding additional research to monitor the flux rate of selenium. Here, we have created a noninvasive and highly efficient genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor, SelFS (Selenium FRET-Sensor), for real-time monitoring of selenium at the cellular and subcellular levels. The construct of the nanosensor contains a selenium-binding protein (SeBP) as the selenium-detecting element inserted between the green fluorescent protein variants enhanced cyan fluorescent protein and Venus. In the presence of selenium, SelFS brings a conformational change, which is seen in the form of FRET. In vitro studies showed that SelFS is highly specific and selective for selenium and stable at an altered pH range from 5.0 to 8.0. SelFS is a flexible and dynamic tool for the detection of selenium in both prokaryotes and eukaryotes in a noninvasive way, with a binding constant (K d) of 0.198 × 10-6 M as compared to its mutants. The developed nanosensor can provide us a reporter tool for a wide range of industrial and environmental applications, which will help us to understand its functions in biological systems.

Directed evolution of the genetically encoded zinc(II) FRET sensor ZapCY1

Zinc(II) ions (Zn2+) play an essential role in living systems, with their delicate concentration balance differing among the various intracellular organelles. The spatiotemporal distribution and homeostasis of Zn2+ can be monitored through photoluminescence imaging using zinc sensors. Among such biosensors, genetically encoded fluorescent sensor proteins are attractive tools owing to their subcellular localization advantage and high biocompatibility. However, the limited fluorescent properties of these proteins, such as their insufficient quantum yield and dynamic range, restrict their practical use. In this study, we developed an expression–screening–directed evolution system and used it to improve ZapCY1, a genetically encoded fluorescence resonance energy transfer (FRET) sensor. After four rounds of directed evolution, the FRET dynamic range of the modified sensor (designated ZapTV-EH) was increased by 1.5–1.7-fold. With its enhanced signal-to-noise ratio and ability to detect a wide Zn2+ concentration range, ZapTV-EH proves to be a better visualization tool for monitoring Zn2+ at the subcellular level. Combined with the simplified subcloning and expression steps and sufficient mutant libraries, this directed evolution system may provide a more simple and efficient way to develop and optimize genetically encoded FRET sensors through high-throughput screening.

A ratiometric fluorescent probe based on FRET for selective monitoring of tungsten in living cells

Tungsten is considered a critical metal due to its unique properties and economic importance. Recent research has linked high tungsten concentrations to pulmonary diseases and increased cancer risk. Tungsten is on the list of critical metals at high risk maintained by the European Union. We have developed a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for the first time to determine tungsten ion efflux mechanisms and measure its concentration and distribution within living cells in real-time. For the construction of the nanosensor, TupA (tungsten binding protein), a highly selective protein, was sandwiched between ECFP and Venus, a FRET pair. SENTUN (SENsor for TUNgsten) was the name given to the newly developed nanosensor. SENTUN was highly specific and selective for tungsten and stable at physiological pH levels. We also created three mutants: SENTUN-118D, SENTUN-118K, and SENTUN-178W, with SENTUN-118K exhibiting the highest affinity for tungsten and its comprehensive range detection. The novelty of this sensor is that it is genetically encoded and can be used for real-time measurement of tungsten levels in living cells.

Real time optical detection of gold in living cells through genetically-encoded probe.

To study the efflux of gold (Au) in living cells, a genetically encoded fluorescence resonance energy transfer (FRET)-based sensor has been developed. The gold-sensing domain GolB from Salmonella typhimurium has been fused to the N- and C-termini of the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus respectively. In living cells, this probe is highly selective and sensitive to gold and it can withstand changes in variable pH ranges. GolSeN-25, the most efficient sensor variant, binds gold with an affinity (Kd) of 0.3 × 10−6 M, covering gold concentrations of nM to μM, and can be used for non-invasive real-time in vivo gold measurement in living cells. A simple and sensitive FRET probe was designed for the detection of gold with high selectivity and can be applied to the analysis of real samples.

Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells.

Simple SummarySpatially and temporally defined H2O2 signatures are essential parts of various signaling pathways. Therefore, monitoring H2O2 dynamics with high spatio–temporal resolution is significantly important to understand how this ubiquitous signaling molecule can control diverse cellular responses. In this study, we designed and characterized a Fluorescence Resonance Energy Transfer (FRET)-based genetically encoded H2O2 sensor that provides a powerful tool to monitor the spatio–temporal dynamics of H2O2 fluxes. We have used this sensor to monitor the flux of H2O2 in live cells under stress conditions. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively and would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.

