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1.  Pitfalls of measuring feeding rate in the fruit fly Drosophila melanogaster 
Nature methods  2008;5(3):214-215.
PMCID: PMC4335172  PMID: 18309307
2.  Single-Cell Genome-Wide Bisulfite Sequencing for Assessing Epigenetic Heterogeneity 
Nature methods  2014;11(8):817-820.
We report a single-cell bisulfite sequencing method (scBS-Seq) capable of accurately measuring DNA methylation at up to 48.4% of CpGs. We observed that ESCs grown in serum or 2i both display epigenetic heterogeneity, with “2i-like” cells present in serum cultures. In silico integration of 12 individual mouse oocyte datasets largely recapitulates the whole DNA methylome, making scBS-Seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.
PMCID: PMC4117646  PMID: 25042786
3.  Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics 
Nature methods  2014;11(8):868-874.
Genome-wide association studies (GWAS) have identified thousands of loci associated wtih complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes involved in the Mendelian disorder long QT syndrome (LQTS). We integrated the LQTS network with GWAS loci from the corresponding common complex trait, QT interval variation, to identify candidate genes that were subsequently confirmed in Xenopus laevis oocytes and zebrafish. We used the LQTS protein network to filter weak GWAS signals by identifying single nucleotide polymorphisms (SNPs) in proximity to genes in the network supported by strong proteomic evidence. Three SNPs passing this filter reached genome-wide significance after replication genotyping. Overall, we present a general strategy to propose candidates in GWAS loci for functional studies and to systematically filter subtle association signals using tissue-specific quantitative interaction proteomics.
PMCID: PMC4117722  PMID: 24952909
4.  All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins 
Nature methods  2014;11(8):825-833.
All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and 2, which show improved brightness and voltage sensitivity, microsecond response times, and produce no photocurrent. We engineered a novel channelrhodopsin actuator, CheRiff, which shows improved light sensitivity and kinetics, and spectral orthogonality to the QuasArs. A co-expression vector, Optopatch, enabled crosstalk-free genetically targeted all-optical electrophysiology. In cultured neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials in dendritic spines, synaptic transmission, sub-cellular microsecond-timescale details of action potential propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In brain slice, Optopatch induced and reported action potentials and subthreshold events, with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without use of conventional electrodes.
PMCID: PMC4117813  PMID: 24952910
5.  Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries 
Nature methods  2014;11(8):834-840.
Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone posttranslational modification (‘PTM’) signatures remains a daunting task in the epigenetics field. Here, we introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semi-synthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how pre-existing PTMs, alone or synergistically, affect further PTM deposition via crosstalk mechanisms. We anticipate that the high-throughput and -sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.
PMCID: PMC4130351  PMID: 24997861
6.  Protein delivery into live cells by incubation with an endosomolytic agent 
Nature methods  2014;11(8):861-867.
We report on how a dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with a particularly high efficiency. By mediating endosomal leakage, dfTAT also delivers proteins into cultured cells after a simple co-incubation procedure. Cytosolic delivery is achieved in most cells in a culture and only a relatively small amount of material remains trapped inside endosomes. Delivery does not require binding interactions between dfTAT and a protein, multiple molecules can be delivered at once, and delivery can be repeated. Remarkably, dfTAT-mediated delivery does not noticeably impact cell viability, proliferation, or gene expression. This new delivery strategy should be extremely useful for cell-based assays, cellular imaging applications, and the ex vivo manipulation of cells.
PMCID: PMC4131206  PMID: 24930129
Cell-penetrating peptide; cytosolic protein delivery; transcription factors
7.  Chemically Defined and Small Molecule-Based Generation of Human Cardiomyocytes 
Nature methods  2014;11(8):855-860.
Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows the elucidation of cardiomyocyte macromolecular and metabolic requirements whilst providing a minimally complex system for the study of maturation and subtype specification.
PMCID: PMC4169698  PMID: 24930130
Human induced pluripotent stem cell; differentiation; cardiomyocyte; heart; chemically defined medium; small molecule
8.  Optimized cell transplantation using adult rag2 mutant zebrafish 
Nature methods  2014;11(8):821-824.
Cell transplantation into adult zebrafish has lagged behind mouse due to the lack of immune compromised models. Here, we have created homozygous rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft zebrafish muscle, blood stem cells, and cancers. rag2E450fs mutant zebrafish are the first immune compromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.
PMCID: PMC4294527  PMID: 25042784
9.  Single molecule–sensitive probes for imaging RNA in live cells 
Nature methods  2009;6(5):347-349.
To visualize native or non-engineered RNA in live cells with single-molecule sensitivity, we developed multiply labeled tetravalent RNA imaging probes (MTRIPs). When delivered with streptolysin O into living human epithelial cancer cells and primary chicken fibroblasts, MTRIPs allowed the accurate imaging of native mRNAs and a non-engineered viral RNA, of RNA co-localization with known RNA-binding proteins, and of RNA dynamics and interactions with stress granules.
PMCID: PMC4297622  PMID: 19349979
10.  Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny 
Nature methods  2013;11(1):106-112.
Although Lgr5+ intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5+ intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5+ cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types.
PMCID: PMC3951815  PMID: 24292484
11.  Single-cell western blotting 
Nature methods  2014;11(7):749-755.
To measure cell-to-cell variation in protein-mediated functions — a hallmark of biological processes — we developed an approach to conduct ~103 concurrent single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30 µm-thick photoactive polyacrylamide gel enables western blotting comprised of: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing. We apply this scWestern to monitor single rat neural stem cell differentiation and responses to mitogen stimulation. The scWestern quantifies target proteins even with off-target antibody binding, multiplexes to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supports analyses of low starting cell numbers (~200) when integrated with fluorescence activated cell sorting. The scWestern thus overcomes limitations in single-cell protein analysis (i.e., antibody fidelity, sensitivity, and starting cell number) and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.
PMCID: PMC4077215  PMID: 24880876
12.  Correlated cryogenic photoactivated localization microscopy and electron cryotomography 
Nature methods  2014;11(7):737-739.
Electron cryotomography (ECT) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated "cryo-PALM"-ECT method for localizing objects within cryotomograms to beyond the diffraction limit of the light microscope, and use it to identify multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.
PMCID: PMC4081473  PMID: 24813625
13.  Simultaneous whole-animal 3D-imaging of neuronal activity using light-field microscopy 
Nature methods  2014;11(7):727-730.
High-speed large-scale 3D imaging of neuronal activity poses a major challenge in neuroscience. Here, we demonstrate intrinsically simultaneous functional imaging of neuronal activity at single neuron resolution for an entire Caenorhabditis elegans as well as for the whole-brain of larval zebrafish. Our technique captures dynamics of spiking neurons in volumes of ~700 μm x 700 μm x 200 μm at 20 Hz and its simplicity makes it an attractive tool for high-speed volumetric calcium imaging.
PMCID: PMC4100252  PMID: 24836920
14.  Bayesian approach to single-cell differential expression analysis 
Nature methods  2014;11(7):740-742.
Single-cell data provides means to dissect the composition of complex tissues and specialized cellular environments. However, the analysis of such measurements is complicated by high levels of technical noise and intrinsic biological variability. We describe a probabilistic model of expression magnitude distortions typical of single-cell RNA sequencing measurements, which enables detection of differential expression signatures and identification of subpopulations of cells in a way that is more tolerant of noise.
PMCID: PMC4112276  PMID: 24836921
15.  Parallel super-resolution imaging 
Nature methods  2013;10(8):709-710.
Massive parallelization of scanning-based super-resolution imaging allows fast imaging of large fields of view.
PMCID: PMC4280568  PMID: 23900251
17.  Deciphering laminar-specific neural inputs with line-scanning fMRI 
Nature methods  2013;11(1):55-58.
Using a line-scanning method during functional magnetic resonance imaging (fMRI) we obtain high temporal (50 ms) and spatial (50 μm) resolution information along the cortical thickness, and show that the laminar position of fMRI onset coincides with distinct neural inputs t in therat somatosensory and motor cortices. This laminar specific fMRI onset allowed the identification of the neural inputs underlying ipsilateral fMRI activation in the barrel cortex due to peripheral denervation-induced plasticity.
PMCID: PMC4276040  PMID: 24240320
18.  Directed evolution approaches to the study of proteins 
Nature methods  2007;4(12):995-997.
PMCID: PMC4255555  PMID: 18049466
19.  Multiscale Representation of Genomic Signals 
Nature methods  2014;11(6):689-694.
Genomic information is encoded on a wide range of distance scales, ranging from tens of base pairs to megabases. We developed a multiscale framework to analyze and visualize the information content of genomic signals. Different types of signals, such as GC content or DNA methylation, are characterized by distinct patterns of signal enrichment or depletion across scales spanning several orders of magnitude. These patterns are associated with a variety of genomic annotations, including genes, nuclear lamina associated domains, and repeat elements. By integrating the information across all scales, as compared to using any single scale, we demonstrate improved prediction of gene expression from Polymerase II chromatin immunoprecipitation sequencing (ChIP-seq) measurements and we observed that gene expression differences in colorectal cancer are not most strongly related to gene body methylation, but rather to methylation patterns that extend beyond the single-gene scale.
PMCID: PMC4040162  PMID: 24727652
20.  High-density single-molecule analysis of cell surface dynamics in C. elegans embryos 
Nature methods  2014;11(6):677-682.
We describe a general, versatile and non-invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in C. elegans embryos. We exploit tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single particle tracking (SPT) analysis. We also introduce and validate a new method called smPReSS that infers exchange rates from quantitative analysis of single molecule photobleaching kinetics, without using SPT. Combining SPT and smPReSS allows spatially and temporally resolved measurements of protein mobility and exchange kinetics. We use these methods (a) to resolve distinct mobility states and spatial variation in exchange rates of the polarity protein Par-6 and (b) to measure spatiotemporal modulation of actin filament assembly and disassembly. The introduction of these methods in a powerful model system offers a promising new avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.
PMCID: PMC4046709  PMID: 24727651
21.  Rapid Adaptive Optical Recovery of Optimal Resolution over LargeVolumes 
Nature methods  2014;11(6):625-628.
Using a de-scanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture and a 14 ms update rate. This permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens, and recover diffraction-limited imaging over large (> 240 μm)3 volumes. We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain.
PMCID: PMC4069208  PMID: 24727653
22.  Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling 
Nature methods  2014;11(6):683-688.
RNA-protein interactions have critical roles in gene regulation. However, high-throughput methods to quantitatively analyze these interactions are lacking. We adapted an Illumina GAIIx sequencer to make several million such measurements with a High-Throughput Sequencing – RNA Affinity Profiling (HiTS-RAP) assay. Millions of cDNAs are sequenced, bound by the E. coli replication terminator protein Tus, and transcribed in situ, whereupon Tus halts transcription leaving RNA stably attached to its template DNA. The binding of fluorescently-labeled protein is then quantified in the sequencer. We used HiTS-RAP to measure the affinity of mutagenized libraries of GFP-binding and NELF-E binding aptamers to their respective targets and thereby identified regions in both aptamers that are critical for their RNA-protein interaction. We show that mutations additively affect binding affinity of the NELF-E binding aptamer, whose interaction depends mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depends primarily on secondary structure.
PMCID: PMC4073888  PMID: 24809628
23.  Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas 
Nature methods  2014;11(6):649-652.
Despite its many favorable properties as a sample support for biological electron microscopy, graphene is not widely used because its hydrophobicity precludes reliable protein deposition. We describe a method to modify graphene using a low-energy hydrogen plasma, which reduces hydrophobicity without degrading the graphene lattice. We show that the use of plasma-treated graphene enables better control of protein distribution in ice for electron cryo-microscopy and improved image quality by reducing radiation-induced sample motion.
PMCID: PMC4141966  PMID: 24747813
24.  Chronic, Wireless Recordings of Large Scale Brain Activity in Freely Moving Rhesus Monkeys 
Nature methods  2014;11(6):670-676.
Advances in techniques for recording large-scale brain activity contribute to both the elucidation of neurophysiological principles and the development of brain-machine interfaces (BMIs). Here we describe a neurophysiological paradigm for performing tethered and wireless large-scale recordings based on movable volumetric three-dimensional (3D) multielectrode implants. This approach allowed us to isolate up to 1,800 units per animal and simultaneously record the extracellular activity of close to 500 cortical neurons, distributed across multiple cortical areas, in freely behaving rhesus monkeys. The method is expandable, in principle, to thousands of simultaneously recorded channels. It also allows increased recording longevity (5 consecutive years), and recording of a broad range of behaviors, e.g. social interactions, and BMI paradigms in freely moving primates. We propose that wireless large-scale recordings could have a profound impact on basic primate neurophysiology research, while providing a framework for the development and testing of clinically relevant neuroprostheses.
PMCID: PMC4161037  PMID: 24776634

Results 1-25 (611)