Showing papers in "Nature Methods in 2008"
TL;DR: Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors.
Abstract: We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41–52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 × 10 5 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices. The mRNA population specifies a cell’s identity and helps to govern its present and future activities. This has made transcriptome analysis a general phenotyping method, with expression microarrays of many kinds in routine use. Here we explore the possibility that transcriptome analysis, transcript discovery and transcript refinement can be done effectively in large and complex mammalian genomes by ultra-high-throughput sequencing. Expression microarrays are currently the most widely used methodology for transcriptome analysis, although some limitations persist. These include hybridization and cross-hybridization artifacts 1–3 , dye-based detection issues and design constraints that preclude or seriously limit the detection of RNA splice patterns and previously unmapped genes. These issues have made it difficult for standard array designs to provide full sequence comprehensiveness (coverage of all possible genes, including unknown ones, in large genomes) or transcriptome comprehensiveness (reliable detection of all RNAs of all prevalence classes, including the least abundant ones that are physiologically relevant). Other
12,293 citations
TL;DR: This work compares and evaluates the differences in physicochemical properties of common fluorescent labels, focusing on traditional organic dyes and QDs, to provide a better understanding of the advantages and limitations of both classes of chromophores.
Abstract: Suitable labels are at the core of Luminescence and fluorescence imaging and sensing. One of the most exciting, yet also controversial, advances in label technology is the emerging development of quantum dots (QDs)--inorganic nanocrystals with unique optical and chemical properties but complicated surface chemistry--as in vitro and in vivo fluorophores. Here we compare and evaluate the differences in physicochemical properties of common fluorescent labels, focusing on traditional organic dyes and QDs. Our aim is to provide a better understanding of the advantages and limitations of both classes of chromophores, to facilitate label choice and to address future challenges in the rational design and manipulation of QD labels.
3,399 citations
TL;DR: These techniques are described and illustrated with examples highlighting current capabilities and limitations of single-molecule force spectroscopy.
Abstract: Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.
2,155 citations
TL;DR: Lifeact, a 17-amino-acid peptide, is described, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.
Abstract: Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.
2,036 citations
TL;DR: A practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes, is provided.
Abstract: Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.
1,929 citations
TL;DR: This approach shows that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability.
Abstract: Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.
1,753 citations
TL;DR: A new generation of non-Sanger-based sequencing technologies has delivered on its promise of sequencing DNA at unprecedented speed, thereby enabling impressive scientific achievements and novel biological applications.
Abstract: A new generation of non-Sanger-based sequencing technologies has delivered on its promise of sequencing DNA at unprecedented speed, thereby enabling impressive scientific achievements and novel biological applications. However, before stepping into the limelight, next-generation sequencing had to overcome the inertia of a field that relied on Sanger-sequencing for 30 years.
1,736 citations
TL;DR: The enhancement mechanisms responsible for the extreme sensitivity of the WGM biosensor are described, its current implementations and applications are reviewed, and its future possibilities are discussed.
Abstract: Optical label-free detectors, such as the venerable surface plasmon resonance (SPR) sensor, are generally favored for their ability to obtain quantitative data on intermolecular binding. However, before the recent introduction of resonant microcavities that use whispering gallery mode (WGM) recirculation, sensitivity to single binding events had not materialized. Here we describe the enhancement mechanisms responsible for the extreme sensitivity of the WGM biosensor, review its current implementations and applications, and discuss its future possibilities.
1,621 citations
TL;DR: Using error-correcting DNA barcodes, one run of a massively parallel pyrosequencer is constructed that characterized nearly as many 16S rRNA genes as have been sequenced to date by Sanger sequencing.
Abstract: We constructed error-correcting DNA barcodes that allow one run of a massively parallel pyrosequencer to process up to 1,544 samples simultaneously. Using these barcodes we processed bacterial 16S rRNA gene sequences representing microbial communities in 286 environmental samples, corrected 92% of sample assignment errors, and thus characterized nearly as many 16S rRNA genes as have been sequenced to date by Sanger sequencing.
1,301 citations
TL;DR: This work created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables.
Abstract: We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.
1,153 citations
TL;DR: A massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, is developed, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that the understanding of transcriptional complexity is far from complete.
Abstract: We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)+ transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.
TL;DR: A simple illumination method of fluorescence microscopy for molecular imaging yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.
Abstract: We describe a simple illumination method of fluorescence microscopy for molecular imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.
TL;DR: This work developed highly photostable variants of mOrange and TagRFP that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.
Abstract: All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.
TL;DR: In this article, the authors present a system and methods for quantitative three-dimensional mapping of refractive index in living or non-living cells, tissues, or organisms using a phase-shifting laser interferometric microscope with variable illumination angle.
Abstract: The present invention relates to systems and methods for quantitative three-dimensional mapping of refractive index in living or non-living cells, tissues, or organisms using a phase-shifting laser interferometric microscope with variable illumination angle. A preferred embodiment provides tomographic imaging of cells and multicellular organisms, and time-dependent changes in cell structure and the quantitative characterization of specimen-induced aberrations in high-resolution microscopy with multiple applications in tissue light scattering.
TL;DR: By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.
Abstract: We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to approximately 60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.
TL;DR: A light microscope that generates images with translationally invariant 30 × 30 × 75nm resolution over a depth of several micrometers enabling 3D sub-diffraction resolution without compromising speed or sensitivity is reported.
Abstract: Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 x 30 x 75 nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.
Karolinska Institutet1, University of Oxford2, University of Toronto3, Centre national de la recherche scientifique4, University of California, Berkeley5, Los Alamos National Laboratory6, Tsinghua University7, University of Science and Technology of China8, Weizmann Institute of Science9, Scripps Research Institute10, Novartis11, Argonne National Laboratory12, Yeshiva University13, Brookhaven National Laboratory14, Rutgers University15, Case Western Reserve University16, University of California, San Francisco17, Columbia University18, Max Delbrück Center for Molecular Medicine19, New York University20
TL;DR: This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
Abstract: In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
TL;DR: Far-field fluorescence nanoscopy with ordinary fluorophores is introduced based on switching the majority of them to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state.
Abstract: We introduce far-field fluorescence nanoscopy with ordinary fluorophores based on switching the majority of them to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state. Continuous widefield illumination by a single laser and a continuously operating camera yielded dual-color images of rhodamine- and fluorescent protein-labeled (living) samples, proving a simple yet powerful super-resolution approach.
TL;DR: QuEST (Quantitative Enrichment of Sequence Tags) as mentioned in this paper is a statistical framework based on the Kernel Density Estimation approach, which utilizes ChIP-Seq data to determine positions where protein complexes come into contact with DNA.
Abstract: Molecular interactions between protein complexes and DNA carry out essential gene regulatory functions. Uncovering such interactions by means of chromatin-immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) has recently become the focus of intense interest. We here introduce QuEST (Quantitative Enrichment of Sequence Tags), a powerful statistical framework based on the Kernel Density Estimation approach, which utilizes ChIP-Seq data to determine positions where protein complexes come into contact with DNA. Using QuEST, we discovered several thousand binding sites for the human transcription factors SRF, GABP and NRSF at an average resolution of about 20 base-pairs. MEME-based motif analyses on the QuEST-identified sequences revealed DNA binding by cofactors of SRF, providing evidence that cofactor binding specificity can be obtained from ChIP-Seq data. By combining QuEST analyses with gene ontology (GO) annotations and expression data, we illustrate how general functions of transcription factors can be inferred.
TL;DR: It is demonstrated that the fusion of human glutaredoxin-1 to roGFP2 facilitates specific real-time equilibration between the sensor protein and the glutathione redox couple, which facilitated the observation of redox changes associated with growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation.
Abstract: Dynamic analysis of redox-based processes in living cells is now restricted by the lack of appropriate redox biosensors. Conventional redox-sensitive GFPs (roGFPs) are limited by undefined specificity and slow response to changes in redox potential. In this study we demonstrate that the fusion of human glutaredoxin-1 (Grx1) to roGFP2 facilitates specific real-time equilibration between the sensor protein and the glutathione redox couple. The Grx1-roGFP2 fusion protein allowed dynamic live imaging of the glutathione redox potential (E(GSH)) in different cellular compartments with high sensitivity and temporal resolution. The biosensor detected nanomolar changes in oxidized glutathione (GSSG) against a backdrop of millimolar reduced glutathione (GSH) on a scale of seconds to minutes. It facilitated the observation of redox changes associated with growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation.
TL;DR: A set of improvements are described to the standard Illumina protocols to make the library preparation more reliable in a high-throughput environment, to reduce bias, tighten insert size distribution and reliably obtain high yields of data.
Abstract: The Wellcome Trust Sanger Institute is one of the world's largest genome centers, and a substantial amount of our sequencing is performed with 'next-generation' massively parallel sequencing technologies: in June 2008 the quantity of purity-filtered sequence data generated by our Genome Analyzer (Illumina) platforms reached 1 terabase, and our average weekly Illumina production output is currently 64 gigabases. Here we describe a set of improvements we have made to the standard Illumina protocols to make the library preparation more reliable in a high-throughput environment, to reduce bias, tighten insert size distribution and reliably obtain high yields of data.
TL;DR: The current state of native mass spectrometry technology is reviewed and several important biological applications are discussed, including high-throughput interactomics studies and current experimental challenges.
Abstract: Native mass spectrometry is an emerging technology that allows the topological investigation of intact protein complexes with high sensitivity and a theoretically unrestricted mass range. This unique tool provides complementary information to established technologies in structural biology, and also provides a link to high-throughput interactomics studies, which do not generate information on exact protein complex-composition, structure or dynamics. Here I review the current state of native mass spectrometry technology and discuss several important biological applications. I also describe current experimental challenges in native mass spectrometry, encouraging readers to contribute to solutions.
TL;DR: A fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs) is described and it is shown that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering.
Abstract: The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems. NOTE: In the version of this article initially published online, the name of one individual was misspelled in the Acknowledgments. The second sentence of the Acknowledgments paragraph should read, “We thank I. Cheesman for helpful discussions.” The error has been corrected for all versions of the article.
TL;DR: An economical, efficient, single-step method for SNP discovery, validation and characterization that uses deep sequencing of reduced representation libraries (RRLs) from specified target populations and may be applied to any species with at least a partially sequenced genome.
Abstract: High-density single-nucleotide polymorphism (SNP) arrays have revolutionized the ability of genome-wide association studies to detect genomic regions harboring sequence variants that affect complex traits. Extensive numbers of validated SNPs with known allele frequencies are essential to construct genotyping assays with broad utility. We describe an economical, efficient, single-step method for SNP discovery, validation and characterization that uses deep sequencing of reduced representation libraries (RRLs) from specified target populations. Using nearly 50 million sequences generated on an Illumina Genome Analyzer from DNA of 66 cattle representing three populations, we identified 62,042 putative SNPs and predicted their allele frequencies. Genotype data for these 66 individuals validated 92% of 23,357 selected genome-wide SNPs, with a genotypic and sequence allele frequency correlation of r = 0.67. This approach for simultaneous de novo discovery of high-quality SNPs and population characterization of allele frequencies may be applied to any species with at least a partially sequenced genome.
TL;DR: An analytical single-particle tracking method and tool, multiple-target tracing (MTT), that takes advantage of the high spatial resolution provided by single-fluorophore sensitivity to generate dynamic maps at high densities of tracked particles, thereby providing global representation of molecular dynamics in cell membranes.
Abstract: Although the highly dynamic and mosaic organization of the plasma membrane is well-recognized, depicting a resolved, global view of this organization remains challenging. We present an analytical single-particle tracking (SPT) method and tool, multiple-target tracing (MTT), that takes advantage of the high spatial resolution provided by single-fluorophore sensitivity. MTT can be used to generate dynamic maps at high densities of tracked particles, thereby providing global representation of molecular dynamics in cell membranes. Deflation by subtracting detected peaks allows detection of lower-intensity peaks. We exhaustively detected particles using MTT, with performance reaching theoretical limits, and then reconnected trajectories integrating the statistical information from past trajectories. We demonstrate the potential of this method by applying it to the epidermal growth factor receptor (EGFR) labeled with quantum dots (Qdots), in the plasma membrane of live cells. We anticipate the use of MTT to explore molecular dynamics and interactions at the cell membrane.
TL;DR: Multicolor three-dimensional stochastic optical reconstruction microscopy (STORM) is demonstrated as a tool to quantitatively probe cellular structures and their interactions to enhance understanding of molecular processes in cells.
Abstract: The ability to directly visualize nanoscopic cellular structures and their spatial relationship in all three dimensions will greatly enhance our understanding of molecular processes in cells. Here we demonstrated multicolor three-dimensional (3D) stochastic optical reconstruction microscopy (STORM) as a tool to quantitatively probe cellular structures and their interactions. To facilitate STORM imaging, we generated photoswitchable probes in several distinct colors by covalently linking a photoswitchable cyanine reporter and an activator molecule to assist bioconjugation. We performed 3D localization in conjunction with focal plane scanning and correction for refractive index mismatch to obtain whole-cell images with a spatial resolution of 20-30 nm and 60-70 nm in the lateral and axial dimensions, respectively. Using this approach, we imaged the entire mitochondrial network in fixed monkey kidney BS-C-1 cells, and studied the spatial relationship between mitochondria and microtubules. The 3D STORM images resolved mitochondrial morphologies as well as mitochondria-microtubule contacts that were obscured in conventional fluorescence images.
TL;DR: The construction of a library of hypomorphic alleles of essential genes and a high-throughput growth competition assay to measure fitness with unprecedented sensitivity are described to dramatically increase the breadth and precision with which quantitative genetic analysis can be performed in yeast.
Abstract: Functional genomic studies in Saccharomyces cerevisiae have contributed enormously to our understanding of cellular processes. Their full potential, however, has been hampered by the limited availability of reagents to systematically study essential genes and the inability to quantify the small effects of most gene deletions on growth. Here we describe the construction of a library of hypomorphic alleles of essential genes and a high-throughput growth competition assay to measure fitness with unprecedented sensitivity. These tools dramatically increase the breadth and precision with which quantitative genetic analysis can be performed in yeast. We illustrate the value of these approaches by using genetic interactions to reveal new relationships between chromatin-modifying factors and to create a functional map of the proteasome. Finally, by measuring the fitness of strains in the yeast deletion library, we addressed an enigma regarding the apparent prevalence of gene dispensability and found that most genes do contribute to growth.
TL;DR: The genetically encoded calcium indicator TN-XXL allows repeated imaging of response properties from individual, identified neurons in vivo, which will be crucial for gaining new insights into cellular mechanisms of plasticity, regeneration and disease.
Abstract: Neurons in the nervous system can change their functional properties over time. At present, there are no techniques that allow reliable monitoring of changes within identified neurons over repeated experimental sessions. We increased the signal strength of troponin C-based calcium biosensors in the low-calcium regime by mutagenesis and domain rearrangement within the troponin C calcium binding moiety to generate the indicator TN-XXL. Using in vivo two-photon ratiometric imaging, we show that TN-XXL exhibits enhanced fluorescence changes in neurons of flies and mice. TN-XXL could be used to obtain tuning curves of orientation-selective neurons in mouse visual cortex measured repeatedly over days and weeks. Thus, the genetically encoded calcium indicator TN-XXL allows repeated imaging of response properties from individual, identified neurons in vivo, which will be crucial for gaining new insights into cellular mechanisms of plasticity, regeneration and disease.
TL;DR: A methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput–compatible format is described.
Abstract: Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.
TL;DR: In the version of this correspondence initially published, the two previously published datasets analyzed were labeled with incorrect references in Figure 1b.
Abstract: Nat. Methods 5, 374–375 (2008); corrected after print 29 May 2008. In the version of this correspondence initially published, the two previously published datasets analyzed were labeled with incorrect references in Figure 1b. Reference 2 should be associated with the second column (80 sites), and reference 3 should be associated with the third column (96 sites).