A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells.

Nitrate (NO3–) is a critical source of nitrogen (N) available to microorganisms and plants. Nitrate sensing activates signaling pathways in the plant system that impinges upon, developmental, molecular, metabolic, and physiological responses locally, and globally. To sustain, the high crop productivity and high nutritional value along with the sustainable environment, the study of rate-controlling steps of a metabolic network of N assimilation through fluxomics becomes an attractive strategy. To monitor the flux of nitrate, we developed a non-invasive genetically encoded fluorescence resonance energy transfer (FRET)-based tool named “FLIP-NT” that monitors the real-time uptake of nitrate in the living cells. The developed nanosensor is suitable for real-time monitoring of nitrate flux in living cells at subcellular compartments with high spatio-temporal resolution. The developed FLIP-NT nanosensor was not affected by the pH change and have specificity for nitrate with an affinity constant (Kd) of ∼5 μM. A series of affinity mutants have also been generated to expand the physiological detection range of the sensor protein with varying Kd values. It has been found that this sensor successfully detects the dynamics of nitrate fluctuations in bacteria and yeast, without the disruption of cellular organization. This FLIP-NT nanosensor could be a very important tool that will help us to advance the understanding of nitrate signaling.

High-performance FRET biosensors for single-cell and in vivo lead detection

Forms of lead (Pb) have been insidiously invading human life for thousands of years without obvious signs of their considerable danger to human health. Blood lead level (BLL) is the routine measure used for diagnosing the degree of lead intoxication, although it is unclear whether there is any safe range of BLL. To develop a practical detection tool for living organisms, we engineered a genetically encoded fluorescence resonance energy transfer (FRET)-based Pb2+ biosensor, ‘Met-lead 1.44 M1’, with excellent performance. Met-lead 1.44 M1 has an apparent dissociation constant (Kd) of 25.97 nM, a detection limit (LOD) of 10 nM (2.0 ppb/0.2 μg/dL), and an enhancement dynamic ratio of nearly ~ 5-fold upon Pb2+ binding. The 10 nM sensitivity of Met-lead 1.44 M1 is five times below the World Health Organization-permitted level of lead in tap water (10 ppb; WHO, 2017), and fifteen times lower than the maximum BLL for children (3 μg/dL). We deployed Met-lead 1.44 M1 to measure Pb2+ concentrations in different living models, including two general human cell lines and one specific line, induced pluripotent stem cell (iPSC)-derived cardiomyocytes, as well as in widely used model species in plant (Arabidopsis thaliana) and animal (Drosophila melanogaster) research. Our results suggest that this new biosensor is suitable for lead toxicological research in vitro and in vivo, and will pave the way toward potential applications for both low BLL measures and rapid detection of environmental lead in its divalent form.

Ratiometric imaging of flux dynamics of cobalt with an optical sensor

Cobalt (Co2+) is a vital micronutrient needed for growth and development by all life types. In humans, cobalt is cytotoxic and genotoxic associated with various pathological and physiological conditions such as allergic dermatitis, pneumonia, interstitial fibrosis, alveolitis, myocardiopathy, rhinitis and lung cancer. In this study, we present a genetically encoded fluorescence resonance energy transfer (FRET)-based Cobalt Optical Sensor (CobOS) to determine this metal ion’s trafficking scheme consisting of uptake/efflux mechanisms and measuring the real time free-ion concentration and distribution within living cells. CobOS comprise of a cobalt-sensing domain CbiKP from Desulfovibrio vulgaris Hildenborough fused with the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus at N- and C-terminus respectively. When studied for its specificity and selectivity, CobOS provides maximum ratiometric readout for cobalt ions, thereby establishing its effectiveness as a FRET cobalt sensor. The sensor is resistant to pH changes and remains unaffected by adding other metal ions that are biologically significant. CobOS-86n, the most efficient sensor variant, binds cobalt with an affinity (Kd) of 0.86 × 10−6 M covering cobalt concentrations of 50 nM to 200 μM and can be of practical interest for real time measurement of in vivo cobalt levels in Escherichia coli (E. coli), yeast and mammalian cells non-invasively using widefield confocal fluorescent microscopy.

Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor.

