Showing papers in "Nature Methods in 2006"

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).

Abstract: We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.

Direct observation of individual endogenous protein complexes in situ by proximity ligation

Abstract: Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.

Genetically encoded fluorescent indicator for intracellular hydrogen peroxide

Abstract: We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.

Detection and localization of single molecular recognition events using atomic force microscopy

Abstract: Because of its piconewton force sensitivity and nanometer positional accuracy, the atomic force microscope (AFM) has emerged as a powerful tool for exploring the forces and the dynamics of the interaction between individual ligands and receptors, either on isolated molecules or on cellular surfaces. These studies require attaching specific biomolecules or cells on AFM tips and on solid supports and measuring the unbinding forces between the modified surfaces using AFM force spectroscopy. In this review, we describe the current methodology for molecular recognition studies using the AFM, with an emphasis on strategies available for preparing AFM tips and samples, and on procedures for detecting and localizing single molecular recognition events.

3' utr seed matches, but not overall identity, are associated with rnai off-targets

Abstract: Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3' untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

Channelrhodopsin-2 and optical control of excitable cells

Abstract: Electrically excitable cells are important in the normal functioning and in the pathophysiology of many biological processes. These cells are typically embedded in dense, heterogeneous tissues, rendering them difficult to target selectively with conventional electrical stimulation methods. The algal protein Channelrhodopsin-2 offers a new and promising solution by permitting minimally invasive, genetically targeted and temporally precise photostimulation. Here we explore technological issues relevant to the temporal precision, spatial targeting and physiological implementation of ChR2, in the context of other photostimulation approaches to optical control of excitable cells.

Feeder-independent culture of human embryonic stem cells.

Abstract: We recently reported the development of TeSR1, a serum-free, animal product–free medium that supports the derivation and long-term feeder-independent culture of human embryonic stem cells1. Although the derivation of new human embryonic stem cell lines in those defined conditions offered an important proof of principle, the costs of some of the defined components in that culture system made it impractical for everyday research use. Here we describe modifications to the medium (mTeSR1) that include the use of animal-sourced proteins (bovine serum albumin (BSA) and Matrigel) and cloned zebrafish basic fibroblast growth factor (zbFGF). We include a simple protocol that allows purification of up to 100 mg zbFGF in less than three days (Fig. 1), an amount sufficient to make 1,000 l of mTeSR1 medium. The modifications presented here make mTeSR1 practical for routine research use, and the protocols presented are those currently used in our laboratory for standard human embryonic stem cell culture. *Note: In the version of this Protocol initially published, the references were numbered incorrectly. The error has been corrected in the HTML and PDF versions of the article.

A comprehensive library of fluorescent transcriptional reporters for Escherichia coli

Abstract: E. coli is widely used for systems biology research; there exists a need, however, for tools that can be used to accurately and comprehensively measure expression dynamics in individual living cells. To address this we present a library of transcriptional fusions of gfp to each of about 2,000 different promoters in E. coli K12, covering the great majority of the promoters in the organism. Each promoter fusion is expressed from a low-copy plasmid. We demonstrate that this library can be used to obtain highly accurate dynamic measurements of promoter activity on a genomic scale, in a glucose-lactose diauxic shift experiment. The library allowed detection of about 80 previously uncharacterized transcription units in E. coli, including putative internal promoters within previously known operons, such as the lac operon. This library can serve as a tool for accurate, high-resolution analysis of transcription networks in living E. coli cells.

Nonblinking and long-lasting single-molecule fluorescence imaging

Abstract: Photobleaching and blinking of fluorophores pose fundamental limitations on the information content of single-molecule fluorescence measurements. Photoinduced blinking of Cy5 has hampered many previous investigations using this popular fluorophore. Here we show that Trolox in combination with the enzymatic oxygen-scavenging system eliminates Cy5 blinking, dramatically reduces photobleaching and improves the signal linearity at high excitation rates, significantly extending the applicability of single-molecule fluorescence techniques.

