High-performance probes for light and electron microscopy.

Abstract:

:We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.

journal_name

Nat Methods

journal_title

Nature methods

authors

Viswanathan S,Williams ME,Bloss EB,Stasevich TJ,Speer CM,Nern A,Pfeiffer BD,Hooks BM,Li WP,English BP,Tian T,Henry GL,Macklin JJ,Patel R,Gerfen CR,Zhuang X,Wang Y,Rubin GM,Looger LL

doi

10.1038/nmeth.3365

subject

Has Abstract

pub_date

2015-06-01 00:00:00

pages

568-76

issue

6

eissn

1548-7091

issn

1548-7105

pii

nmeth.3365

journal_volume

12

pub_type

杂志文章
  • Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP.

    abstract::The commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstr...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.1258

    authors: Biteen JS,Thompson MA,Tselentis NK,Bowman GR,Shapiro L,Moerner WE

    更新日期:2008-11-01 00:00:00

  • A streamlined platform for high-content functional proteomics of primary human specimens.

    abstract::Achieving information content of satisfactory breadth and depth remains a formidable challenge for proteomics. This problem is particularly relevant to the study of primary human specimens, such as tumor biopsies, which are heterogeneous and of finite quantity. Here we present a functional proteomics strategy that uni...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth778

    authors: Jessani N,Niessen S,Wei BQ,Nicolau M,Humphrey M,Ji Y,Han W,Noh DY,Yates JR 3rd,Jeffrey SS,Cravatt BF

    更新日期:2005-09-01 00:00:00

  • An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.

    abstract::We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.4396

    authors: Corces MR,Trevino AE,Hamilton EG,Greenside PG,Sinnott-Armstrong NA,Vesuna S,Satpathy AT,Rubin AJ,Montine KS,Wu B,Kathiria A,Cho SW,Mumbach MR,Carter AC,Kasowski M,Orloff LA,Risca VI,Kundaje A,Khavari PA,Montine TJ,

    更新日期:2017-10-01 00:00:00

  • The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells.

    abstract::Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, terme...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.2895

    authors: Petschnigg J,Groisman B,Kotlyar M,Taipale M,Zheng Y,Kurat CF,Sayad A,Sierra JR,Mattiazzi Usaj M,Snider J,Nachman A,Krykbaeva I,Tsao MS,Moffat J,Pawson T,Lindquist S,Jurisica I,Stagljar I

    更新日期:2014-05-01 00:00:00

  • Quantifying domain-ligand affinities and specificities by high-throughput holdup assay.

    abstract::Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.3438

    authors: Vincentelli R,Luck K,Poirson J,Polanowska J,Abdat J,Blémont M,Turchetto J,Iv F,Ricquier K,Straub ML,Forster A,Cassonnet P,Borg JP,Jacob Y,Masson M,Nominé Y,Reboul J,Wolff N,Charbonnier S,Travé G

    更新日期:2015-08-01 00:00:00

  • Multiomics sequencing goes spatial.

    abstract::Microfluidic channels provide a means to deliver barcodes encoding spatial information to a tissue, which allows co-profiling of gene expression and proteins of interest in a spatially resolved manner. ...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/s41592-020-01043-w

    authors: Tang L

    更新日期:2021-01-01 00:00:00

  • Cell-type-specific signaling networks in heterocellular organoids.

    abstract::Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simu...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/s41592-020-0737-8

    authors: Qin X,Sufi J,Vlckova P,Kyriakidou P,Acton SE,Li VSW,Nitz M,Tape CJ

    更新日期:2020-03-01 00:00:00

  • Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes.

    abstract::The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sens...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.2773

    authors: Thestrup T,Litzlbauer J,Bartholomäus I,Mues M,Russo L,Dana H,Kovalchuk Y,Liang Y,Kalamakis G,Laukat Y,Becker S,Witte G,Geiger A,Allen T,Rome LC,Chen TW,Kim DS,Garaschuk O,Griesinger C,Griesbeck O

    更新日期:2014-02-01 00:00:00

  • High-speed volumetric imaging of neuronal activity in freely moving rodents.

    abstract::Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope (MiniLFM) capable of capturing neuronal network activity within a volume of 700 × 600 × 360 µm3 at 16 Hz in the hippocampus of freely moving ...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/s41592-018-0008-0

    authors: Skocek O,Nöbauer T,Weilguny L,Martínez Traub F,Xia CN,Molodtsov MI,Grama A,Yamagata M,Aharoni D,Cox DD,Golshani P,Vaziri A

    更新日期:2018-06-01 00:00:00

  • Current challenges in open-source bioimage informatics.

    abstract::We discuss the advantages and challenges of the open-source strategy in biological image analysis and argue that its full impact will not be realized without better support and recognition of software engineers' contributions to the biological sciences and more support of this development model from funders and instit...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.2082

    authors: Cardona A,Tomancak P

    更新日期:2012-06-28 00:00:00

  • A complete data processing workflow for cryo-ET and subtomogram averaging.

    abstract::Electron cryotomography is currently the only method capable of visualizing cells in three dimensions at nanometer resolutions. While modern instruments produce massive amounts of tomography data containing extremely rich structural information, data processing is very labor intensive and the results are often limited...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/s41592-019-0591-8

    authors: Chen M,Bell JM,Shi X,Sun SY,Wang Z,Ludtke SJ

    更新日期:2019-11-01 00:00:00

  • Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo.

    abstract::Glial cells have been identified as key signaling components in the brain; however, methods to investigate their structure and function in vivo have been lacking. Here, we describe a new, highly selective approach for labeling astrocytes in intact rodent neocortex that allows in vivo imaging using two-photon microscop...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth706

    authors: Nimmerjahn A,Kirchhoff F,Kerr JN,Helmchen F

    更新日期:2004-10-01 00:00:00

  • Metabolic biotinylation of cell surface receptors for in vivo imaging.

    abstract::We have developed a versatile, potent technique for imaging cells in culture and in vivo by expressing a metabolically biotinylated cell-surface receptor and visualizing it with labeled streptavidin moieties. The recombinant reporter protein, which incorporates a biotin acceptor peptide (BAP) between an N-terminal sig...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth875

    authors: Tannous BA,Grimm J,Perry KF,Chen JW,Weissleder R,Breakefield XO

    更新日期:2006-05-01 00:00:00

  • Combining tumor genome simulation with crowdsourcing to benchmark somatic single-nucleotide-variant detection.

    abstract::The detection of somatic mutations from cancer genome sequences is key to understanding the genetic basis of disease progression, patient survival and response to therapy. Benchmarking is needed for tool assessment and improvement but is complicated by a lack of gold standards, by extensive resource requirements and b...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.3407

    authors: Ewing AD,Houlahan KE,Hu Y,Ellrott K,Caloian C,Yamaguchi TN,Bare JC,P'ng C,Waggott D,Sabelnykova VY,ICGC-TCGA DREAM Somatic Mutation Calling Challenge participants.,Kellen MR,Norman TC,Haussler D,Friend SH,Stolovitzky G,Ma

    更新日期:2015-07-01 00:00:00

  • Neutron-encoded mass signatures for multiplexed proteome quantification.

    abstract::We describe a protein quantification method called neutron encoding that exploits the subtle mass differences caused by nuclear binding energy variation in stable isotopes. These mass differences are synthetically encoded into amino acids and incorporated into yeast and mouse proteins via metabolic labeling. Mass spec...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.2378

    authors: Hebert AS,Merrill AE,Bailey DJ,Still AJ,Westphall MS,Strieter ER,Pagliarini DJ,Coon JJ

    更新日期:2013-04-01 00:00:00

  • Doxycycline-dependent photoactivated gene expression in eukaryotic systems.

    abstract::High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activ...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.1340

    authors: Cambridge SB,Geissler D,Calegari F,Anastassiadis K,Hasan MT,Stewart AF,Huttner WB,Hagen V,Bonhoeffer T

    更新日期:2009-07-01 00:00:00

  • The Caenorhabditis elegans Lifespan Machine.

    abstract::The measurement of lifespan pervades aging research. Because lifespan results from complex interactions between genetic, environmental and stochastic factors, it varies widely even among isogenic individuals. The actions of molecular mechanisms on lifespan are therefore visible only through their statistical effects o...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.2475

    authors: Stroustrup N,Ulmschneider BE,Nash ZM,López-Moyado IF,Apfeld J,Fontana W

    更新日期:2013-07-01 00:00:00

  • Quantitative analysis of gene expression in a single cell by qPCR.

    abstract::We developed a quantitative PCR method featuring a reusable single-cell cDNA library immobilized on beads for measuring the expression of multiple genes in a single cell. We used this method to analyze multiple cDNA targets (from several copies to several hundred thousand copies) with an experimental error of 15.9% or...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.1338

    authors: Taniguchi K,Kajiyama T,Kambara H

    更新日期:2009-07-01 00:00:00

  • Tracking transmitter-gated P2X cation channel activation in vitro and in vivo.

    abstract::We present a noninvasive approach to track activation of ATP-gated P2X receptors and potentially other transmitter-gated cation channels that show calcium fluxes. We genetically engineered rat P2X receptors to carry calcium sensors near the channel pore and tested this as a reporter for P2X(2) receptor opening. The me...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth1144

    authors: Richler E,Chaumont S,Shigetomi E,Sagasti A,Khakh BS

    更新日期:2008-01-01 00:00:00

  • A large-scale evaluation of computational protein function prediction.