(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.

Monitoring the Heavy Metal Lead Inside Living Drosophila with a FRET-Based Biosensor.

The harmful impact of the heavy metal lead on human health has been known for years. However, materials that contain lead remain in the environment. Measuring the blood lead level (BLL) is the only way to officially evaluate the degree of exposure to lead. The so-called “safe value” of the BLL seems to unreliably represent the secure threshold for children. In general, lead’s underlying toxicological mechanism remains unclear and needs to be elucidated. Therefore, we developed a novel genetically encoded fluorescence resonance energy transfer (FRET)-based lead biosensor, Met-lead, and applied it to transgenic Drosophila to perform further investigations. We combined Met-lead with the UAS-GAL4 system to the sensor protein specifically expressed within certain regions of fly brains. Using a suitable imaging platform, including a fast epifluorescent or confocal laser-scanning/two-photon microscope with high resolution, we recorded the changes in lead content inside fly brains ex vivo and in vivo and at different life stages. The blood–brain barrier was found to play an important role in the protection of neurons in the brain against damage due to the heavy metal lead, either through food or microinjection into the abdomen. Met-lead has the potential to be a powerful tool for the sensing of lead within living organisms by employing either a fast epi-FRET microscope or high-resolution brain imaging.

Visualization of thiamine in living cells using genetically encoded fluorescent nanosensor

Thiamine (vitamin B1) plays a fundamental role in energy metabolism. Its deficiency in humans manifest in the form of serious diseases like Beriberi and Wernicke–Korsakoff syndrome. Monitoring of thiamine within living cells in real time is of acute interest in medicine. Fluorescent indicator protein for thiamine (FLIPT) is a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor developed in this study that allows real time monitoring of thiamine levels within living cells. Nanosensor is constructed by sandwiching thiamine binding protein (thiB) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), a FRET pair. Definite conformational change in the sensory domain thiB is depicted as a change in the level of this attuned solute under in vitro and in vivo conditions. This turn out when thiamine binds to sensory domain that brings the donor-acceptor pair in vicinity resulting in increased FRET ratio. Henceforth, any variation in thiamine concentration changes the resultant FRET ratio. The constructed nanosensor was very specific to thiamine and stable to pH within physiological range. The calculated affinity (Kd) of FLIPT is 529 nM. Chimeric protein was expressed in Escherichia coli (E. coli), yeast and mammalian cells to monitor the thiamine levels. Thus, FLIPT is a genetically encoded FRET based nanosensor that allows real time monitoring of thiamine levels within living cells in a non-invasive manner.

FRET-based nanosensors for monitoring and quantification of alcohols in living cells.

Odorants constitute a small and chemically diverse group of molecules with ethanol functioning as a key odorant that induces reproductive toxicity and adverse chronic effects on the liver. Analytical tools designed so far for the detection of odorant molecules are relatively invasive. Therefore, a tool that can measure the corresponding rate changes of ethanol concentration in real-time is highly desirable. Here in this work, we report a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for in vivo quantification of ethanol at the cellular level with high spatial and temporal resolution. A human odorant-binding protein (hOBPIIa) was flanked by fluorescent proteins ECFP (Enhanced Cyan Fluorescent Protein) and Venus at the N- and C-terminus respectively. The constructed FRET nanosensor was named the fluorescent indicator protein for odorants (FLIPO). FLIPO allows in vitro and in vivo determination of FRET changes in a concentration-dependent manner. The developed nanosensor is highly specific to ethanol, stable to pH changes and provides rapid detection rate response. FLIPO-42 is the most efficient nanosensor created that measures ethanol with an apparent affinity (Kd) of 4.16 μM and covers the physiological range of 500 nM to 12 μM ethanol measurement. FLIPO-42 can measure ethanol dynamics in bacterial, yeast and mammalian cells non-invasively in real time which proves its efficacy as a sensing device in both prokaryotic and eukaryotic systems. Taken together, a prototype for a set of nanosensors was established, potentially enabling the monitoring of dynamic changes of ethanol and investigate its uptake and metabolism with subcellular resolution in vivo and ex vivo. Furthermore, the advent of a set of novel nanosensors will provide us with the tools for numerous medical, scientific, industrial and environmental applications which would help to illuminate their role in biological systems.

