FRET-based Sensor Research Articles

Development of a BRET-Based Molecular Tension Sensor to Study Altered Tensions in Disease Pathogenesis

Alteration of mechanical forces is an emerging factor in diseases like cancer. Changes in macroscopic stiffness in disease are accompanied by a wealth of changes in a cell's tensional homeostasis at a molecular level where mechanotransduction signaling pathways are aberrantly activated. However, the lack of tools to measure tensions at a molecular level in the cellular environment has crippled the identification of mechanosensing proteins and characterization of magnitudes of physiologic force in normal and disease contexts. We have developed a new bioluminescence resonance energy transfer (BRET)-based molecular tension sensor that responds predictably to changes in forces and exhibits an enhanced dynamic range compared to other FRET-based molecular tension sensors. We show that our tension sensor exhibits a distance-dependent change in BRETthrough the use of rigid alpha-helical linkers of varying length. As a proof of concept, we inserted our sensor into the canonical mechanosensing focal adhesion protein vinculin, and observed expected tension changes. We also encoded it into the mechanosensing cell-surface receptor Notch. Upon presentation of Notch's ligand on a neighboring cell, we detected a decrease in BRET signal corresponding to a stretching of the sensor due to the force induced by endocytosis of the ligand. This signal can be measured in bulk by a luminometer and in individual cells via imaging. We anticipate our BRET-based molecular tension sensor will be well suited to identify novel mechanosensing proteins and characterize the magnitude of change in tension sensed during disease pathogenesis.

Advances in methods for analyzing IP3 signaling and understanding of coupled Ca2+ and IP3 oscillations

Inositol 1,4,5-trisphosphate (IP3) is an important intracellular messenger produced by phospholipase C via the activation of G-protein-coupled receptor- or receptor-tyrosine-kinase-mediated pathways, and is involved in numerous responses to hormones, neurotransmitters, and growth factors through the releases of Ca2+ from intracellular stores via IP3 receptors. IP3-mediated Ca2+ signals often exhibit complex spatial and temporal organizations, such as Ca2+ oscillations. Recently, new methods have become available to measure IP3 concentration ([IP3]) using AlphaScreen technology, fluorescence polarization, and competitive ligand binding assay (CFLA). These methods are useful for the high throughput screening in drug discovery. Calcium ions generate versatile intracellular signals such as Ca2+ oscillations and waves. Fluorescent sensors molecules to monitor changes in [IP3] in single living cells are crucial to study the mechanism for the spatially and temporally regulated Ca2+ signals. In particular, FRET-based IP3 sensors are useful for the quantitative monitoring intracellular [IP3], and allowed to uncovered the oscillatory IP3 dynamics in association with Ca2+ oscillations. A mathematical model of coupled Ca2+ and IP3 oscillations predicts that Ca2+ oscillations are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations. These model predictions have also been confirmed experimentally. At present, however, usefulness of FRET-based IP3 sensors are limited by their relatively small change in fluorescence. Development of novel IP3 sensors with improve dynamic range would be important for understanding the regulatory mechanism of Ca2+ signaling and for in vivo IP3 imaging.

Tensile test of isolated single stress fiber expressing FRET-based tension sensor

Tensile test of isolated single stress fiber expressing FRET-based tension sensor

Measurement of stress distribution in stress fibers using FRET-based tension sensor

Measurement of stress distribution in stress fibers using FRET-based tension sensor

An effective signal amplifying strategy for copper (II) sensing by using in situ fluorescent proteins as energy donor of FRET

Fluorescence resonance energy transfer (FRET) is a reliable, sensitive, and robust assay method for detection of many biological targets. However, it generally needs an externally-introduced label to form the donor-acceptor pair, which could alter the accuracy of the detection. To address this issue, we report herein the FRET-based reactive copper ion sensors by using in situ fluorescent proteins (FP) of human urine or blood serum as the energy donor. Using Bull Serum Albumin (BSA) as model proteins, the sensor exhibits a remarkably fluorescence enhancement when BSA binds to the surface of copper clusters (Cu NCs) base on electrostatic interaction. Conversely, low fluorescence enhancement is observed without using BSA. As proof-of-principle, this positive approach is directly applied to detect Cu2+ using urine as the energy donor, accompanying with a signal enhancement by two factors and low detection limit of 0.5 μM Cu2+. Moreover, the proposed sensor could be applied in other complex environments, such as blood serum or cell culture medium. Consequently, the effectiveness, simplicity and diversity of our proposed strategy enable the development of a class of probes toward complex human environment for rapid, sensitive, and selective detection of targets.

