Multicolor Whole-Cell Bacterial Sensing Using a Synchronous Fluorescence Spectroscopy-Based Approach

The wide collection of currently available fluorescent proteins (FPs) offers new possibilities for multicolor reporter gene-based studies of bacterial functions. However, the simultaneous use of multiple FPs is often limited by the bleed-through of their emission spectra. Here we introduce an original approach for detection and separation of multiple overlapping fluorescent signals from mixtures of bioreporters strains. The proposed method relies on the coupling of synchronous fluorescent spectroscopy (SFS) with blind spectral decomposition achieved by the Canonical Polyadic (CP) decomposition (also known as Candecomp/Parafac) of three-dimensional data arrays. Due to the substantial narrowing of FP emission spectra and sensitive detection of multiple FPs in a one-step scan, SFS reduced spectral overlap and improved the selectivity of the CP unmixing procedure. When tested on mixtures of labeled E. coli strains, the SFS/CP approach could easily extract the contribution of at least four overlapping FPs. Furthermore, it allowed to simultaneously monitor the expression of three iron responsive genes and pyoverdine production in P. aeruginosa. Implemented in a convenient microplate format, this multiplex fluorescent reporter method provides a useful tool to study complex processes with different variables in bacterial systems.     

Improved donor/acceptor BRET couples for monitoring β-arrestin recruitment to G protein-coupled receptors

We report highly sensitive bioluminescence resonance energy transfer (BRET) assays with optimized donor/acceptor couples. We combined the energy donors Renilla luciferase (Rluc) and the Rluc8 variant with the energy acceptors yellow fluorescent protein, the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well-established assays measuring ligand-induced β-arrestin (βARR) intramolecular rearrangements and recruitment to G protein-coupled receptors. We show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing βARR assays and offer new options for the development of future BRET assays.     

Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer

Despite the fact that numerous studies suggest the existence of receptor multiprotein complexes, visualization and monitoring of the dynamics of such protein assemblies remain a challenge. In this study, we established appropriate conditions to consider spatiotemporally resolved images of such protein assemblies using bioluminescence resonance energy transfer (BRET) in mammalian living cells. Using covalently linked Renilla luciferase and yellow fluorescent proteins, we depicted the time course of dynamic changes in the interaction between the V2-vasopressin receptor and β-arrestin induced by a receptor agonist. The protein-protein interactions were resolved at the level of subcellular compartments (nucleus, plasma membrane, or endocytic vesicules) and in real time within tens-of-seconds to tens-of-minutes time frame. These studies provide a proof of principle as well as experimental parameters and controls required for high-resolution dynamic studies using BRET imaging in single cells.     

A rapid, sensitive, and selective bioluminescence resonance energy transfer (BRET)-based nucleic acid sensing system

Here we report the design of a bioluminescence resonance energy transfer (BRET)-based sensing system that could detect nucleic acid target in 5 min with high sensitivity and selectivity. The sensing system is based on adjacent binding of oligonucleotide probes labeled with Renilla luciferase (Rluc) and quantum dot (Qd) on the nucleic acid target. Here Rluc, a bioluminescent protein that generates light by a chemical reaction, is employed as an energy donor, and a quantum dot is used as an energy acceptor. Bioluminescence emission of Rluc overlaps with the Qd absorption whereas the emission of Qd is shifted from the emission of Rluc allowing for monitoring of BRET. In the presence of target, the labeled probes bind adjacently in a head-to-head fashion leading to BRET from Rluc to Qd upon addition of a substrate coelenterazine. The sensing system could detect target nucleic acid in buffer as well as in Escherichia coli cellular matrix in 5 min with a detection limit of 0.54 pmol. The ability to detect target nucleic acid rapidly in a cellular matrix with high sensitivity will prove highly beneficial in biomedical and environmental applications.     

Dual fluorescent protein reporters for studying cell behaviors in vivo

Fluorescent proteins (FPs) are useful tools for visualizing live cells and their behaviors. Protein domains that mediate subcellular localization have been fused to FPs to highlight cellular structures. FPs fused with histone H2B incorporate into chromatin allowing visualization of nuclear events. FPs fused to a glycosylphosphatidylinositol anchor signal sequence label the plasma membrane, highlighting cellular shape. Thus, a reporter gene containing both types of FP fusions would allow for effective monitoring of cell shape, movement, mitotic stage, apoptosis, and other cellular activities. Here, we report a binary color‐coding system using four differently colored FP reporters that generates 16 distinct color codes to label the nuclei and plasma membranes of live cells in culture and in transgenic mice. As an initial test of this system in vivo, the promoter of the human Ubiquitin C (UBC) gene was used to widely express one of the color‐code reporters. Widespread expression of the reporter was attained in embryos; however, both male and female transgenic mice were infertile. In contrast, the promoter of the mouse Oct4/Pou5f1 gene linked to two different color‐code reporters specifically labeled blastocysts, primordial germ cells, and postnatal germ cells, and these mice were fertile. Time‐lapse movies of fluorescently‐labeled primordial germs cells demonstrate the utility of the color‐code system to visualize cell behaviors. This set of new FP reporters should be a useful tool for labeling distinct cell populations and studying their behaviors in complex tissues in vivo. genesis 47:708–717, 2009. © 2009 Wiley‐Liss, Inc.     

Analysis of in vitro SUMOylation using bioluminescence resonance energy transfer (BRET)

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.     

Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels

Multiplexed bioluminescence resonance energy transfer (BRET) assays were developed to monitor the activation of several functional transient receptor potential (TRP) channels in live cells and in real time. We probed both TRPV1 intramolecular rearrangements and its interaction with Calmodulin (CaM) under activation by chemical agonists and temperature. Our BRET study also confirmed that: (1) capsaicin and heat promoted distinct transitions, independently coupled to channel gating, and that (2) TRPV1 and Ca²⁺-bound CaM but not Ca²⁺-free CaM were preassociated in resting live cells, while capsaicin activation induced both the formation of more TRPV1/CaM complexes and conformational changes. The BRET assay, based on the interaction with Calmodulin, was successfully extended to TRPV3 and TRPV4 channels. We therefore developed a full-spectral three-color BRET assay for analyzing the specific activation of each of the three TRPV channels in a single sample. Such key improvement in BRET measurement paves the way for the simultaneous monitoring of independent biological pathways in live cells.     

Detecting Protein–Protein Interactions in Living Cells: Development of a Bioluminescence Resonance Energy Transfer Assay to Evaluate the PSD-95/NMDA Receptor Interaction

The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein–protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.     

Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer

Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC₄ and its metabolites LTD₄ and LTE₄ stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC₄ is formed by addition of glutathione to LTA₄, catalyzed by the integral membrane protein, LTC₄ synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114–150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57–88. The functional significance of LTCS homo-oligomer formation is currently being investigated.     

Maturation models of fluorescent proteins are necessary for unbiased estimates of promoter activity

Fluorescent proteins (FPs) are a powerful tool to quantitatively monitor gene expression. The dynamics of a promoter and its regulation can be inferred from fluorescence data. The interpretation of fluorescent data, however, is strongly dependent on the maturation of FPs since different proteins mature in distinct ways. We propose a novel approach for analyzing fluorescent reporter data by incorporating maturation dynamics in the reconstruction of promoter activities. Our approach consists of developing and calibrating mechanistic maturation models for distinct FPs. These models are then used alongside a Bayesian approach to estimate promoter activities from fluorescence data. We demonstrate by means of targeted experiments in Escherichia coli that our approach provides robust estimates and that accounting for maturation is, in many cases, essential for the interpretation of gene expression data.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

Cellular BRET assay suggests a conformational rearrangement of preformed TrkB/Shc complexes following BDNF-dependent activation

We developed a cellular Bioluminescent Resonance Energy Transfer (BRET) assay based on the interaction of TrkB fused to Renilla luciferase with the intracellular adaptor protein Shc fused to Enhanced Yellow Fluorescent Protein (EYFP). The TrkB agonist Brain Derived Neurotrophic Factor (BDNF) induced a maximum BRET signal as of 10 min with an EC₅₀ value of 1.4 nM, similar to the other endogenous agonists NT-3 and NT-4/5, 1.5 nM and 0.34 nM, respectively. Interestingly, measure of the BRET signal with increasing expression of Shc-EYFP, in the presence or absence of BDNF, suggested a conformational change of preformed TrkB/Shc complexes rather than Shc recruitment. Furthermore, the Y516F TrkB mutant deficient to bind Shc as well as the kinase-dead K572R TrkB mutant was unable to respond to BDNF and exhibited a lower basal BRET signal than that of the wild-type TrkB receptor, again suggesting a preformed complex with constitutive activity. The double YY706/707FF TrkB mutant in the kinase activation loop also showed reduced basal activity but surprisingly kept its capacity to enhance BDNF-induced interaction with Shc, though with less efficacy. The Trk selective kinase inhibitors K252a and BMS-9 blocked BDNF-induced BRET signal with similar potency (100–150 nM), the preferential c-Met inhibitor PF-2341006 being one order of magnitude less potent. Remarkably, in the absence of BDNF, K252a and BMS-9 also reduced basal activity to the level of the Y516F TrkB mutant, suggesting that these compounds were able to reduce the TrkB constitutive activity. BRET responses of mutants and to kinase inhibitors thus reveal a complex level of interaction between TrkB and Shc and suggest that this BRET assay could be of great utility to test blockers of TrkB signalling in a physiologically relevant context.     

Real-time monitoring of ubiquitination in living cells by BRET

Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiological environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioluminescence resonance energy transfer (BRET), we measured the ubiquitination of beta-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addition to allowing the detection of basal and GPCR-regulated ubiquitination of beta-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.     

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a ''donor'' luciferase enzyme to an ''acceptor'' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.     

Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells

Bioluminescence resonance energy transfer (BRET) is an increasingly popular technique for studying protein-protein interactions in live cells. It is particularly suitable for real-time monitoring of such interactions, however, the timescale over which assays can be carried out is currently relatively short (minutes) due to substrate instability. We present a new derivation of the BRET technology, termed 'extended BRET' (eBRET), which now enables protein-protein interactions to be monitored in real-time for many hours. This capability has significant benefits for investigating cellular function over extended timescales, as we have illustrated using the agonist-induced G-protein coupled receptor/beta-arrestin interaction. The potential for studying the modulation of such interactions by agonists, antagonists, inhibitors, dominant negative mutants and co-expressed accessory proteins is substantial. Furthermore, the advantages of eBRET have important implications for the development of high-throughput BRET screening systems, an ever-expanding area of interest for the pharmaceutical industry.     

Sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase

Background

Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99.

Principal Findings

Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate.

Significance

We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time.     

Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells

A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100 Å of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.