Latest Articles Include:
- Method of the Year 2010
- Nat Meth 8(1):1 (2011)
Nature Methods | Editorial Method of the Year 2010 Journal name:Nature MethodsVolume: 8,Page:1Year published:(2011)DOI:doi:10.1038/nmeth.f.321Published online20 December 2010 With the capacity to control cellular behaviors using light and genetically encoded light-sensitive proteins, optogenetics has opened new doors for experimentation across biological fields. View full text Additional data - The Author file: Erik Jorgensen
- Nat Meth 8(1):3 (2011)
Nature Methods | This Month The author file: Erik Jorgensen * Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:3Year published:(2011)DOI:doi:10.1038/nmeth0111-3Published online29 December 2010 Fluorescent proteins can be located in electron micrographs. View full text Additional data - Negative space
- Nat Meth 8(1):5 (2011)
Nature Methods | This Month Negative space * Bang Wong1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:5Year published:(2011)DOI:doi:10.1038/nmeth0111-5Published online29 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Negative space, also known as whitespace, refers to the unmarked areas of the page. Collectively, it is the margins and the gaps between text blocks and images. Whitespace is as much a part of a composition as the titles, words and pictures. The Swiss typographer Jan Tschichold calls whitespace 'the lungs of a good design'1. In addition to giving elements breathing room, judicious use of whitespace can dramatically improve the visual appeal and effectiveness of figures, posters and slides. The term whitespace stems from the printing practice in which white paper is generally used. Margins and gaps that separate blocks of text make it easier to access written material because they provide a visual structure. Well-planned negative space balances the positive (nonwhite) space and is key to aesthetic. Asian art makes wide use of negative space to create harmony and to add dimension to flat silkscreen prints. View full text Figures at a glance * Figure 1: Empty space defines the shape of an object. (,) Ribbon diagram of a protein () and with the negative space masked in black (). * Figure 2: Whitespace can be used to structure content. () An example of a scientific poster. () A space study reveals that contents in sections 1–6 are scattered and whitespace is fragmented. () An example of consolidated whitespace organizing contents. Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Bang Wong is the creative director of the Broad Institute of the Massachusetts Institute of Technology and Harvard and an adjunct assistant professor in the Department of Art as Applied to Medicine at The Johns Hopkins University School of Medicine. Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Preassembled zinc-finger arrays for rapid construction of ZFNs
- Nat Meth 8(1):7 (2011)
Nature Methods | Correspondence Preassembled zinc-finger arrays for rapid construction of ZFNs * Seokjoong Kim1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Mi Jung Lee2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hyojin Kim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mijin Kang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jin-Soo Kim1 Contact Jin-Soo Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Page:7Year published:(2011)DOI:doi:10.1038/nmeth0111-7aPublished online29 December 2010 To the Editor: Since the publication of our Correspondence1 and the reply of Joung et al.2, we improved zinc-finger nuclease (ZFN) modular assembly. ZFNs are artificial restriction enzymes3 composed of tailor-made zinc-finger DNA-binding arrays and the FokI nuclease domain, which can induce site-specific mutations4 and large chromosomal deletions5 in higher eukaryotic cells and organisms. View full text Author information * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Primary authors * These authors contributed equally to this work. * Seokjoong Kim & * Mi Jung Lee Affiliations * Department of Chemistry, Seoul National University, Seoul, South Korea. * Seokjoong Kim, * Hyojin Kim & * Jin-Soo Kim * ToolGen, Inc., Biotechnology Incubating Center, Seoul National University, Seoul, South Korea. * Mi Jung Lee & * Mijin Kang Competing financial interests M.J.L. and M.K. are employees of ToolGen, Inc. J.-S.K. holds stock in ToolGen, Inc. Corresponding author Correspondence to: * Jin-Soo Kim Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (268K) Supplementary Figures 1–2, Supplementary Tables 1–2, Supplementary Methods Additional data - Live-cell dSTORM with SNAP-tag fusion proteins
- Nat Meth 8(1):7-9 (2011)
Nature Methods | Correspondence Live-cell dSTORM with SNAP-tag fusion proteins * Teresa Klein1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Löschberger1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sven Proppert1 Search for this author in: * NPG journals * PubMed * Google Scholar * Steve Wolter1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sebastian van de Linde1 Search for this author in: * NPG journals * PubMed * Google Scholar * Markus Sauer1 Contact Markus Sauer Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:7–9Year published:(2011)DOI:doi:10.1038/nmeth0111-7bPublished online29 December 2010 To the Editor: In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores1. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores2, 3. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione3. View full text Subject terms: * Biophysics Author information * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Department of Biotechnology and Biophysics, Julius Maximilians University Wuerzburg, Wuerzburg, Germany. * Teresa Klein, * Anna Löschberger, * Sven Proppert, * Steve Wolter, * Sebastian van de Linde & * Markus Sauer Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Markus Sauer Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (652K) Supplementary Figures 1–4, Supplementary Methods Additional data - Programming molecular instruments
- Nat Meth 8(1):11 (2011)
Nature Methods | Research Highlights Programming molecular instruments * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:11Year published:(2011)DOI:doi:10.1038/nmeth0111-11Published online29 December 2010 Small conditional RNAs prove their mettle in multiplexed mRNA imaging and show promise as potential cancer therapeutics. View full text Subject terms: * Small RNAs Additional data - The birth of a ribosome
- Nat Meth 8(1):12-13 (2011)
Nature Methods | Research Highlights The birth of a ribosome * Allison Doerr Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:12–13Year published:(2011)DOI:doi:10.1038/nmeth0111-12aPublished online29 December 2010 A team of researchers applied a 'discovery single-particle profiling' experimental strategy to visualize the assembly of the ribosome via time-resolved electron microscopy. View full text Subject terms: * Imaging Additional data - A model brain
- Nat Meth 8(1):12-13 (2011)
Nature Methods | Research Highlights A model brain * Natalie de Souza Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:12–13Year published:(2011)DOI:doi:10.1038/nmeth0111-12bPublished online29 December 2010 A predictive model of intercellular metabolic interactions in the human brain is reported. View full text Subject terms: * Systems Biology Additional data - News in brief
- Nat Meth 8(1):13 (2011)
Nature Methods | Research Highlights News in brief Journal name:Nature MethodsVolume: 8,Page:13Year published:(2011)DOI:doi:10.1038/nmeth0111-13Published online29 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Video-rate stimulated Raman scattering Stimulated Raman scattering (SRS) microscopy is a label-free optical imaging technique based on the detection of signature molecular bond vibrations. Saar et al. now report technical developments that greatly increase the SRS imaging speed, allowing video-rate SRS microscopy. These advances facilitated SRS imaging of the skin and of small molecule drug penetration into the skin of living mice and humans, highlighting the potential utility of SRS for clinical imaging. Saar, B.G.et al. Science330, 1368–1370 (2010). View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - The dyes that came in from the cold
- Nat Meth 8(1):14 (2011)
Nature Methods | Research Highlights The dyes that came in from the cold * Michael Eisenstein Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:14Year published:(2011)DOI:doi:10.1038/nmeth0111-14Published online29 December 2010 Three groups achieve room-temperature, single-molecule detection of nonfluorescent, photon-absorbing compounds. View full text Subject terms: * Single molecule Additional data - Unraveling synapse diversity