Challenging FRET-based E-Cadherin force measurements in Drosophila

Mechanical forces play a critical role during embryonic development. Cellular and tissue wide forces direct cell migration, drive tissue morphogenesis and regulate organ growth. Despite the relevance of mechanics for these processes, our knowledge of the dynamics of mechanical forces in living tissues remains scarce. Recent studies have tried to address this problem with the development of tension sensors based on Förster resonance energy transfer (FRET). These sensors are integrated into force bearing proteins and allow the measurement of mechanical tensions on subcellular structures. Here, we developed such a FRET-based sensor to measure E-Cadherin tensions in different Drosophila tissues in and ex vivo. Similar to previous studies, we integrated the sensor module into E-cadherin. We assessed the sensitivity of the sensor by measuring dynamic, developmental processes and mechanical modifications in three Drosophila tissues: the wing imaginal disc, the amnioserosa cells and the migrating border cells. However, these assays revealed that the sensor is not functional to measure the magnitude of tensions occurring in any of the three tissues. Moreover, we encountered technical problems with the measurement of FRET, which might represent more general pitfalls with FRET sensors in living tissues. These insights will help future studies to better design and control mechano-sensing experiments.

Live imaging using a FRET glucose sensor reveals glucose delivery to all cell types in the Drosophila brain

All complex nervous systems are metabolically separated from circulation by a blood-brain barrier (BBB) that prevents uncontrolled leakage of solutes into the brain. Thus, all metabolites needed to sustain energy homeostasis must be transported across this BBB. In invertebrates, such as Drosophila, the major carbohydrate in circulation is the disaccharide trehalose and specific trehalose transporters are expressed by the glial BBB. Here we analyzed whether glucose is able to contribute to energy homeostasis in Drosophila. To study glucose influx into the brain we utilized a genetically encoded, FRET-based glucose sensor expressed in a cell type specific manner. When confronted with glucose all brain cells take up glucose within two minutes. In order to characterize the glucose transporter involved, we studied Drosophila Glut1, the homologue of which is primarily expressed by the BBB-forming endothelial cells and astrocytes in the mammalian nervous system. In Drosophila, however, Glut1 is expressed in neurons and is not found at the BBB. Thus, Glut1 cannot contribute to initial glucose uptake from the hemolymph. To test whether gap junctional coupling between the BBB forming cells and other neural cells contributes to glucose distribution we assayed these junctions using RNAi experiments and only found a minor contribution of gap junctions to glucose metabolism. Our results provide the entry point to further dissect the mechanisms underlying glucose distribution and offer new opportunities to understand brain metabolism.

Erratum to: Dynamic monitoring of Gi/o-protein-mediated decreases of intracellular cAMP by FRET-based Epac sensors.

Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by Gs- and Gi/o-proteins, is a central issue in biomedical research. The classical biochemical method to monitor increases in intracellular cAMP concentrations consists of a radioactive multicellular assay, which is well established, highly sensitive, and reproducible, but precludes continuous spatial and temporal assessment of cAMP levels in single living cells. For this purpose, Forster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor Gs-induced cAMP increases and it has remained elusive whether Epac sensors can reliably detect decreased intracellular cAMP levels as well. In this study, we systematically optimize experimental strategies employing FRET-based cAMP sensors to monitor Gi/o-mediated cAMP reductions. FRET experiments with adrenergic α2A or μ opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon Gi/o-coupled receptor activation in single living cells, and this effect can be reversed by selective receptor antagonists. Moreover, pre-treatment with forskolin or 3-isobutyl-1-methylxanthine (IBMX) to artificially increase basal cAMP levels was not required to monitor Gi/o-coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.

Dynamic monitoring of Gi/o-protein-mediated decreases of intracellular cAMP by FRET-based Epac sensors

Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by Gs- and Gi/o-proteins, is a central issue in biomedical research. The classical biochemical method to monitor increases in intracellular cAMP concentrations consists of a radioactive multicellular assay, which is well established, highly sensitive, and reproducible, but precludes continuous spatial and temporal assessment of cAMP levels in single living cells. For this purpose, Förster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor Gs-induced cAMP increases and it has remained elusive whether Epac sensors can reliably detect decreased intracellular cAMP levels as well. In this study, we systematically optimize experimental strategies employing FRET-based cAMP sensors to monitor Gi/o-mediated cAMP reductions. FRET experiments with adrenergic α2A or μ opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon Gi/o-coupled receptor activation in single living cells, and this effect can be reversed by selective receptor antagonists. Moreover, pre-treatment with forskolin or 3-isobutyl-1-methylxanthine (IBMX) to artificially increase basal cAMP levels was not required to monitor Gi/o-coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.

In vivo imaging of Ca2+ accumulation during cotton fiber initiation using fluorescent indicator YC3.60.