Integrated Platform for Monitoring Single-cell MAPK Kinetics in Computer-controlled Temporal Stimulations

Extracellular response kinase (ERK) is one of the key regulator of cell fate, such as proliferation, differentiation and cell migration. Here, we propose a novel experimental pipeline to learn ERK kinetics by temporal growth factor (GF) stimulation. High signal-to-noise ratio of genetically encoded Fluorescence resonance energy transfer (FRET) biosensor enables to get a large number of single-cell ERK activity at each time point, while computer-controlled microfluidics fine-tune the temporal stimulation. Using this platform, we observed that static Epidermal growth factor (EGF) stimulation led to transient ERK activation with a significant cell-to-cell variation, while dynamic stimulation of 3′ EGF pulse led to faster adaptation kinetics with no discrepancy. Multiple EGF pulses retriggered ERK activity with respect to frequency of stimulation. We also observed oscillation of ERK activity of each cell at basal state. Introducing of Mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, was not only dropping the average of basal activity for 7.5%, but also diminishing oscillatory behavior. Activity level raised up when inhibitor was removed, followed by transient peak of ERK kinetics. We expect this platform to probe Mitogen-associated protein kinase (MAPK) signaling network for systems biology research at single cellular level.

Supragranular Pyramidal Cells Exhibit Early Metabolic Alterations in the 3xTg-AD Mouse Model of Alzheimer's Disease.

The impairment of cerebral glucose utilization is an early and predictive biomarker of Alzheimer’s disease (AD) that is likely to contribute to memory and cognition disorders during the progression of the pathology. Yet, the cellular and molecular mechanisms underlying these metabolic alterations remain poorly understood. Here we studied the glucose metabolism of supragranular pyramidal cells at an early presymptomatic developmental stage in non-transgenic (non-Tg) and 3xTg-AD mice, a mouse model of AD replicating numerous hallmarks of the disease. We performed both intracellular glucose imaging with a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose biosensor and transcriptomic profiling of key molecular elements of glucose metabolism with single-cell multiplex RT-PCR (scRT-mPCR). We found that juvenile pyramidal cells exhibit active glycolysis and pentose phosphate pathway at rest that are respectively enhanced and impaired in 3xTg-AD mice without alteration of neuronal glucose uptake or transcriptional modification. Given the importance of glucose metabolism for neuronal survival, these early alterations could initiate or at least contribute to the later neuronal dysfunction of pyramidal cells in AD.

Investigating the Impact of Altered Subunit Expression on Sweet Taste Receptor Surface Trafficking and Signaling

The sweet taste receptor is a heterodimeric G protein‐coupled receptor (GPCR), consisting of the subunits Tas1R2 (T1R2) and Tas1R3 (T1R3). Expression of this receptor has been demonstrated in various tissues throughout the body, including the pancreas, small intestine and adipose tissue. This receptor is thought to play a role in maintaining the body's energy homeostasis, and may potentially be a target in the treatment of metabolic disorders such as diabetes and obesity. Some studies show that the two sweet taste receptor subunits are expressed in unequal quantities in pancreatic β cells and adipocytes, however the impact of this unequal expression on receptor function is not fully understood. This study aims to determine the impact of varying the degree of expression of the two subunits on receptor surface trafficking and downstream signaling pathways. To address this aim, sweet taste receptor‐expressing stable cell lines were generated in AD293 cells by sequential selection of vectors, and in HEK293 TRex cells using bicistronic vectors and the FlpIn system. Receptor expression was characterized through real‐time PCR and biotinylation pull‐down experiments, while receptor signaling was determined in live cells using a genetically encoded Fluorescence Resonance Energy Transfer (FRET) sensor and a Bioluminescence Resonance Energy Transfer (BRET) sensor. Our results show that expression of both receptor subunits is predominantly intracellular, and that co‐expression of both subunits does not improve surface expression. Co‐expression of both subunits was, however, found to be necessary for signaling, as cell lines expressing only one subunit did not show functional responses to aspartame. The magnitude of these functional responses was found to be greatest when subunit expression was closest to 1:1, suggesting that the sweet taste receptor is most efficacious as a 1:1 heterodimer. Cell lines expressing both subunits were found to signal through Gi, as well as undergo extracellular signal‐regulated kinase (ERK) phosphorylation, demonstrating the ability of this receptor to couple to multiple signaling pathways. While some studies using mouse sweet taste receptors have reported Gs signaling at high concentrations of sweeteners, this could not be replicated in this study using human receptors, suggesting that signaling pathways may be species‐specific. Overall, this study shows that co‐expression of both sweet taste receptor subunits is necessary for signaling but not for surface trafficking and that this receptor is most efficacious when both subunits are expressed at equal levels.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Real-Time Image-Based Approach to Distinguish and Discriminate Apoptosis from Necrosis.