- Nat Meth 8(1):16 (2011)
Nature Methods | Research Highlights Unraveling synapse diversity * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:16Year published:(2011)DOI:doi:10.1038/nmeth0111-16Published online29 December 2010 Array tomography opens the door to the large-scale exploration of molecular diversity of individual brain synapses. View full text Subject terms: * Neuroscience Additional data - RNA-based reprogramming
- Nat Meth 8(1):18 (2011)
Nature Methods | Research Highlights RNA-based reprogramming * Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:18Year published:(2011)DOI:doi:10.1038/nmeth0111-18Published online29 December 2010 RNA molecules can both induce pluripotency and direct differentiation. View full text Subject terms: * Stem Cells Additional data - Light tools
- Nat Meth 8(1):19-22 (2011)
Nature Methods | News Feature Light tools * Monya Baker1 Contact Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:19–22Year published:(2011)DOI:doi:10.1038/nmeth.f.322Published online20 December 2010 Optogenetics grows from an idea into a discipline. Monya Baker reports. View full text Additional data Affiliations * Monya Baker is technology editor for Nature and Nature Methods Corresponding author Correspondence to: * Monya Baker - Optogenetics: controlling cell function with light
- Nat Meth 8(1):24-25 (2011)
Nature Methods | Primer Optogenetics: controlling cell function with light * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:24–25Year published:(2011)DOI:doi:10.1038/nmeth.f.323Published online20 December 2010 A brief description of the basic steps required to control cellular function with optogenetics is presented. View full text Additional data - Optogenetics
- Nat Meth 8(1):26-29 (2011)
Nature Methods | Commentary Optogenetics * Karl Deisseroth1 Contact Karl Deisseroth Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:26–29Year published:(2011)DOI:doi:10.1038/nmeth.f.324Published online20 December 2010 Optogenetics is a technology that allows targeted, fast control of precisely defined events in biological systems as complex as freely moving mammals. By delivering optical control at the speed (millisecond-scale) and with the precision (cell type–specific) required for biological processing, optogenetic approaches have opened new landscapes for the study of biology, both in health and disease. View full text Figures at a glance * Figure 1: Graphical illustration of 'optogenetics' emerging in the scientific literature. Demonstration of single-component optogenetic control of neurons with microbial opsins4 was followed by corresponding optogenetic terminology2 in October 2006, and corresponding optogenetic control of freely moving mammals using microbial opsins and the fiberoptic neural interface9, 10. Also marked are identifications of bacteriorhodopsin3, halorhodopsin5 and channelrhodopsin6, all of which were much later (2005–2010) shown to function as fast, single-component optogenetic tools in neurons. Numbers indicate only publications searchable by 'optogenetics' or derivatives thereof on 1 December 2010. * Figure 2: Principle of optogenetics in neuroscience. Targeted excitation (as with a blue light–activated channelrhodopsin) or inhibition (as with a yellow light–activated halorhodopsin), conferring cellular specificity and even projection specificity not feasible with electrodes while maintaining high temporal (action-potential scale) precision. Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Karl Deisseroth is at the Howard Hughes Medical Institute, Department of Bioengineering and Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Karl Deisseroth Additional data - From cudgel to scalpel: toward precise neural control with optogenetics
- Nat Meth 8(1):30-34 (2011)
Nature Methods | Commentary From cudgel to scalpel: toward precise neural control with optogenetics * Simon Peron1 Search for this author in: * NPG journals * PubMed * Google Scholar * Karel Svoboda1 Contact Karel Svoboda Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:30–34Year published:(2011)DOI:doi:10.1038/nmeth.f.325Published online20 December 2010 Optogenetics is routinely used to activate and inactivate genetically defined neuronal populations in vivo. A second optogenetic revolution will occur when spatially distributed and sparse neural assemblies can be precisely manipulated in behaving animals. View full text Figures at a glance * Figure 1: Manipulating neural assemblies with light. () Mapping neural assemblies, for example, using calcium imaging and two-photon laser scanning microscopy, in populations of neurons. () Silencing neurons based on their response type. () Activating neurons to elicit specific activity patterns. Stippled border indicates expression of both excitatory (cyan) and inhibitory (yellow) transducers. * Figure 2: Methods for two-photon photostimulation with ChR2. () Photostimulation with a stationary diffraction limited excitation volume in a two-photon microscope produces subthreshold excitation. () Photostimulation with a diffraction-limited excitation volume scanned rapidly over the cell of interest, here with a spiral pattern. () Photostimulation with extended excitation volumes, defined by temporal focusing () or temporal focusing combined with generalized phase contrast (), to excite one () or multiple () neurons. Transducer expression is indicated with a cyan border; cells attaining desired response are indicated in yellow. * Figure 3: Effects of dense packing of neural element on the precision of photostimulation. () Stimulation of the targeted cell (target) and undesired stimulation (undesired response). () Volume electron microscopy reconstruction showing 600 axons in a 9.1 μm × 9.0 μm × 4.1 μm volume of cortical tissue (hippocampal CA1 stratum radiatum; reconstruction provided by D.B. Chklovskii; see ref. 18). * Figure 4: Hypothetical scheme to manipulate distributed, sparse assemblies. () Neurons that are active during a well-defined behavioral epoch are selected for expression by illuminating with diffuse light. () Neurons expressing the optogenetic transducer. () Activation of the transducer in specific assemblies using diffuse light. Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Simon Peron and Karel Svoboda are at the Howard Hughes Medical Institute Janelia Farm Research Campus, Ashburn, Virginia, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Karel Svoboda Additional data - The promise of optogenetics in cell biology: interrogating molecular circuits in space and time
- Nat Meth 8(1):35-38 (2011)
Nature Methods | Commentary The promise of optogenetics in cell biology: interrogating molecular circuits in space and time * Jared E Toettcher1 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher A Voigt2 Search for this author in: * NPG journals * PubMed * Google Scholar * Orion D Weiner3 Search for this author in: * NPG journals * PubMed * Google Scholar * Wendell A Lim4 Contact Wendell A Lim Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:35–38Year published:(2011)DOI:doi:10.1038/nmeth.f.326Published online20 December 2010 Optogenetic modules offer cell biologists unprecedented new ways to poke and prod cells. The combination of these precision perturbative tools with observational tools, such as fluorescent proteins, may dramatically accelerate our ability to understand the inner workings of the cell. View full text Figures at a glance * Figure 1: Modes of light-regulated biochemistry. () Protein activity can be put directly under light control by fusion to light-responsive domains or residues (green). Upon stimulation with light (gold arrow), allosteric inhibition is removed, leading to activation. (,) Protein activity can be indirectly controlled using light-dependent anchoring to a subcellular compartment () or scaffolding (). * Figure 2: In vivo biochemistry: from component lists to signal processing. () Cell regulatory networks are comprised of cascades of interacting proteins as well as feedback and feed-forward loops. Typically, they are stimulated by extracellular ligands or pharmacological agents and observed by following protein levels or pathway activity (such as a transcriptional response). () Classical chemical and genetic perturbations block or enhance individual nodes to identify phenotype changes. (,) Light-gated inputs can be used to specifically and precisely perturb activation at distinct nodes in a pathway (inputs 1–4, labeled I1 to I4) (), using a rich set of temporal inputs including fixed levels of activation and frequency-modulated signals (). * Figure 3: Reversibility and spatial precision. () Optogenetics allows the investigator to apply spatially restricted light inputs (orange). However, diffusion of protein activity (red) from the site of activation destroys the applied spatial pattern. () Faithful spatial patterns can be maintained by coupling local activation with global inactivation (green), either by implementing an inactivating light wavelength or a short-lived active state. Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Jared E. Toettcher is at the Department of Cellular and Molecular Pharmacology and the Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, USA. * Christopher A. Voigt is at the Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USA. * Orion D. Weiner is at the Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, USA. * Wendell A. Lim is at the Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Wendell A Lim Additional data - Channelrhodopsin engineering and exploration of new optogenetic tools
- Nat Meth 8(1):39-42 (2011)
Nature Methods | Commentary Channelrhodopsin engineering and exploration of new optogenetic tools * Peter Hegemann1 Contact Peter Hegemann Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Möglich1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:39–42Year published:(2011)DOI:doi:10.1038/nmeth.f.327Published online20 December 2010 Rhodopsins from microalgae and eubacteria are powerful tools for manipulating the function of neurons and other cells, but these tools still have limitations. We discuss engineering approaches that can help advance optogenetics. View full text Figures at a glance * Figure 1: A simplified kinetic model of channelrhodopsin function. The model includes two closed states; one prevails after dark adaptation (DA state), whereas the other (LA state) is only occupied after several hundred milliseconds in the light. Light absorption and subsequent isomerization of the retinal chromophore (red) converts both dark states into open conducting states, O1 and O2. * Figure 2: Structural model of a channelrhodopsin. () Three-dimensional computer model of ChR2. Mutations of the amino acid residues shown in stick representation are known to substantially influence absorption, conductance (without selectivity change), kinetics and ion selectivity, as indicated for each residue. The retinal moiety is shown in yellow, residues conserved in all four known channelrhodopsins are colored blue, and residues that differ in various channelrhodopsins are colored gray. Oxygen, nitrogen and sulfur atoms are colored red, blue and dark yellow, respectively. Graphics are based on the coordinates of H. salinarum bacteriorhodopsin28 and were drawn with Pymol (Schrödinger). () Structures of the two possible chromophore dark state isomers: the all-trans, 15-anti form and the 13-cis, 15-syn form. Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Peter Hegemann and Andreas Möglich are at Humboldt Universität zu Berlin, Department of Biology, Biophysics, Berlin, Germany. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Peter Hegemann Additional data - Zinc-finger nucleases
- Nat Meth 8(1):43 (2011)
Nature Methods | Methods to Watch Zinc-finger nucleases * Natalie de Souza Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:43Year published:(2011)DOI:doi:10.1038/nmeth.f.328Published online20 December 2010 Genome-engineering tools with improved design and efficiency will become widely used. View full text Additional data - Targeted proteomics
- Nat Meth 8(1):43 (2011)
Nature Methods | Methods to Watch Targeted proteomics * Allison Doerr Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:43Year published:(2011)DOI:doi:10.1038/nmeth.f.329Published online20 December 2010 Targeted analysis of proteins on a broad scale with mass spectrometry is becoming a reality. View full text Additional data - Torrents of sequence
- Nat Meth 8(1):44 (2011)
Nature Methods | Methods to Watch Torrents of sequence * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:44Year published:(2011)DOI:doi:10.1038/nmeth.f.330Published online20 December 2010 In 2011, we will see the arrival of new and improved sequencing technologies. View full text Additional data - Seamless delivery
- Nat Meth 8(1):44 (2011)
Nature Methods | Methods to Watch Seamless delivery * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:44Year published:(2011)DOI:doi:10.1038/nmeth.f.331Published online20 December 2010 The payoffs for efficient cargo delivery into living cells make the development of better methods worthwhile. View full text Additional data - Single-molecule structure determination
- Nat Meth 8(1):45 (2011)
Nature Methods | Methods to Watch Single-molecule structure determination * Allison Doerr Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:45Year published:(2011)DOI:doi:10.1038/nmeth.f.332Published online20 December 2010 X-ray free-electron lasers may enable single-molecule structure determination. View full text Additional data - Adaptive optics for biological imaging
- Nat Meth 8(1):45 (2011)
Nature Methods | Methods to Watch Adaptive optics for biological imaging * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:45Year published:(2011)DOI:doi:10.1038/nmeth.f.333Published online20 December 2010 The use of adaptive optics to correct light distortions promises to greatly improve the imaging quality of thick biological tissues. View full text Additional data - Networking to understand disease
- Nat Meth 8(1):46 (2011)
Nature Methods | Methods to Watch Networking to understand disease * Natalie de Souza Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:46Year published:(2011)DOI:doi:10.1038/nmeth.f.334Published online20 December 2010 The application of systems approaches to human disease will continue to expand. View full text Additional data - Fast 3D super-resolution fluorescence microscopy
- Nat Meth 8(1):46 (2011)
Nature Methods | Methods to Watch Fast 3D super-resolution fluorescence microscopy * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Page:46Year published:(2011)DOI:doi:10.1038/nmeth.f.335Published online20 December 2010 High-speed fluorescence imaging in all three dimensions at nanometer resolution will resolve, in finer detail, the workings of the living cell. View full text Additional data - Screening: the age of fishes
- Nat Meth 8(1):47-51 (2011)
Nature Methods | Technology Feature Screening: the age of fishes * Monya Baker1 Contact Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:47–51Year published:(2011)DOI:doi:10.1038/nmeth0111-47Published online29 December 2010 Advances in microfluidics and imaging, combined with some high-profile studies, are increasing interest in whole-organism screening. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Monya Baker is technology editor for Nature and Nature Methods Corresponding author Correspondence to: * Monya Baker Additional data - Zinc-finger nucleases transition to the CoDA
- Nat Meth 8(1):53-55 (2011)
Nature Methods | News and Views Zinc-finger nucleases transition to the CoDA * David J Segal1 Contact David J Segal Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:53–55Year published:(2011)DOI:doi:10.1038/nmeth0111-53Published online29 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Potent and easily produced custom zinc-finger nucleases will be game-changers for the field, at a time when the field itself is changing. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * David J. Segal is at the Genome Center and Department of Biochemistry and Molecular Medicine, University of California–Davis, Davis, California, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * David J Segal Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - At last, functional glycomics
- Nat Meth 8(1):55-57 (2011)
Nature Methods | News and Views At last, functional glycomics * Joseph Zaia1 Contact Joseph Zaia Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:55–57Year published:(2011)DOI:doi:10.1038/nmeth0111-55Published online29 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Microarrays made from naturally expressed glycolipids help winnow function from heterogeneous glycan structures. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Joseph Zaia is at Boston University, Boston, Massachusetts, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Joseph Zaia Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Seeing is believing
- Nat Meth 8(1):57-58 (2011)
Nature Methods | News and Views Seeing is believing * Jahar Bhattacharya1 Contact Jahar Bhattacharya Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:57–58Year published:(2011)DOI:doi:10.1038/nmeth0111-57Published online29 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Stabilization with a suction window permits in vivo imaging of the mouse lung vasculature with video-rate two-photon microscopy. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Jahar Bhattacharya is at the Lung Biology Lab, Department of Medicine, Columbia University Medical Center, New York, New York, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Jahar Bhattacharya Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Assemblies: the good, the bad, the ugly
- Nat Meth 8(1):59-60 (2011)
Nature Methods | Commentary Assemblies: the good, the bad, the ugly * Ewan Birney1 Contact Ewan Birney Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 8,Pages:59–60Year published:(2011)DOI:doi:10.1038/nmeth0111-59Published online29 December 2010 The low cost of short-read sequencing has motivated the development of de novo assemblies from only short-read data; impressively, assemblies for large mammalian genomes are now available. However, this is still a developing field, and these de novo assemblies have many artifacts, as do all de novo assemblies. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Ewan Birney is at the European Molecular Biology Laboratory–European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, UK. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Ewan Birney Additional data - Limitations of next-generation genome sequence assembly
- Nat Meth 8(1):61-65 (2011)
Nature Methods | Perspective Limitations of next-generation genome sequence assembly * Can Alkan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Saba Sajjadian1 Search for this author in: * NPG journals * PubMed * Google Scholar * Evan E Eichler1 Contact Evan E Eichler Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:61–65Year published:(2011)DOI:doi:10.1038/nmeth.1527Published online21 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg High-throughput sequencing technologies promise to transform the fields of genetics and comparative biology by delivering tens of thousands of genomes in the near future. Although it is feasible to construct de novo genome assemblies in a few months, there has been relatively little attention to what is lost by sole application of short sequence reads. We compared the recent de novo assemblies using the short oligonucleotide analysis package (SOAP), generated from the genomes of a Han Chinese individual and a Yoruban individual, to experimentally validated genomic features. We found that de novo assemblies were 16.2% shorter than the reference genome and that 420.2 megabase pairs of common repeats and 99.1% of validated duplicated sequences were missing from the genome. Consequently, over 2,377 coding exons were completely missing. We conclude that high-quality sequencing approaches must be considered in conjunction with high-throughput sequencing for comparative genomics an! alyses and studies of genome evolution. View full text Subject terms: * Genetics and genomics Author information * Abstract * Author information * Supplementary information Affiliations * Department of Genome Sciences, University of Washington School of Medicine and Howard Hughes Medical Institute, Seattle, Washington, USA. * Can Alkan, * Saba Sajjadian & * Evan E Eichler Contributions C.A. and E.E.E. conceived the study and wrote the manuscript. C.A. and S.S. analyzed the data. Competing financial interests E.E.E. is a scientific advisory board member of Pacific Biosciences. Corresponding author Correspondence to: * Evan E Eichler Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Table 1 (408K) Contamination found in reported human new sequence insertions from the genomes of two individuals. * Supplementary Table 3 (5M) Analysis of nonredundant autosomal genes in the YH genome assembly. * Supplementary Table 5 (2M) Assigned positions of duplicated sequences (YH) to the NCBI build 36 assembly. Text files * Supplementary Table 4 (12M) Analysis of nonredundant autosomal coding exons in the YH genome. NOTE: This is a tab-delimited text file with 171,751 rows of data. Confirm that all data will load into your application before proceeding. PDF files * Supplementary Text and Figures (404K) Supplementary Figures 1–2, Supplementary Table 2, Supplementary Note Additional data - Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)
- Nat Meth 8(1):67-69 (2011)
Nature Methods | Brief Communication Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA) * Jeffry D Sander1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth J Dahlborg1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mathew J Goodwin1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lindsay Cade4 Search for this author in: * NPG journals * PubMed * Google Scholar * Feng Zhang5 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Cifuentes6 Search for this author in: * NPG journals * PubMed * Google Scholar * Shaun J Curtin7 Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica S Blackburn1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Stacey Thibodeau-Beganny1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Yiping Qi5 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J Pierick5 Search for this author in: * NPG journals * PubMed * Google Scholar * Ellen Hoffman6 Search for this author in: * NPG journals * PubMed * Google Scholar * Morgan L Maeder1, 2, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Cyd Khayter1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Deepak Reyon9 Search for this author in: * NPG journals * PubMed * Google Scholar * Drena Dobbs9 Search for this author in: * NPG journals * PubMed * Google Scholar * David M Langenau1, 3, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert M Stupar7 Search for this author in: * NPG journals * PubMed * Google Scholar * Antonio J Giraldez6 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel F Voytas5 Search for this author in: * NPG journals * PubMed * Google Scholar * Randall T Peterson4, 10, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Jing-Ruey J Yeh4, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * J Keith Joung1, 2, 3, 8 Contact J Keith Joung Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:67–69Year published:(2011)DOI:doi:10.1038/nmeth.1542Received17 June 2010Accepted16 November 2010Published online12 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations. View full text Subject terms: * Molecular biology Author information * Author information * Supplementary information Affiliations * Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. * Jeffry D Sander, * Elizabeth J Dahlborg, * Mathew J Goodwin, * Jessica S Blackburn, * Stacey Thibodeau-Beganny, * Morgan L Maeder, * Cyd Khayter, * David M Langenau & * J Keith Joung * Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA. * Jeffry D Sander, * Elizabeth J Dahlborg, * Mathew J Goodwin, * Stacey Thibodeau-Beganny, * Morgan L Maeder, * Cyd Khayter & * J Keith Joung * Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA. * Jeffry D Sander, * Jessica S Blackburn, * David M Langenau & * J Keith Joung * Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, USA. * Lindsay Cade, * Randall T Peterson & * Jing-Ruey J Yeh * Department of Genetics, Cell Biology and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA. * Feng Zhang, * Yiping Qi, * Christopher J Pierick & * Daniel F Voytas * Genetics Department, Yale University School of Medicine, New Haven, Connecticut, USA. * Daniel Cifuentes, * Ellen Hoffman & * Antonio J Giraldez * Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota, USA. * Shaun J Curtin & * Robert M Stupar * Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA. * Morgan L Maeder, * David M Langenau & * J Keith Joung * Department of Genetics, Development and Cell Biology and Bioinformatics and Computational Biology Program, Iowa State University, Ames, Iowa, USA. * Deepak Reyon & * Drena Dobbs * Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. * Randall T Peterson & * Jing-Ruey J Yeh * Broad Institute, Cambridge, Massachusetts, USA. * Randall T Peterson Contributions J.D.S. and J.K.J. conceived of the CoDA engineering method; J.D.S., S.J.C., D.M.L., R.M.S., A.J.G., D.F.V., R.T.P., J.-R.J.Y. and J.K.J. designed research; J.D.S., E.J.D., M.J.G., L.C., F.Z., D.C., S.J.C., J.S.B., S.T.-B., Y.Q., C.J.P., E.H., M.L.M. and C.K. performed experiments; J.D.S., D.R. and D.D. identified potential genomic CoDA target sites; and J.D.S., R.M.S., D.F.V., R.T.P., J.-R.J.Y. and J.K.J. wrote the paper. Competing financial interests F.Z. and D.F.V. are paid for work performed for Cellectis Plant Sciences. Corresponding author Correspondence to: * J Keith Joung Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–5, Supplementary Tables 1–5 and Supplementary Discussion Additional data - SAINT: probabilistic scoring of affinity purification–mass spectrometry data
- Nat Meth 8(1):70-73 (2011)
Nature Methods | Brief Communication SAINT: probabilistic scoring of affinity purification–mass spectrometry data * Hyungwon Choi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Brett Larsen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhen-Yuan Lin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ashton Breitkreutz2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dattatreya Mellacheruvu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Damian Fermin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhaohui S Qin3, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Mike Tyers2, 4, 5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Anne-Claude Gingras2, 4 Contact Anne-Claude Gingras Search for this author in: * NPG journals * PubMed * Google Scholar * Alexey I Nesvizhskii1, 7 Contact Alexey I Nesvizhskii Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:70–73Year published:(2011)DOI:doi:10.1038/nmeth.1541Received28 June 2010Accepted09 November 2010Published online05 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We present 'significance analysis of interactome' (SAINT), a computational tool that assigns confidence scores to protein-protein interaction data generated using affinity purification–mass spectrometry (AP-MS). The method uses label-free quantitative data and constructs separate distributions for true and false interactions to derive the probability of a bona fide protein-protein interaction. We show that SAINT is applicable to data of different scales and protein connectivity and allows transparent analysis of AP-MS data. View full text Figures at a glance * Figure 1: Probability model in SAINT. (,) Interaction data in the presence () and absence () of control purifications. Schematic of the experimental AP-MS procedure is shown at the top and a spectral count interaction table is illustrated at the bottom. Ctrl, control; rep, replicate; freq, frequency. () Modeling spectral count distributions for true and false interactions. For the interaction between prey i and bait j, SAINT uses all relevant data for the two proteins, as shown in the column of the bait (green) and the data in the row of the prey (orange) in and . () Probability is calculated for each replicate by application of Bayes rule, and a summary probability is calculated for the interaction pair (i,j). * Figure 2: Analysis of TIP49 and DUB datasets. () Benchmarking of filtered interactions in the TIP49 dataset by the overlap with interactions previously reported in BioGRID and iRefWeb databases. () Co-annotation of interaction partners to common GO terms in 'biological processes' in the TIP49 dataset. () Benchmarking against BioGRID and iRefWeb in the DUB dataset. () Co-annotation to GO terms in the DUB dataset. Author information * Author information * Supplementary information Affiliations * Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA. * Hyungwon Choi, * Dattatreya Mellacheruvu, * Damian Fermin & * Alexey I Nesvizhskii * Centre for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada. * Brett Larsen, * Zhen-Yuan Lin, * Ashton Breitkreutz, * Mike Tyers & * Anne-Claude Gingras * Department of Biostatistics and Center for Statistical Genetics, University of Michigan, Ann Arbor, Michigan, USA. * Zhaohui S Qin * Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. * Mike Tyers & * Anne-Claude Gingras * Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK. * Mike Tyers * Centre for Systems Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK. * Mike Tyers * Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA. * Alexey I Nesvizhskii * Present address: Department of Biostatistics and Bioinformatics, Emory University, Atlanta, Georgia, USA. * Zhaohui S Qin Contributions H.C. and A.I.N. developed, implemented and tested the SAINT method; H.C. wrote the software; B.L., A.B., Z.-Y.L., A.-C.G. and M.T. generated data for the initial SAINT modeling and provided feedback on the model performance; D.M. and D.F. assisted with data analysis and processing; Z.S.Q. contributed to statistical model development; H.C., A.-C.G. and A.I.N. wrote the manuscript; A.I.N. and A.-C.G. conceived the study; A.I.N. directed the project with input from A.-C.G. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Anne-Claude Gingras or * Alexey I Nesvizhskii Supplementary information * Author information * Supplementary information Excel files * Supplementary Table 1 (1008K) Data for the TIP49 dataset. () List of all detected interactions and scores from PP-NSAF, CompPASS and SAINT. () All interactions in control purifications were included in a separate table after merging of 35 technical replicate purifications into 9 purifications. () Table of technical replicates of control purifications. () GO terms enrichment in top scoring interactions for each scoring method. * Supplementary Table 2 (3M) Data for the DUB dataset. () List of all detected interactions and scores from CompPASS and SAINT. (–) GO terms enrichment in top scoring interactions for each scoring method. * Supplementary Table 3 (100K) Data for the CDC23 dataset. List of all detected interactions with SAINT scores and results reported by t-test. Zip files * Supplementary Software (2M) PDF files * Supplementary Text and Figures (240K) Supplementary Figure 1 Additional data - Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures
- Nat Meth 8(1):74-79 (2011)
Nature Methods | Article Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures * Yannick Doyon1 Contact Yannick Doyon Search for this author in: * NPG journals * PubMed * Google Scholar * Thuy D Vo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew C Mendel1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shon G Greenberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jianbin Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Danny F Xia1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey C Miller1 Search for this author in: * NPG journals * PubMed * Google Scholar * Fyodor D Urnov1 Search for this author in: * NPG journals * PubMed * Google Scholar * Philip D Gregory1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael C Holmes1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:74–79Year published:(2011)DOI:doi:10.1038/nmeth.1539Received04 June 2010Accepted05 November 2010Published online05 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Zinc-finger nucleases (ZFNs) drive efficient genome editing by introducing a double-strand break into the targeted gene. Cleavage is induced when two custom-designed ZFNs heterodimerize upon binding DNA to form a catalytically active nuclease complex. The importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization. Here we report the development and application of a yeast-based selection system designed to functionally interrogate the ZFN dimer interface. We identified critical residues involved in dimerization through the isolation of cold-sensitive nuclease domains. We used these residues to engineer ZFNs that have superior cleavage activity while suppressing homodimerization. The improvements were portable to orthogonal domains, allowing the concomitant and independent cleavage of two loci using two different ZFN pairs. These ZFN architectures provide a general means! for obtaining highly efficient and specific genome modification. View full text Subject terms: * Molecular biology * Genetics and genomics Figures at a glance * Figure 1: Isolation of FokI cold-sensitive mutants. () Schematic of two independent single-strand annealing (SSA) reporter constructs containing a homodimer site for the CCR5-L ZFN that were integrated in the yeast genome. The PHO5 SSA reporter contains the positive selection cassette natMX conferring dominant resistance to nourseothricin. The MEL1 SSA reporter contains the URA3 gene for negative selection using 5-fluoroorotic acid (5-FOA), in addition to the kanMX cassette conferring dominant resistance to geneticin (G418). A DNA double-stranded break induced by a functional homodimeric ZFN will result in reconstitution of the reporter genes and elimination of both positive and negative selection markers. PHO, 5′ fragment of PHO5; HO5, 3′ fragment of PHO5; MEL, 5′ fragment of MEL1; and EL1, 3′ fragment of MEL1. () Schematic depicting random mutagenesis of the FokI nuclease domain and assembly of the library of mutants. CYC1t, transcription terminator of the CYC1 gene. S. cerevisiae HIS3 gene was included for selectio! n of transformants in yeast. () Relative activity after induction of the reporter strain transformed with the indicated mutant vectors at three different temperatures compared to the wild type. In the leftmost lane (−), the reporter strain was transformed with an expression vector lacking a zinc-finger protein domain. () ZFN expression was monitored by western blot with an antibody to Flag (anti-Flag). Blots with antibody to histone H3 (anti-H3) were used as controls for loading. * Figure 2: Isolated mutations cluster to the FokI dimer interface. () Location of the mutations in the three-dimensional structure of the wild-type FokI dimer19 interface amino acids 470–540. Mutated residues are shown in gold with oxygen atoms in red and nitrogen atoms in blue, and numbers refer to the mutations described in Supplementary Table 1. () Alternative representation of the dimer interface corresponding to an approximate 100° turn around the x axis. () Molecular modeling of the putative salt-bridge interaction between Arg537 and Asp496 in the obligate heterodimeric interface. * Figure 3: Enhanced activity of the ELD:KKK and ELD:KKR architecture. () Autoradiograms showing results of a Cel-1 assay6, 17, 21 to determine the frequency of ZFN-induced indels, conducted on genomic DNA from K562 cells collected 3 and 20 d after transfection with the indicated KDR ZFN constructs (400 ng). () Western blots showing ZFN expression. The NFκB p65 antibody was used as a loading control. () Autoradiograms showing results of a Cel-1 assay to determine the frequency of ZFN-induced indels, conducted on genomic DNA from K562 cells collected 3 d after transfection with the indicated NR3C1 ZFN constructs (80 and 400 ng). () Autoradiograms showing results of a PCR-based assay9 to determine ZFN-driven tag integration frequency, conducted on genomic DNA from K562 cells collected 3 d after transfection with the indicated NR3C1 ZFN constructs (80 and 400 ng). The percentage of BamHI-sensitive DNA resulting from targeted integration (TI) of the donor is indicated below each lane. Arrows denote specific cleavage products. Asterisks indicate a ! nonspecific amplification product present in each lane. WT, wild type. * Figure 4: Improved activity of new ZFN mutants in primary cells. () Autoradiograms showing results of a Cel-1 assay to determine the frequency of ZFN-induced indels, conducted on genomic DNA from PBMCs collected 3 d and 10 d after transfection with the indicated NR3C1 ZFN constructs (100 ng, 200 ng and 400 ng). Arrows denote specific cleavage products. () Mean values (± s.e.m.) of the relative activities of the indicated ZFNs from six independent transfections into PBMCs as determined by Cel-1 assays conducted on genomic DNA collected at day 3 after transfection and using 400 ng of ZFN expression vector. P values were calculated using the two-sample t-test. * Figure 5: Preservation of the obligate heterodimer specificity. () Flow cytometry data for human K562 cells after transfection with the indicated NR3C1 ZFN constructs (80 ng) and stained with antibodies to γ-H2AX. The percentage of positive cells is shown on days 2 and 3 after transfection. Data from two independent transfections are shown. () Autoradiograms showing results of a Cel-1 assay to determine the frequency of ZFN-induced indels at the intended target (NR3C1) and two homodimer off-target sites, found in noncoding regions of RCSD1 and SREBF2 genes, conducted on genomic DNA from K562 cells collected 3 d after transfection. Arrows denote specific cleavage products. *, nonspecific cutting by the Cel-1 nuclease. **, nonspecific amplification product present in each lane. () Western blots showing ZFN expression. The NFκB p65 antibody was used as a loading control. () Autoradiograms showing results of a Cel-1 assay to determine the frequency of ZFN-induced indels, conducted on genomic DNA from K562 cells collected 3 d after transfec! tion with the indicated NR3C1 ZFN constructs (400 ng). Arrows denote specific cleavage products. () ZFN expression monitored as in . Author information * Abstract * Author information * Supplementary information Affiliations * Sangamo BioSciences, Richmond, California, USA. * Yannick Doyon, * Thuy D Vo, * Matthew C Mendel, * Shon G Greenberg, * Jianbin Wang, * Danny F Xia, * Jeffrey C Miller, * Fyodor D Urnov, * Philip D Gregory & * Michael C Holmes Contributions Y.D. and S.G.G. isolated the cold-sensitive mutants. Y.D., T.D.V., M.C.M., J.W. and D.F.X. characterized the engineered domains. Y.D., J.C.M. and M.C.H. designed the experiments. Y.D., F.D.U., P.D.G. and M.C.H. wrote the manuscript. Competing financial interests All authors are employees of Sangamo BioSciences. Corresponding author Correspondence to: * Yannick Doyon Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–15, Supplementary Tables 1–2 and Supplementary Notes 1–2 Additional data - Protein localization in electron micrographs using fluorescence nanoscopy
- Nat Meth 8(1):80-84 (2011)
Nature Methods | Article Protein localization in electron micrographs using fluorescence nanoscopy * Shigeki Watanabe1 Search for this author in: * NPG journals * PubMed * Google Scholar * Annedore Punge2 Search for this author in: * NPG journals * PubMed * Google Scholar * Gunther Hollopeter1 Search for this author in: * NPG journals * PubMed * Google Scholar * Katrin I Willig2 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert John Hobson1 Search for this author in: * NPG journals * PubMed * Google Scholar * M Wayne Davis1 Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan W Hell2 Search for this author in: * NPG journals * PubMed * Google Scholar * Erik M Jorgensen1 Contact Erik M Jorgensen Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:80–84Year published:(2011)DOI:doi:10.1038/nmeth.1537Received03 June 2010Accepted20 October 2010Published online21 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection! protein in electron micrographs. View full text Subject terms: * Cell biology Figures at a glance * Figure 1: Correlative fluorescence and electron microscopy using histone H2B fusion proteins. (–) Confocal image (), STED image () and electron micrograph () from the same thin GMA section (120 nm) from a worm expressing histone H2B–Citrine. () Correlative STED microscopy and electron micrographs showing histone H2B–Citrine (overlay of the images in and ). The images in – show an intestinal cell nucleus. (–) Sum TIRF image (; represents all the photons detected by the camera during the experiment), PALM image () and electron micrograph () from a thin GMA section (70 nm) from a worm expressing histone H2B–tdEos. () Correlative PALM and electron micrographs showing histone H2B–tdEos (overlay of the images in and ). The images in – show a muscle cell nucleus. Scale bars, 3 μm (–) and 1 μm (–). * Figure 2: Correlative fluorescence and electron microscopy using TOM20 fusion proteins. (–) Confocal image (), STED image () and electron micrograph () from the same GMA thin section (120 nm) of a worm expressing TOM20–Citrine. () Correlative STED and electron micrographs showing TOM20–Citrine (overlay of the images in and ). (–) Sum TIRF image (), PALM image () and electron micrograph () from a thin LR White section (70 nm) of a worm expressing TOM20–tdEos. () Correlative PALM and electron micrographs showing TOM20–tdEos (overlay of the images in and ). PALM images of sections from a worm expressing TOM20–tdEos are from tissue embedded in LR White; all other samples were embedded in GMA. Scale bars, 1 μm (–) and 2 μm (–). * Figure 3: Correlative fluorescence and electron microscopy using α-liprin fusion proteins. (–) Confocal image (), STED image () and electron micrograph () of the same thin GMA section (70 nm) from a worm expressing α-liprin–Citrine. () Correlative STED microscopy and electron micrographs showing α-liprin–Citrine (overlay of the images in and ). (–) Sum TIRF image (), PALM image () and electron micrograph () from a thin section (70 nm) of a section of a worm expressing α-liprin–Dendra. Asterisk in marks a region of predominant background signal, which was discarded by emission time threshold. () Correlative PALM and electron micrographs showing α-liprin–Dendra (overlay of the images in and ). SV, synaptic vesicle. Scale bars, 500 nm. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biology and Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah, USA. * Shigeki Watanabe, * Gunther Hollopeter, * Robert John Hobson, * M Wayne Davis & * Erik M Jorgensen * Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. * Annedore Punge, * Katrin I Willig & * Stefan W Hell Contributions S.W. and E.M.J. conceived and designed experiments. G.H., R.J.H. and M.W.D. provided strains and advice. S.W. optimized the methods, prepared the samples and performed PALM imaging. A.P. and K.I.W. performed STED imaging. S.W., S.W.H. and E.M.J. wrote the manuscript. S.W.H. and E.M.J. provided funding. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Erik M Jorgensen Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–5, Supplementary Table 1, Supplementary Notes 1–5 Additional data - Shotgun glycomics: a microarray strategy for functional glycomics