Targeting and tracing antigens in live cells with fluorescent nanobodies

Abstract: We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes

Abstract: MicroRNAs (miRNAs) are 20-23 nucleotide (nt) RNA molecules that regulate gene expression post-transcriptionally. A key step toward understanding the function of the hundreds of miRNAs identified in animals is to determine their expression during development. Here we performed a detailed analysis of conditions for in situ detection of miRNAs in the zebrafish embryo using locked nucleic acid (LNA)-modified DNA probes and report expression patterns for 15 miRNAs in the mouse embryo.

Fluorescence cross-correlation spectroscopy in living cells.

Abstract: Cell biologists strive to characterize molecular interactions directly in the intracellular environment. The intrinsic resolution of optical microscopy, however, allows visualization of only coarse subcellular localization. By extracting information from molecular dynamics, fluorescence cross-correlation spectroscopy (FCCS) grants access to processes on a molecular scale, such as diffusion, binding, enzymatic reactions and codiffusion, and has become a valuable tool for studies in living cells. Here we review basic principles of FCCS and focus on seminal applications, including examples of intracellular signaling and trafficking. We consider FCCS in the context of fluorescence resonance energy transfer and multicolor imaging techniques and discuss application strategies and recent technical advances.

A guide through present computational approaches for the identification of mammalian microRNA targets.

Abstract: Computational microRNA (miRNA) target prediction is a field in flux. Here we present a guide through five widely used mammalian target prediction programs. We include an analysis of the performance of these individual programs and of various combinations of these programs. For this analysis we compiled several benchmark data sets of experimentally supported miRNA–target gene interactions. Based on the results, we provide a discussion on the status of target prediction and also suggest a stepwise approach toward predicting and selecting miRNA targets for experimental testing.

Probing the role of multicellular organization in three-dimensional microenvironments

Abstract: Successful application of living cells in regenerative medicine requires an understanding of how tissue structure relates to organ function. There is growing evidence that presentation of extracellular cues in a three-dimensional (3D) context can fundamentally alter cellular responses. Thus, microenvironment studies that previously were limited to adherent two-dimensional (2D) cultures may not be appropriate for many cell types. Here we present a method for the rapid formation of reproducible, high-resolution 3D cellular structures within a photopolymerizable hydrogel using dielectrophoretic forces. We demonstrate the parallel formation of >20,000 cell clusters of precise size and shape within a thin 2-cm(2) hydrogel and the maintenance of high cell viability and differentiated cell markers over 2 weeks. By modulating cell-cell interactions in 3D clusters, we present the first evidence that microscale tissue organization regulates bovine articular chondrocyte biosynthesis. This platform permits investigation of tissue architecture in other multicellular processes, from embryogenesis to regeneration to tumorigenesis.

Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.

Abstract: Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.

BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions.

Abstract: The most important biotechnological advances made in the 20th century involved methods that converted single DNA molecules into populations of identical DNA molecules. The first wave of techniques used cells (cloning)1 and the second used PCR2. Cloning was advantageous because the populations arising from individual molecules were inherently separated. In contrast, each template required individual compartments (tubes) for PCR-based methods if separate products were desired. Emulsion PCR overcame this disadvantage by miniaturizing the compartments so that millions of templates could be individually amplified within a single tube3. BEAMing (beads, emulsions, amplification and magnetics) is a process built on emulsion PCR that (i) includes beads within the compartments and (ii) ensures that one strand of the PCR product is bound to the beads4. After amplification, each compartment contains a bead that is coated with thousands of copies of the single DNA molecule originally present. These beads can be recovered with a magnet or by centrifugation. Beads obtained via BEAMing accurately reflect the DNA diversity present in template populations and this method can be used to determine what fraction of a DNA population contains a specific mutation5. Because each bead contains thousands of molecules of the identical sequence, the signal to noise ratio obtained by hybridization or enzymatic assays is extremely high. Millions of beads can be analyzed within minutes using flow cytometry or optical scanning instruments. The DNA bound to beads also provides excellent templates for high-throughput sequencing. In this protocol we describe detailed methods for BEAMing, including a new technique for simultaneously generating 192 emulsions suitable for BEAMing.

Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy

Abstract: Lipid bodies have an important role in energy storage and lipid regulation. Here we show that lipid bodies are a major source of contrast in third-harmonic generation (THG) microscopy of cells and tissues. In hepatocytes, micrometer-sized lipid bodies produce a THG signal 1-2 orders of magnitude larger than other structures, which allows one to image them with high specificity. THG microscopy with approximately 1,200 nm excitation can be used to follow the distribution of lipid bodies in a variety of unstained samples including insect embryos, plant seeds and intact mammalian tissue (liver, lung). We found that epi-THG imaging is possible in weakly absorbing tissues because bulk scattering redirects a substantial fraction of the forward-generated harmonic light toward the objective. Finally, we show that the combination of THG microscopy with two-photon and second-harmonic imaging provides a new tool for exploring the interactions between lipid bodies, extracellular matrix and fluorescent compounds (vitamin A, NADH and others) in tissues.

Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET)

Abstract: Bioluminescence resonance energy transfer (BRET) is a straightforward biophysical technique for studying protein-protein interactions. It requires: (1) that proteins of interest and suitable controls be labeled with either a donor or acceptor molecule, (2) placement of these labeled proteins in the desired environment for assessing their potential interaction, and (3) use of suitable detection instrumentation to monitor resultant energy transfer. There are now several possible applications, combinations of donor and acceptor molecules, potential assay environments and detection system perturbations. Therefore, this review aims to demystify and clarify the important aspects of the BRET methodology that should be considered when using this technique.

Amplification of complex gene libraries by emulsion PCR

Abstract: 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK. 2Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. 3Institut de Science et d’Ingénierie Supramoléculaires (ISIS), 8 allée Gaspard Monge, BP 70028, 67083 Strasbourg Cedex, France. 4Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, Givat Ram, 91904, Jerusalem, Israel. Correspondence should be addressed to A.D.G. (griffiths@isis.u-strasbg.fr) or D.S.T. (tawfik@weizmann.ac.il).

Minimizing the risk of reporting false positives in large-scale RNAi screens

Abstract: Large-scale RNA interference (RNAi)-based analyses, very much as other ‘omic’ approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.

CAGE: cap analysis of gene expression

Abstract: 1Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), Yokohama Institute 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan. 2 Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa Wako, Saitama, 351-0198, Japan. 3 K.K. Dnaform, Tsukuba Branch, 3-1 Chuo 8-chome, Ami Machi, Inashiki Gun, Ibaraki, 300-0332, Japan. 4Present address: Vaxine Pty Ltd., Department of Diabetes and Endocrinology, Flinders Medical Centre, Bedford Park, Southern Australia 5042, Australia. Correspondence should be addressed to Y.H. (rgscerg@gsc.riken.jp), P.C. (rgscerg@gsc.riken.jp) or M.H. (matthias. harbers@dnaforminc.com).

A highly sensitive protein-protein interaction assay based on Gaussia luciferase

Abstract: Protein-fragment complementation assays (PCAs) provide a general strategy to study the dynamics of protein-protein interactions in vivo and in vitro. The full potential of PCA requires assays that are fully reversible and sensitive at subendogenous protein expression levels. We describe a new assay that meets these criteria, based on the Gaussia princeps luciferase enzyme, demonstrating chemical reversal, and induction and inhibition of a key interaction linking insulin and TGFβ signaling.

High-throughput RNAi screening by time-lapse imaging of live human cells.

Abstract: RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.

Whole-genome genotyping with the single-base extension assay

Abstract: We describe an efficient, accurate and robust whole-genome genotyping (WGG) assay based on a two-color, single-base extension (SBE), single-nucleotide polymorphism (SNP)-scoring step. We report genotyping results for biallelic International HapMap quality control (QC) SNPs using a single probe per locus. We show scalability, throughput and accuracy of the system by resequencing homozygous loci from our 100k Human-1 Genotyping BeadChip.

An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells

Abstract: Tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry. Although TAP was instrumental in elucidating the yeast cellular machinery, in mammalian cells the method suffers from a low overall yield. We designed several dual-affinity tags optimized for use in mammalian cells and compared the efficiency of each tag to the conventional TAP tag. A tag based on protein G and the streptavidin-binding peptide (GS-TAP) resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. This allows purification of protein complexes that were hitherto not amenable to TAP and use of less starting material, leading to higher success rates and enabling systematic interaction proteomics projects. Using the well-characterized Ku70-Ku80 protein complex as an example, we identified both core elements as well as new candidate effectors.

