Nature Methods

Next-generation high-density self-assembling functional protein arrays.

Publication Date: 2008 May 11 PMID: 18469824
Authors: Ramachandran, N. - Raphael, J. V. - Hainsworth, E. - Demirkan, G. - Fuentes, M. G. - Rolfs, A. - Hu, Y. - Labaer, J.
Journal: Nat Methods

We developed a high-density self-assembling protein microarray, based on the nucleic acid programmable protein array (NAPPA) concept, to display thousands of proteins that are produced and captured in situ from immobilized cDNA templates. We arrayed up to 1,000 unique human cDNAs and obtained high yields of protein expression and capture with minimal variation and good reproducibility. This method will enable various experimental approaches to study protein function in high throughput.

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Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples.

Publication Date: 2008 May 11 PMID: 18469823
Authors: Juette, M. F. - Gould, T. J. - Lessard, M. D. - Mlodzianoski, M. J. - Nagpure, B. S. - Bennett, B. T. - Hess, S. T. - Bewersdorf, J.
Journal: Nat Methods

Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 x 30 x 75nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.

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Real-time imaging of the intracellular glutathione redox potential.

Publication Date: 2008 May 11 PMID: 18469822
Authors: Gutscher, M. - Pauleau, A. L. - Marty, L. - Brach, T. - Wabnitz, G. H. - Samstag, Y. - Meyer, A. J. - Dick, T. P.
Journal: Nat Methods

Dynamic analysis of redox-based processes in living cells is now restricted by the lack of appropriate redox biosensors. Conventional redox-sensitive GFPs (roGFPs) are limited by undefined specificity and slow response to changes in redox potential. In this study we demonstrate that the fusion of human glutaredoxin-1 (Grx1) to roGFP2 facilitates specific real-time equilibration between the sensor protein and the glutathione redox couple. The Grx1-roGFP2 fusion protein allowed dynamic live imaging of the glutathione redox potential (E(GSH)) in different cellular compartments with high sensitivity and temporal resolution. The biosensor detected nanomolar changes in oxidized glutathione (GSSG) against a backdrop of millimolar reduced glutathione (GSH) on a scale of seconds to minutes. It facilitated the observation of redox changes associated with growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation.

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Improving the photostability of bright monomeric orange and red fluorescent proteins.

Publication Date: 2008 May 4 PMID: 18454154
Authors: Shaner, N. C. - Lin, M. Z. - McKeown, M. R. - Steinbach, P. A. - Hazelwood, K. L. - Davidson, M. W. - Tsien, R. Y.
Journal: Nat Methods

All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

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SNP genotyping: six technologies that keyed a revolution.

Publication Date: 2008 May PMID: 18446158
Authors: Perkel, J.
Journal: Nat Methods

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Super-resolution light microscopy goes live.

Publication Date: 2008 May PMID: 18446157
Authors: Gustafsson, M. G.
Journal: Nat Methods

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Metastasis: two assays explore the two roads traveled.

Publication Date: 2008 May PMID: 18446156
Authors: Bielenberg, D. R.
Journal: Nat Methods

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BAC to the future: functional genomics in mammals.

Publication Date: 2008 May PMID: 18446155
Authors: Roguev, A. - Krogan, N. J.
Journal: Nat Methods

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Unexpected failure rates for modular assembly of engineered zinc fingers.

Publication Date: 2008 May PMID: 18446154
Authors: Ramirez, C. L. - Foley, J. E. - Wright, D. A. - Muller-Lerch, F. - Rahman, S. H. - Cornu, T. I. - Winfrey, R. J. - Sander, J. D. - Fu, F. - Townsend, J. A. - Cathomen, T. - Voytas, D. F. - Joung, J. K.
Journal: Nat Methods

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Two-photon dual-color imaging using fluorescent proteins.

Publication Date: 2008 May PMID: 18446153
Authors: Kawano, H. - Kogure, T. - Abe, Y. - Mizuno, H. - Miyawaki, A.
Journal: Nat Methods

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Straightforward ladder sequencing of peptides using a Lys-N metalloendopeptidase.

Publication Date: 2008 May PMID: 18425140
Authors: Taouatas, N. - Drugan, M. M. - Heck, A. J. - Mohammed, S.
Journal: Nat Methods

We introduce a method for sequencing peptides by mass spectrometry using a metalloendopeptidase that cleaves proteins at the amino side of lysine (Lys-N). When analyzed by electron transfer dissociation (ETD)-based mass spectrometric sequencing, Lys-N-digested peptides that contain a single lysine residue produce spectra dominated by c-type fragment ions, providing simple ladders for sequence determination. This method should be a valuable strategy for de novo sequencing and the analysis of post-translational modifications.

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Modeling lymphangiogenesis in a three-dimensional culture system.