Recent cell biology studies reveal that a cell can die through multiple pathways via distinct signaling mechanisms. Among these, apoptosis and necrosis are two distinct cell death pathways, and their detection and discrimination is vital in the drug discovery process and in understanding diverse biological processes. Although sensitive assays for apoptosis and necrosis are available, it is extremely difficult to adapt any of these methods to discriminate apoptosis-inducing stimuli from necrosis-inducing stimuli because of the acquisition of secondary necrosis by apoptotic cells when they are not phagocytosed. Essentially, any assay for discriminating apoptosis and necrosis needs to be carried out in real-time kinetic mode. Caspase 3 or 7 activation is observed in the majority of apoptotic cell death. Similarly, the absence of caspase 3/7 activation and cell membrane leakage are the two prominent indicators for necrotic cell death or necroptosis. The programmed form of necrosis, called pyroptosis, is also accompanied by membrane leakage and most often associated with activation of specific caspases such as caspase 1, 4, or 11, but not through caspase 3/7 activation. Here, a robust and sensitive real-time method is described to distinguish and discriminate apoptosis from necrosis. The assay utilizes stable integration of a genetically encoded fluorescence resonance energy transfer (FRET) probe for caspase 3/7 activation and the mitochondrion-targeted DsRed to identify necrotic cells. Caspase activation is determined by cleavage of the FRET probe; loss of soluble FRET probe with retention of mitochondrial red fluorescence indicates necrosis. This unit describes an important protocol for the generation of sensor cells expressing both probes, followed by detailed analysis of apoptosis and necrosis by microscopy imaging, confocal imaging, high-throughput imaging, and flow cytometry. © 2018 by John Wiley & Sons, Inc.

Tracking the Activity of mTORC1 in Living Cells Using Genetically Encoded FRET-based Biosensor TORCAR.

Mechanistic target of rapamycin complex 1 (mTORC1) is a highly conserved serine/threonine protein kinase that responds to multiple distinct signals (e.g., growth factors, amino acids, stress, and energy level) and coordinates cell growth and proliferation. The underlying molecular mechanisms by which these stimuli regulate the activity of mTORC1 are still not fully understood. The spatial compartmentalization of mTORC1 signaling has been suggested as an important mechanism for mTORC1 to achieve the signal specificity and efficiency. To examine the spatial regulation of the activity of mTORC1 in live cells, we describe a protocol using a newly developed molecular tool, a genetically encoded fluorescence resonance energy transfer (FRET)-based mTORC1 activity reporter, TORCAR. When expressed in the cell, TORCAR acts as a surrogate substrate of mTORC1, and exhibits a change in FRET in response to phosphorylation by mTORC1. Genetically targeting TORCAR to specific subcellular locations further allows for the characterization of spatial compartmentalized mTORC1 signaling. © 2016 by John Wiley & Sons, Inc.

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters.

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z' factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC50) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening.

Fluorescence Lifetime Imaging of Apoptosis.

Genetically encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools for analyzing cell signaling and function at single-cell resolution in standard 2D cell cultures, but these reporters rarely have been applied to 3D environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in light absorption complicate this analysis method in 3D settings. Herein we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter. This microscopic imaging technology allowed us to identify treatment-induced apoptosis in single breast cancer cells in environments ranging from 2D cell culture, spheroids with cancer and bone marrow stromal cells, and living mice with orthotopic human breast cancer xenografts. Using this imaging strategy, we showed that combined metabolic therapy targeting glycolysis and glutamine pathways significantly reduced overall breast cancer metabolism and induced apoptosis. We also determined that distinct subpopulations of bone marrow stromal cells control the resistance of breast cancer cells to chemotherapy, suggesting heterogeneity of treatment responses of malignant cells in different bone marrow niches. Overall, this study establishes FLIM with phasor analysis as an imaging tool for apoptosis in cell-based assays and living mice, enabling real-time, cellular-level assessment of treatment efficacy and heterogeneity.