Non-tip-focused Ca 2+ gradient indicated by genetically expressing a FRET-based calcium sensor YC3.60 was established in spherical expanding cotton fibers, which is vital for cotton fiber initiation. Cotton fiber is a single cell elongated from ovule epidermis. It is not only the most important natural fiber used in the textile industry but also an ideal model for studying cell differentiation and elongation. Before linear cell growth, cotton fibers undergo spherical expansion at the beginning of initiation. Ca2+, as an important secondary messenger, plays a central role in polarized cell growth including cotton fiber elongation. However, the role of Ca2+ in fiber initiation is far from well understood. In this paper, through ovule culture we demonstrate that Ca2+ is crucial for fiber initiation. Using transgenic cotton expressing the fluorescent Ca2+ indicator YC3.60, we show cellular and intracellular distribution of Ca2+ in cotton ovule epidermis and fiber cells. In the initiating fiber cell, Ca2+ accumulated mainly at the base of the cell, while in the fast elongating cell, the Ca2+ was enriched in the tip region. This cellular distribution of Ca2+ reported by YC3.60 was confirmed by the staining with a Ca2+-sensitive dye fluo-3/AM. Compared to the fluorescent dye staining, the YC3.60 system can reveal more detailed information on the intracellular distribution without photobleaching. Taken together, our data suggest that Ca2+ plays an important role in spherical expansion of cotton fiber initials.

FRET-Based Sensors Unravel Activation and Allosteric Modulation of the GABAB Receptor

The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABAB, which is a target for various therapeutic applications. It is anobligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation ofthe GABAB receptor remains poorly understood. Here, we have developed FRET-based conformational GABAB sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABAB receptor and should facilitate the screening and identification of new chemicals targeting this receptor.

Imaging ERK and PKA Activation in Single Dendritic Spines during Structural Plasticity

Extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) play important roles in LTP and spine structural plasticity. While fluorescence resonance energy transfer (FRET)-based sensors for these kinases had previously been developed, they did not provide sufficient sensitivity for imaging small neuronal compartments, such as single dendritic spines in brain slices. Here we improved the sensitivity of FRET-based kinase sensors for monitoring kinase activity under two-photon fluorescence lifetime imaging microscopy (2pFLIM). Using these improved sensors, we succeeded in imaging ERK and PKA activation in single dendritic spines during structural long-term potentiation (sLTP) in hippocampal CA1 pyramidal neurons, revealing that the activation of these kinases spreads widely with length constants of more than 10μm. The strategy for improvement of sensors used here should be applicable for developing highly sensitive biosensors for various protein kinases. VIDEO ABSTRACT.

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

Mammalian Diaphanous 1 Mediates a Pathway for E-cadherin to Stabilize Epithelial Barriers through Junctional Contractility

Formins are a diverse class of actin regulators that influence filament dynamics and organization. Several formins have been identified at epithelial adherens junctions, but their functional impact remains incompletely understood. Here, we tested the hypothesis that formins might affect epithelial interactions through junctional contractility. We focused on mDia1, which was recruited to the zonula adherens (ZA) of established Caco-2 monolayers in response to E-cadherin and RhoA. mDia1 was necessary for contractility at the ZA, measured by assays that include a FRET-based sensor that reports molecular-level tension across αE-catenin. This reflected a role in reorganizing F-actin networks to form stable bundles that resisted myosin-induced stress. Finally, we found that the impact of mDia1 ramified beyond adherens junctions to stabilize tight junctions and maintain the epithelial permeability barrier. Therefore, control of tissue barrier function constitutes apathway for cadherin-based contractility to contribute to the physiology of established epithelia.

5D imaging approaches reveal the formation of distinct intracellular cAMP spatial gradients.

Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions. Several lines of evidence suggest that the distribution of cAMP within cells is not uniform. However, to date, no studies have measured the kinetics of 3D cAMP distributions within cells. This is largely due to the low signal-to-noise ratio of FRET-based probes. We previously reported that hyperspectral imaging improves the signal-to-noise ratio of FRET measurements. Here we utilized hyperspectral imaging approaches to measure FRET signals in five dimensions (5D) - three spatial (x, y, z), wavelength (λ), and time (t) - allowing us to visualize cAMP gradients in pulmonary endothelial cells. cAMP levels were measured using a FRET-based sensor (H188) comprised of a cAMP binding domain sandwiched between FRET donor and acceptor - Turquoise and Venus fluorescent proteins. We observed cAMP gradients in response to 0.1 or 1 μM isoproterenol, 0.1 or 1 μM PGE1, or 50 μM forskolin. Forskolin- and isoproterenol-induced cAMP gradients formed from the apical (high cAMP) to basolateral (low cAMP) face of cells. In contrast, PGE1-induced cAMP gradients originated from both the basolateral and apical faces of cells. Data suggest that 2D (x,y) studies of cAMP compartmentalization may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D (x,y,z) studies are required to assess mechanisms of signaling specificity. Results demonstrate that 5D imaging technologies are powerful tools for measuring biochemical processes in discrete subcellular domains. This work was supported by NIH P01HL066299, R01HL058506, S10RR027535, AHA 16PRE27130004 and the Abraham Mitchell Cancer Research Fund.