Genome-scale loss-of-function screening with a lentiviral RNAi library.

Abstract: The discovery that RNA interference (RNAi) is functional in mammalian cells led us to form The RNAi Consortium (TRC) with the goal of enabling large-scale loss-of-function screens through the development of genome-scale RNAi libraries and methodologies for their use. These resources form the basis of a loss-of-function screening platform created at the Broad Institute. Our human and mouse libraries currently contain >135,000 lentiviral clones targeting 27,000 genes. Initial screening efforts have demonstrated that these libraries and methods are practical and powerful tools for high-throughput lentivirus RNAi screens.

A versatile tool for conditional gene expression and knockdown

Abstract: Drug-inducible systems allowing the control of gene expression in mammalian cells are invaluable tools for genetic research, and could also fulfill essential roles in gene- and cell-based therapy. Currently available systems, however, often have limited in vivo functionality because of leakiness, insufficient levels of induction, lack of tissue specificity or prohibitively complicated designs. Here we describe a lentiviral vector-based, conditional gene expression system for drug-controllable expression of polymerase (Pol) II promoter-driven transgenes or Pol III promoter-controlled sequences encoding small inhibitory hairpin RNAs (shRNAs). This system has great robustness and versatility, governing tightly controlled gene expression in cell lines, in embryonic or hematopoietic stem cells, in human tumors xenotransplanted into nude mice, in the brain of rats injected intraparenchymally with the vector, and in transgenic mice generated by infection of fertilized oocytes. These results open up promising perspectives for basic or translational research and for the development of gene-based therapeutics.

Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology

Abstract: For more than a decade the 'neurosphere assay' has been used to define and measure neural stem cell (NSC) behavior, with similar assays now used in other organ systems and in cancer. We asked whether neurospheres are clonal structures whose diameter, number and composition accurately reflect the proliferation, self-renewal and multipotency of a single founding NSC. Using time-lapse video microscopy, coculture experiments with genetically labeled cells, and analysis of the volume of spheres, we observed that neurospheres are highly motile structures prone to fuse even under ostensibly 'clonal' culture conditions. Chimeric neurospheres were prevalent independent of ages, species and neural structures. Thus, the intrinsic dynamic of neurospheres, as conventionally assayed, introduces confounders. More accurate conditions (for example, plating a single cell per miniwell) will be crucial for assessing clonality, number and fate of stem cells. These cautions probably have implications for the use of 'cytospheres' as an assay in other organ systems and with other cell types, both normal and neoplastic.

Protein complex expression by using multigene baculoviral vectors.

Abstract: Elucidation of the molecular basis of protein-interaction networks, in particular in higher eukaryotes, is hampered by insufficient quantities of endogenous multiprotein complexes. Present recombinant expression methods often require considerable investment in both labor and materials before multiprotein expression, and after expression and biochemical analysis these methods do not provide flexibility for expressing an altered multiprotein complex. To meet these demands, we have recently introduced MultiBac, a modular baculovirus-based system specifically designed for eukaryotic multiprotein expression 1 . Here we describe new transfer vectors and a combination of DNA recombination–based methods, which further facilitate the generation of multigene cassettes for protein coexpression (Fig. 1), thus providing a flexible platform for generation of protein expression vectors and their rapid regeneration for revised expression studies. Genes encoding components of a multiprotein complex are inserted into a suite of compatible transfer vectors by homologous recombination. These progenitor constructs are then rapidly joined in the desired combination by Cre-loxP–mediated in vitro plasmid fusion. Protocols for integration of the resulting multigene expression cassettes into the MultiBac baculoviral genome are provided that rely on Tn7 transposition and/or Cre-loxP reaction carried out in vivo in Escherichia coli cells tailored for this purpose. Detailed guidelines for multigene virus generation and amplification, cell culture maintenance and protein production are provided, together with data illustrating the simplicity and remarkable robustness of the present method for multiprotein expression using a composite MultiBac baculoviral vector.

A monovalent streptavidin with a single femtomolar biotin binding site

Abstract: Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 104-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.