    abstract::Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report th...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.2340

    authors: Radivojac P,Clark WT,Oron TR,Schnoes AM,Wittkop T,Sokolov A,Graim K,Funk C,Verspoor K,Ben-Hur A,Pandey G,Yunes JM,Talwalkar AS,Repo S,Souza ML,Piovesan D,Casadio R,Wang Z,Cheng J,Fang H,Gough J,Koskinen P,Törö

    更新日期:2013-03-01 00:00:00

  • Targeted chromosome elimination from ES-somatic hybrid cells.

    abstract::To engineer a stem cell genome, we developed a technology for targeted elimination of chromosomes from mouse embryonic stem (ES)-somatic hybrid cells. Here we demonstrate the use of a universal chromosome elimination cassette (CEC) for elimination of a single embryonic stem cell (ESC)-derived chromosome 11 or 12, and ...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth973

    authors: Matsumura H,Tada M,Otsuji T,Yasuchika K,Nakatsuji N,Surani A,Tada T

    更新日期:2007-01-01 00:00:00

  • Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval.

    abstract::Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades res...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/s41592-020-0816-x

    authors: Xu F,Ma D,MacPherson KP,Liu S,Bu Y,Wang Y,Tang Y,Bi C,Kwok T,Chubykin AA,Yin P,Calve S,Landreth GE,Huang F

    更新日期:2020-05-01 00:00:00

  • In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts.

    abstract::Here we integrated multiphoton laser scanning microscopy and the registration of second harmonic generation images of collagen fibers to overcome difficulties in tracking stromal cell-matrix interactions for several days in live mice. We show that the matrix-modifying hormone relaxin increased tumor-associated fibrobl...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.1295

    authors: Perentes JY,McKee TD,Ley CD,Mathiew H,Dawson M,Padera TP,Munn LL,Jain RK,Boucher Y

    更新日期:2009-02-01 00:00:00

  • Molecular engineering: Unnatural design.

    abstract::Researchers designed an enzyme to carry out the Diels-Alder reaction, an activity not found in nature. ...

    journal_title:Nature methods

    pub_type: 评论,杂志文章

    doi:10.1038/nmeth0910-671

    authors: Doerr A

    更新日期:2010-09-01 00:00:00

  • Rapid, optimized interactomic screening.

    abstract::We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screening method that com...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.3395

    authors: Hakhverdyan Z,Domanski M,Hough LE,Oroskar AA,Oroskar AR,Keegan S,Dilworth DJ,Molloy KR,Sherman V,Aitchison JD,Fenyö D,Chait BT,Jensen TH,Rout MP,LaCava J

    更新日期:2015-06-01 00:00:00

  • Improved and expanded Q-system reagents for genetic manipulations.

    abstract::The Q system is a repressible binary expression system for transgenic manipulations in living organisms. Through protein engineering and in vivo functional tests, we report here variants of the Q-system transcriptional activator, including QF2, for driving strong and ubiquitous expression in all Drosophila tissues. Ou...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.3250

    authors: Riabinina O,Luginbuhl D,Marr E,Liu S,Wu MN,Luo L,Potter CJ

    更新日期:2015-03-01 00:00:00

  • 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 o...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth846

    authors: Szulc J,Wiznerowicz M,Sauvain MO,Trono D,Aebischer P

    更新日期:2006-02-01 00:00:00

  • Fluorogenic DNA sequencing in PDMS microreactors.

    abstract::We developed a multiplex sequencing-by-synthesis method combining terminal phosphate-labeled fluorogenic nucleotides (TPLFNs) and resealable polydimethylsiloxane (PDMS) microreactors. In the presence of phosphatase, primer extension by DNA polymerase using nonfluorescent TPLFNs generates fluorophores, which are confin...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.1629

    authors: Sims PA,Greenleaf WJ,Duan H,Xie XS

    更新日期:2011-06-12 00:00:00

  • Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms.

    abstract::We established a conditional site-specific recombination system based on dimerizable Cre recombinase-mediated recombination in the apicomplexan parasite Toxoplasma gondii. Using a new single-vector strategy that allows ligand-dependent, efficient removal of a gene of interest, we generated three knockouts of apicomple...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.2301

    authors: Andenmatten N,Egarter S,Jackson AJ,Jullien N,Herman JP,Meissner M

    更新日期:2013-02-01 00:00:00

  • FaST linear mixed models for genome-wide association studies.

    abstract::We describe factored spectrally transformed linear mixed models (FaST-LMM), an algorithm for genome-wide association studies (GWAS) that scales linearly with cohort size in both run time and memory use. On Wellcome Trust data for 15,000 individuals, FaST-LMM ran an order of magnitude faster than current efficient algo...

    journal_title:Nature methods

    pub_type: 杂志文章

    doi:10.1038/nmeth.1681

    authors: Lippert C,Listgarten J,Liu Y,Kadie CM,Davidson RI,Heckerman D

    更新日期:2011-09-04 00:00:00