Glucokinase Mediated Glucosensing in Hypothalamic Neurons

Glucosensing is the ability of specialized cells to detect changes in extracellular glucose concentration and convert this information into a change in membrane potential; this allows glucose levels to influence the activity of cells that regulate glucose-altering or glucose-dependent physiological processes. Two major glucosensing cellular populations are pancreatic and hypothalamic cells; the mechanism of glucosensing in pancreatic beta cells is established, but that of hypothalamic neurons remains controversial. Beta cells glucosense by expressing a specialized type of hexokinase (the rate-limiting enzyme in glucose metabolism) called glucokinase (GCK). GCK, unlike standard hexokinase, is not saturated at physiological levels of glucose, allowing glucose metabolism in cells expressing GCK to increase when the extracellular glucose concentration is raised. This increase in metabolism is then converted into a membrane depolarization. Furthermore, S-nitrosylation can enhance the activity of GCK, and therefore enhance glucose metabolism and glucosensing. Here we use GT1-7 cells to explore whether a similar mechanism of glucosensing exists in hypothalamic neurons. The addition of a glucose concentration at which GCK is sensitive (and standard hexokinase is saturated) leads to an increase in cellular metabolism, as measured by NADH autofluorescence; but only in the presence of isoproterenol, a beta-adrenergic receptor agonist which increases intracellular calcium to activate neuronal nitric oxide synthase and facilitate S-nitrosylation. We also use a FRET-based GCK sensor to measure GCK activation under low and high glucose concentrations and in the presence of isoproterenol. These studies reveal the presence of a functional GCK and the sensitivity of GCK to post-translational/signaling cascade modulation. Further, the lack of metabolic response to glucose alone may be linked to high basal activity of GCK in this serum-grown cell line. These results point to a functional, receptor-potentiated glucose sensing mechanism in neurons that is mediated through post-translational activation of GCK.

In Situ Measurements of Protein Forces and Intracellular Ca2+ under Fluid Shear Stimuli

During traumatic brain injury, brain cells experience transient shear stresses that alter the forces in structural proteins. An increase in intracellular Ca2+ is seen universally in cells subjected to shear forces, but how are mechanical forces coupled to the Ca2+ influx is not well understood. We used a microfluidic chamber driven with a high-speed pressure servo to generate controlled fluid shear stress to cultured astrocytes, and simultaneously measured the intracellular responses using FRET-based force sensors actinin-cpstFRET and Ca2+ probes jRCaMP1h. We found that fluid shear generated non-uniform stresses in actinin. The time dependent force distribution is highly sensitive to the feature of the stimulus such as amplitude, duration rise time, and the frequencies of stimulation. In cells with weaker stress fibers, a rapid shear pulse (23 dyn/cm2, 2 ms rise time, 400 ms duration) produced an immediate and long-lasting increase in actinin stress at the upstream end of the cell and minimal changes at the downstream end. In contrast, a slow ramp to the same amplitude caused a minimal, and a more uniform increase in actinin stresses. Simultaneous Ca2+ imaging showed that the initial Ca2+ rise began at the upstream end of the cell where there was high strain, and it propagated to the entire cell within ∼4 s. Moreover, the Ca2+ peaked much faster (∼4 s) at the front end and slower in the center of cell body (∼10 s). This behavior occurred in ∼40% cells. In cells with abundant strong actin bundles, there was minimal response to shear stress. The actinin stress increased transiently and uniformly over the entire cell during the period of stimulation, and the Ca2+ rise appeared starting from the cell body. These results suggest that the force distribution directly alter the initial Ca2+ dynamics in different cellular domains. This work was funded by NINDS.

Critical Evaluation of FRET-Based Biosensor Performance: Implications for Measuring Ion Concentrations

Fluorescent sensors are widely used to visualize, quantify, and reveal the dynamics of small molecules, secondary metabolites, metals, and ions. One of the great promises of such sensors is the ability to quantify cellular signals in precise locations with high temporal resolution, enabling the creation of real-time dynamic maps of cellular signals. Yet this is coupled with the challenge of how to ensure that sensors are measuring what we think they are measuring, developing robust approaches for quantification, and assessing whether sensors are perturbing the underlying biology. This talk will highlight our efforts to develop genetically encoded FRET-based sensors for quantitative mapping of zinc ions in cells. I will discuss approaches for defining whether sensors perturb cellular ions, the importance of carefully defining dynamic range, strategies to compare sensor functionality in vitro, in cells, and in organelles, and the specific challenges associated with quantifying ions in cellular organelles.

HTS-compatible FRET-based conformational sensors clarify membrane receptor activation

Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis.