Fluorescence resonance energy transfer (FRET) analysis demonstrates dimer/oligomer formation of the human breast cancer resistance protein (BCRP/ABCG2) in intact cells

The human breast cancer resistance protein (BCRP/ABCG2) is a half ATP-binding cassette (ABC) efflux transporter that plays an important role in drug resistance and disposition. Although BCRP is believed to function as a homodimer or homooligomer, this has not been demonstrated in vivo in intact cells. Therefore, in the present study, we investigated dimer/oligmer formation of BCRP in intact cells. Wild-type BCRP and the mutant C603A were attached to cyan or yellow fluorescence protein and expressed in HEK293 cells by transient transfection. Protein levels, cell surface expression, and efflux activities of wild-type and mutant BCRP were determined by immunoblotting, 5D3 antibody binding, and flow cytometric efflux assay, respectively. Dimer/oligomer formation of BCRP in intact cells was analyzed using fluorescence resonance energy transfer (FRET) microscopy. Wild-type BCRP and C603A were expressed in HEK293 cells at comparable levels. C603A was predominantly expressed in the plasma membrane as was wild-type protein. Furthermore, C603A retained the same mitoxantrone efflux activity and the ability of dimer/oligmer formation as wild-type BCRP. Finally, cross-linking experiments yielded data consistent with the FRET analysis. In conclusion, we have, for the first time, demonstrated that BCRP can form a dimer/oligomer in vivo in intact cells using the FRET technique. We have also shown that Cys⁶⁰³ alone does not seem to be essential for dimer/oligomer formation of BCRP.     

Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET)

Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9 ± 0.4% (n = 36), 4.5 ± 0.4% (n = 39), and 8.3 ± 0.6% (n = 44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7 ± 1.1% (n = 39) and 9.0 ± 0.9% (n = 37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.     

Fluorescence resonance energy transfer (FRET): theory and experiments

Fluorescence resonance energy transfer (FRET) is a powerful biophysical technique permitting macromolecular interactions between fluorescent molecules in close contact with each other to be analysed. Studies of the kinetics of association/dissociation between biological macromolecules may be carried out using this technique. Distances of interaction between donor-acceptor may be estimated by the FRET approach.     

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha–beta–PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA–PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha–alpha and alpha–PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha–alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)₂.     

Fluorescence resonance energy transfer (FRET) based molecular detection of a genetically modified PCB degrader in soil

Genetic analysis of the location of a mini-Tn5 promoted insertion of the LB400 bph operon in the rhizosphere coloniser Pseudomonas fluorescens F113rifPCB, allowed the development of a specific PCR detection system based on the unique DNA sequence at this insertion site. Real time PCR using both SYBR green chemistry and Fluorescence Resonance Energy Transfer probes allowed the precise identification of the recombinant strain and its quantitative detection in soil microcosms over a (bacteria/g) range of five orders of magnitude. This new assay can detect the genetically modified microorganism from soil in less than 90 min and at levels below the detection limits of standard PCR or cultivable counts on selective media.     

Fluorescence resonance energy transfer (FRET) using ssDNA binding fluorescent dye

There is a need for simple and inexpensive methods for genotyping single nucleotide polymorphisms (SNPs) and short insertion/deletion variations (InDels). In this work, I demonstrate that a single-stranded DNA (ssDNA) binding dye can be used as a donor fluorophore for fluorescence resonance energy transfer (FRET). The method presented is a homogenous assay in which detection is based on the FRET from the fluorescence of the ssDNA dye bound to the unmodified detection primer to the fluorescent nucleotide analog incorporated into this detection primer during cyclic template directed primer extension reaction. Collection of the FRET emission spectrum with a scanning fluorescence spectrophotometer allows powerful data analysis. The fluorescence emission signal is modified by the optical properties of the assay vessel. This seems to be a completely neglected parameter. By proper selection of the optical properties of the assay plate one can improve the detection of the fluorescence emission signal.     

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.     

The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis

The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle. Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis. However, the intracellular site of this protein interaction is still unclear. To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19. Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19. This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures. The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging. Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction. Results were confirmed and quantified by donor fluorescence photobleaching experiments. PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19. We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells. These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane.     

Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling

Background  

Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET) to visualize pathogen-initiated signaling events in human cells.     

In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair

An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP2)-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2Apro) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2Apro from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research.     

Spectral imaging and its applications in live cell microscopy

In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct fluorescent molecules at the same time in the same sample. However, the spectral properties of the fluorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods. Spectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging. This method also offers advantages for fast multicolor time-lapse microscopy and fluorescence resonance energy transfer measurements in living samples. Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples.     

Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channel

Acid-sensing ion channels (ASICs) are believed to be homo- or heteromeric complexes, which have been verified by classical methods such as co-immunoprecipitation or electrophysiological assays. However, the exact subunit combinations of ASICs in living cells have not been established yet. Here, we apply assays based on fluorescence resonance energy transfer (FRET) between GFP color mutants CFP and YFP to investigate ASIC assembly directly in living cells. Homomerization as well as heteromerization of different combinations of ASIC subunits were found. In addition, our results suggest the formation of heteromeric 1a/2a channels of stoichiometry consisting of at least two 1a subunits and two 2a subunits. Similar stoichiometry was observed from heteromeric 1a/2b and 2a/2b channels. Our results imply that these heteromeric ASIC channels contain at least four subunits.     

Fluorescence lifetime imaging microscopy (FLIM) detects stimulus-dependent phosphorylation of the low density lipoprotein receptor-related protein (LRP) in primary neurons

The low-density lipoprotein receptor-related protein (LRP) is a large, endocytic receptor involved in intracellular signalling. LRP acts as a co-receptor with the PDGF-receptor (PDGF-r) for platelet-derived growth factor (PDGF). PDGF-r and Src-kinases induce tyrosine-phosphorylation of LRP. We used fluorescence lifetime imaging microscopy (FLIM) to specifically detect LRP phosphorylation, measure its extent and localization in intact cells, and assess its effects upon LRP-APP interaction. Robust phosphorylation of LRP throughout the cell was observed after overexpression of Src-kinase. This depended on LRP's distal NPXY domain. By contrast, activation of the PDGF-r resulted in phosphorylation of the subpopulation of LRP at or near the cell surface. PDGF activation triggered phosphorylation of endogenous LRP in primary neurons. LRP is also a trafficking receptor for the Alzheimer-related molecule amyloid-precursor-protein (APP). PDGF stimulation did not affect LRP-APP interactions. This approach allows exquisite subcellular resolution of specific LRP post-translational changes and protein-protein interactions of endogenous proteins in intact cells.     

Fluorescence resonance energy transfer (FRET)-based specific labeling of Cryptosporidium oocysts for detection in environmental samples.

BACKGROUND: Accurate detection and quantification of Cryptosporidium oocysts in water are a challenge to the water industry. This article demonstrates a way to fluorescently label Cryptosporidium oocysts, based on fluorescence resonance energy transfer (FRET). Labeled oocysts can then be applied to environmental waters and their movement followed by flow cytometric detection and enumeration of the FRET-labeled oocysts, as demonstrated here with environmental water samples. METHODS: Cryptosporidium oocysts were labeled with three fluorochromes, FITC, Texas red, and Cy7, that through FRET yielded a Stokes shift of approximately 272 nm with excitation from a standard argon laser emitting at 488 nm. Defined flow cytometric settings and gatings were used to select FITC/green (530-nm), Texas red/red (650-nm), and Cy7/infrared (780-nm) fluorescing particles with light scatter properties similar to oocysts. Water concentrates were seeded with 10 tri-labeled oocysts and were analyzed using flow cytometry. Unseeded water concentrates were also analyzed. RESULTS: Analysis of unseeded water concentrates detected no autofluorescent particle similar to the labeled oocysts. Labeled oocysts were detected successfully with up to 85% recovery in water concentrates spiked with 10 tri-labeled oocysts. CONCLUSIONS: Low numbers of FRET-labeled oocysts can be quantified and clearly distinguished from autofluorescing background in environmental water concentrates.     

Intensity-based energy transfer measurements in digital imaging microscopy

Investigation of protein-protein associations is important in understanding structure and function relationships in living cells. Using Förster-type resonance energy transfer between donor and acceptor labeled monoclonal antibodies we can assess the cell surface topology of membrane proteins against which the antibodies were raised. In our current work we elaborated a quantitative image microscopic technique based on the measurement of fluorescence intensities to calculate the energy transfer efficiency on a pixel-by-pixel basis. We made use of the broad excitation and emission spectrum of cellular autofluorescence for background correction of images. In addition to the reference autofluorescence images (UV background) we recorded three fluorescent images (donor, acceptor and energy transfer signal) of donor-acceptor double labeled samples, and corrected for spectral spillage of the directly excited donor and acceptor fluorescence into the energy transfer image. After careful image registration we were able to calculate the energy transfer efficiency on a pixel-by-pixel basis. In this paper, we also present a critical comparison between results obtained with this method and other approaches (photobleaching and flow cytometric energy transfer measurements).     

Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance energy transfer (FRET)

The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of these sites, we studied FRET in a series of mutants bearing pyrene iodoacetamide at different positions, and we measured the distances from each of the sites to the donor. Our results demonstrate the presence of a high affinity binding site at the enzyme periphery. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217-220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site. This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand.     

Bidirectional fluorescence resonance energy transfer (FRET) in mutated and chemically modified yellow fluorescent protein (YFP)

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Fo?rster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.