- Nat Meth 8(1):85-90 (2011)
Nature Methods | Article Shotgun glycomics: a microarray strategy for functional glycomics * Xuezheng Song1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yi Lasanajak1 Search for this author in: * NPG journals * PubMed * Google Scholar * Baoyun Xia1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jamie Heimburg-Molinaro1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeanne M Rhea2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hong Ju1 Search for this author in: * NPG journals * PubMed * Google Scholar * Chunmei Zhao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ross J Molinaro2 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard D Cummings1 Contact Richard D Cummings Search for this author in: * NPG journals * PubMed * Google Scholar * David F Smith1 Contact David F Smith Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:85–90Year published:(2011)DOI:doi:10.1038/nmeth.1540Received30 August 2010Accepted10 November 2010Published online05 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells an! d is a strategy for focusing structural analyses on functionally important glycans. View full text Subject terms: * Biochemistry Figures at a glance * Figure 1: Schematic for shotgun glycomics. Glycans are released chemically or enzymatically from glycoproteins, and GSLs are modified directly. The fluorescently labeled products (labeled with 'tag') are separated, quantified and printed to create microarrays available for interrogation with GBPs. * Figure 2: Fluorescent derivatization of GSLs for shotgun glycomics. () The derivatization of GSLs with a bifunctional linker. () C18-HPLC profiles of AOAB derivatization of GM1, GD1a, GT1b and BBG mixture detected by fluorescence. () MALDI-TOF spectra of GM1-AOAB, GD1a-AOAB and GT1b-AOAB purified by HPLC as shown in . The spectra were acquired in the reflective negative mode. ([M-H]− represents negative molecular ions generated by loss of a proton). () The normal-phase HPLC profiles of crude ODA treatment of BBG-PNPA conjugates without precipitation, the precipitate and the filtrate of BBG-PNPA mixture after addition of acetonitrile. * Figure 3: Binding assay on the BBG–GSL-AOAB microarray prepared from two-dimensional HPLC separation. (,) Analysis of 0.1 μg ml−1 CTSB () and anti-GD1a at 1:20 dilution of ascites fluid () on the BBG-GSL-AOAB microarray. Error bars, s.d. (n = 4 replicates). (,) Structural characterization of bound fractions 9 () and 24 () by MS and MS/MS. * Figure 4: Binding of sera from individuals with Lyme disease and control sera on the BBG microarray. () Comparison of IgG binding of sera from individuals with Lyme disease and control sera (tested at 1:100 dilution). The average RFUs were normalized in each serum sample by setting the binding of fraction 33 in control serum and serum from individual with Lyme disease to 100. () Distribution of binding by sera from individual with Lyme disease and control sera over six selected GSL-AOAB fractions (fractions 12, 17, 26, 33, 39 and 40). P values were calculated using Student's t-test, *P < 0.05. () Proposed structural characterization of bound fraction 12 by MS and MS/MS. * Figure 5: The GSL microarray from human erythrocytes and its interrogation with lectins and antibodies. () C18-HPLC profiles of O–blood-type erythrocyte GSL-AOAB. () C18-HPLC profiles of A–blood-type erythrocyte GSL-AOAB. Vertical lines denote each fraction collected in and . (–) The binding of plant lectins Aleuria aurantia lectin (AAL) (1 μg ml−1) (), Ulex eurpoaeus agglutinin 1 (UEA-I) (10 μg ml−1) () and Helix pomatia agglutinin (HPA) (10 μg ml−1) () and antibody to blood group A (10 μg ml−1) (), in which GSL-AOAB fractions 1–23 were from human blood group O erythrocytes, fractions 24–48 were from human blood group A erythrocytes and fractions 49–52 were controls, including the AEAB derivatives of lacto-N-neotetraose (LNnT), lacto-N-fucopentaose III (LNFIII), Lewisy-Lewisx (LeYLeX) and biotin. Inset in is a magnified view for fractions 1–48. Error bars, s.d. (n = 4). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA. * Xuezheng Song, * Yi Lasanajak, * Baoyun Xia, * Jamie Heimburg-Molinaro, * Hong Ju, * Chunmei Zhao, * Richard D Cummings & * David F Smith * Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. * Jeanne M Rhea & * Ross J Molinaro Contributions X.S., R.D.C. and D.F.S. planned the project, and X.S., Y.L., B.X., H.J., C.Z., J.M.R. and R.J.M. carried out the experiments and supplied critical reagents. X.S., Y.L., J.H.-M., R.D.C. and D.F.S. analyzed the data and wrote the manuscript. All authors edited and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Richard D Cummings or * David F Smith Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–6 and Supplementary Tables 1–3 Additional data - Stabilized imaging of immune surveillance in the mouse lung
- Nat Meth 8(1):91-96 (2011)
Nature Methods | Article Stabilized imaging of immune surveillance in the mouse lung * Mark R Looney1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Emily E Thornton2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Debasish Sen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Wayne J Lamm3 Search for this author in: * NPG journals * PubMed * Google Scholar * Robb W Glenny3 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew F Krummel2 Contact Matthew F Krummel Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:91–96Year published:(2011)DOI:doi:10.1038/nmeth.1543Received22 July 2010Accepted18 November 2010Published online12 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Real-time imaging of cellular and subcellular dynamics in vascularized organs requires image resolution and image registration to be simultaneously optimized without perturbing normal physiology. This problem is particularly pronounced in the lung, in which cells may transit at speeds >1 mm s−1 and in which normal respiration results in large-scale tissue movements that prevent image registration. Here we report video-rate, two-photon imaging of a physiologically intact preparation of the mouse lung that is stabilizing and nondisruptive. Using our method, we obtained evidence for differential trapping of T cells and neutrophils in mouse pulmonary capillaries, and observed neutrophil mobilization and dynamic vascular leak in response to stretch and inflammatory models of lung injury in mice. The system permits physiological measurement of motility rates of >1 mm s−1, observation of detailed cellular morphology and could be applied in the future to other organs and tissues! while maintaining intact physiology. View full text Subject terms: * Immunology Figures at a glance * Figure 1: Experimental setup and image stability for intravital imaging of the mouse lung. () Anterior and posterior views of the thoracic suction window fitted with a coverslip. () Side-view rendering of the suction window showing suction chamber, cover slip (green arrows) and vacuum flows (blue arrows near tissue, red arrows toward suction regulator). () Surgical preparation of left thorax with exposed left lung. () Suction window in situ. () Representative images at the indicated depths in a mouse injected with Texas Red–dextran, showing the capillary bed above (left) and below (right) the subpleural alveoli (middle). (,) Still images of CFP fluorescence in an actin-CFP–expressing mouse lung at the indicated times after the start of imaging (color-coded arbitrarily), and a merge of these three images (far right). The plot shows the Pearson's coefficient between time points. Images shown in were captured at 30 fps. In , each frame represents 15 integrated images that are then merged (time points aligned and the Pearson's coefficient from this integration is ! shown). Scale bars, 5 mm (), 10 mm (,) and 50 μm (–). * Figure 2: Perfusion velocities of beads and neutrophils in the lung. () The micrographs show sequential images of individual beads traversing the lung microcirculation (arrowheads), in actin-CFP–expressing mice injected intravenously with red fluorescent microspheres and then imaged at 30 fps. Time elapsed after the first frame is indicated. Scale bar, 50 μm. () Perfusion velocities of individual beads shown as individual dots in small (109 ± 12 μm s−1, mean ± s.e.m. (gray bars), n = 14) and medium-sized blood vessels (280 ± 53 μm s−1, mean ± s.e.m., n = 11; P < 0.001). () The micrograph shows one frame recorded at 30 fps with four representative tracks of neutrophils (colony-stimulating factor 1R (Cfms)-EGFP) and beads inside a vessel of an actin-CFP–expressing mouse injected with fluorescent beads and imaged at 30 fps. Scale bar, 10 μm. () The plots show the average and instantaneous track speeds of neutrophils in small (0.91 ± 0.16 μm s−1, mean ± s.e.m., n = 5) and medium-sized (96.5 ± 37.8 μm s−1, mean ± s.e.m., ! n = 5, *P < 0.05 and **P < 0.001) blood vessels. () Histogram of neutrophil perfusion velocities in a medium-sized blood vessel. Solid line highlights cells that are crawling along the vessel at slow speeds, and dotted line highlights cells with velocities most consistent with flow within vessels. * Figure 3: Perfusion velocities of T cells in the lung. () Average track speeds of naive (2.48 ± 0.49 μm s−1, mean ± s.e.m., n = 4) and activated T cells (0.41 ± 0.07 μm s−1, mean ± s.e.m., n = 4, *P < 0.01) injected into the jugular vein of actin-CFP–expressing mice. () Representative images showing the morphology of naive T cells (CD2 RFP) and T cell blasts (ubiquitin-GFP). Arrows indicate a T cell blast with two leading edges, likely extending into two vascular branches. Scale bar, 10 μm. () Width of the capillary segments containing naive (5.61 ± 0.39 μm, mean ± s.e.m., n = 8) and activated T cells (7.75 ± 0.41 μm, mean ± s.e.m., n = 12, *P < 0.01) are plotted. () Maximal intensity projections of single time points showing the sizes of intravascular naive (left) and activated (right) T cells. Scale bar, 50 μm. Images are from 40 μm z-dimension stack. () Average diameters of naive (7.74 ± 0.23 μm, mean ± s.e.m., n = 12) and activated T cells (11.36 ± 0.40 μm, mean ± s.e.m., n = 12, *P < 0.05, **P < 0! .001 and P < 0.0001). * Figure 4: Imaging inflammation and injury-induced neutrophil dynamics in physiologically intact lungs. () Images of the lung of a LysM-GFP mouse injected with Texas Red–dextran and imaged before (left) and after (right) intratracheal instillation of MIP-2. Scale bar, 50 μm, 40 μm z stack. () Number of GFP+ neutrophils in the imaging field before (16.75 ± 3.06 cells, mean ± s.e.m., n = 4) and after MIP-2 (44.25 ± 4.42 cells, mean ± s.e.m., n = 4, *P < 0.01) intratracheal (IT) instillation. () Number of GFP+ neutrophils in the lung vasculature under continuous suction (n = 4 for each time point). () Representative images of an intravascular GFP+ neutrophil. Scale bar, 10 μm, single z plane at 5:20, 6:40 and 9:20 min:s. () Representative images of a GFP+ neutrophil moving within alveoli. Scale bar, 10 μm, single z plane at 0:40, 5:40 and 9:00 min:s. () Images of the lung of an actin-CFP/c-fms-GFP mouse before and after intratracheal instillation of LPS. Scale bar, 50 μm, 40 μm z stack. () Number of neutrophils per field before (2.75 ± 0.48, mean ± s.e.m., n = 4) an! d after LPS instillation (10.25 ± 1.32, mean ± s.e.m., n = 4, *P < 0.01). () Images of the lung of an actin-CFP mouse injected with Texas Red–dextran and either challenged with intratracheal LPS for 50 min () or subjected to ventilator-induced lung injury for 60 min (). Scale bar, 50 μm, 40 μm z stack. () The plots show the average intensity of fluorescent dextran in the alveolar space at the indicated times after LPS treatment () or ventilator-induced lung injury (). Blue lines are the pretreatment average (n = 3 alveoli), red lines are individual alveoli measured after treatment and black lines are the after treatment average (n = 5 alveoli). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Mark R Looney & * Emily E Thornton Affiliations * Departments of Medicine and Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA. * Mark R Looney * Department of Pathology, University of California, San Francisco, San Francisco, California, USA. * Emily E Thornton, * Debasish Sen & * Matthew F Krummel * Department of Medicine, University of Washington, Seattle, Washington, USA. * Wayne J Lamm & * Robb W Glenny Contributions M.R.L. conceived and designed the experiment, validated and implemented the technique, collected and analyzed data and wrote the manuscript. E.E.T. conceived and designed the experiment, validated and implemented the technique, collected and analyzed data and wrote the manuscript. D.S. implemented the technique and collected and analyzed data. W.J.L. conceived and designed the experiment and validated the technique. R.W.G. conceived and designed the experiment and edited the manuscript. M.F.K. conceived and designed the experiment, provided administrative and financial support and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Matthew F Krummel Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (2M) Ventilation with thoracic suction. Real-time brightfield video of expanding and contracting alveoli during ventilation in the microscope setup at 100A magnification. * Supplementary Movie 2 (5M) Video-rate intravital lung imaging. Video-rate (30 fps, single z plane) two-photon movie of actin-CFP (blue) mouse with unaveraged inspiration and expiration. * Supplementary Movie 3 (192K) Fifteen-frame-averaged intravital lung imaging. Two-photon video of actin-CFP (blue) mouse with averaged (fifteen frames, single z plane) acquisition showing stability and intravascular cellular movement. * Supplementary Movie 4 (244K) Lung circulation with thoracic suction. Two-photon video of wild-type mouse injected with Texas Red dextran (red) marking vasculature. Unlabeled cellular shadows are observed in motion in the labeled vasculature. * Supplementary Movie 5 (2M) Perfusion velocities in the lung. Two-photon video of a single z plane with 1-μm beads (red) flowing through alveolar capillaries of diameter 10–15 μm. Bead tracks are marked with dragon tails. Vasculature marked with actin-CFP (blue). Time is indicated in s:ms. Bead speeds of ~0–400 μm s-1 with an average of ~110 μm s-1 were observed inside capillaries. * Supplementary Movie 6 (9M) Simultaneous imaging of neutrophil and bead velocities in the lung. Video-rate, two-photon movie of a single z plane with i.v. 1-μm beads (red) flowing through vasculature (30 μm diameter) marked with actin-CFP (blue). Neutrophils (c-fms, green) flow through the vessels. Cell (green) and bead (red) tracks are marked with dragon tails. Bead track speed average is 297.6 μm s-1 with a range of 106.8–728.3 μm s-1. * Supplementary Movie 7 (4M) Naive and T-cell blast migration in the lungs. Left-sided movie from an actin-CFP mouse injected with naive CD2-RFP (red) T cells (5 A 107). Right-sided movie from a wild-type mouse injected with T-cell blasts (ubiquitin-GFP, green, 5 A 107). * Supplementary Movie 8 (2M) Neutrophil recruitment into the lung with intratracheal MIP-2. Two-photon video of LysM-GFP marked neutrophils (green) in dextran marked vasculature (red) at baseline (left) and 70 min after intratracheal MIP-2 treatment (right, 5 μg, i.t., 40 μm z stack). * Supplementary Movie 9 (8M) Intravascular neutrophil migratory activity in the lung. Two-photon video of a LysM-GFP neutrophil (green) crawling through dextran marked vasculature (red) at 60 min after MIP-2 treatment (5 μg, i.t.). The center of mass of the intravascular neutrophil is marked with a grey sphere. Flashing yellow arrowheads indicate the alternating leading edge of the intravascular neutrophil. * Supplementary Movie 10 (928K) Extravascular neutrophil migratory activity in the lung. Two-photon video of LysM-GFP neutrophils (green) in dextran (red) marked vasculature in a mouse 60 min after MIP-2 treatment (5 μg, i.t.). The first pass consists of one z plane to show cellular detail with the cell of interest, marked with a white sphere, deforming itself to move between alveolar spaces. The second pass includes 40 μm in z to provide context. * Supplementary Movie 11 (7M) Neutrophil recruitment into the lung after intratracheal LPS. Two-photon video of c-fms–GFP neutrophils (green) in a lung where all cells are marked by actin-CFP (blue) before (left) and 70 min after LPS treatment (5 mg kg-1, i.t., 40 μm z stack). * Supplementary Movie 12 (532K) Lung vascular leak after intratracheal LPS. Two-photon video of lung tissue marked with actin-CFP (blue) showing dextran leak (red) into the extravascular space 50 min after LPS treatment (5 mg kg-1, i.t., 40 μm z stack). * Supplementary Movie 13 (852K) Ventilator-induced lung injury and lung vascular leak. Two-photon video of lung tissue marked with actin-CFP (blue) showing dextran leak (red) into the extravascular space 50 min after induction with high tidal volume lung injury (ventilator-induced lung injury, 40 μm z stack). PDF files * Supplementary Text and Figures (156K) Supplementary Figure 1 Additional data
No comments:
Post a Comment