Publication Date: 2008 May PMID: 18425139
Authors: Bruyere, F. - Melen-Lamalle, L. - Blacher, S. - Roland, G. - Thiry, M. - Moons, L. - Frankenne, F. - Carmeliet, P. - Alitalo, K. - Libert, C. - Sleeman, J. P. - Foidart, J. M. - Noel, A.
Journal: Nat Methods

A lack of appropriate in vitro models of three-dimensional lymph vessel growth hampers the study of lymphangiogenesis. We developed a lymphatic ring assay--a potent, reproducible and quantifiable three-dimensional culture system for lymphatic endothelial cells that reproduces spreading of endothelial cells from a pre-existing vessel, cell proliferation, migration and differentiation into capillaries. In the assay, mouse thoracic duct fragments are embedded in a collagen gel, leading to the formation of lumen-containing lymphatic capillaries, which we assessed by electron microscopy and immunostaining. We developed a computerized method to quantify the lymphatic network. By applying this model to gene-deficient mice, we found evidence for involvement of the matrix metalloproteinase, MMP-2, in lymphangiogenesis. The lymphatic ring assay bridges the gap between two-dimensional in vitro models and in vivo models of lymphangiogenesis, can be used to exploit the potential of existing transgenic mouse models, and rapidly identify regulators of lymphangiogenesis.

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Monovalent, reduced-size quantum dots for imaging receptors on living cells.

Publication Date: 2008 May PMID: 18425138
Authors: Howarth, M. - Liu, W. - Puthenveetil, S. - Zheng, Y. - Marshall, L. F. - Schmidt, M. M. - Wittrup, K. D. - Bawendi, M. G. - Ting, A. Y.
Journal: Nat Methods

We describe a method to generate monovalent quantum dots (QDs) using agarose gel electrophoresis. We passivated QDs with a carboxy-terminated polyethylene-glycol ligand, yielding particles with half the diameter of commercial QDs, which we conjugated to a single copy of a high-affinity targeting moiety (monovalent streptavidin or antibody to carcinoembryonic antigen) to label cell-surface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.

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Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors.

Publication Date: 2008 May PMID: 18425137
Authors: Ai, H. W. - Hazelwood, K. L. - Davidson, M. W. - Campbell, R. E.
Journal: Nat Methods

Fluorescence resonance energy transfer (FRET) with fluorescent proteins is a powerful method for detection of protein-protein interactions, enzyme activities and small molecules in the intracellular milieu. Aided by a new violet-excitable yellow-fluorescing variant of Aequorea victoria GFP, we developed dual FRET-based caspase-3 biosensors. Owing to their distinct excitation profiles, each FRET biosensor can be ratiometrically imaged in the presence of the other.

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Suspended-drop electroporation for high-throughput delivery of biomolecules into cells.

Publication Date: 2008 May PMID: 18408727
Authors: Guignet, E. G. - Meyer, T.
Journal: Nat Methods

We present a high-throughput method that enables efficient delivery of biomolecules into cells. The device consists of an array of 96 suspended electrode pairs, where small sample volumes are top-loaded, electroporated and bottom-ejected into 96-well plates. We demonstrate the use of this suspended-drop electroporation (SDE) device to effectively introduce fluorescent dextran, small interfering RNA (siRNA) or cDNA into primary neurons, differentiated neutrophils and other cell types with conventionally low transfection rates.

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Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.

Publication Date: 2008 May PMID: 18408726
Authors: Shroff, H. - Galbraith, C. G. - Galbraith, J. A. - Betzig, E.
Journal: Nat Methods

We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to approximately 60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.

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Spheroid-based engineering of a human vasculature in mice.

Publication Date: 2008 May PMID: 18391960
Authors: Alajati, A. - Laib, A. M. - Weber, H. - Boos, A. M. - Bartol, A. - Ikenberg, K. - Korff, T. - Zentgraf, H. - Obodozie, C. - Graeser, R. - Christian, S. - Finkenzeller, G. - Stark, G. B. - Heroult, M. - Augustin, H. G.
Journal: Nat Methods

The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

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BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.

Publication Date: 2008 May PMID: 18391959
Authors: Poser, I. - Sarov, M. - Hutchins, J. R. - Heriche, J. K. - Toyoda, Y. - Pozniakovsky, A. - Weigl, D. - Nitzsche, A. - Hegemann, B. - Bird, A. W. - Pelletier, L. - Kittler, R. - Hua, S. - Naumann, R. - Augsburg, M. - Sykora, M. M. - Hofemeister, H. - Zhang, Y. - Nasmyth, K. - White, K. P. - Dietzel, S. - Mechtler, K. - Durbin, R. - Stewart, A. F. - Peters, J. M. - Buchholz, F. - Hyman, A. A.
Journal: Nat Methods

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.

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Improved genetic manipulation of human embryonic stem cells.

Publication Date: 2008 May PMID: 18391958
Authors: Braam, S. R. - Denning, C. - van den Brink, S. - Kats, P. - Hochstenbach, R. - Passier, R. - Mummery, C. L.
Journal: Nat Methods

Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth requirements to standardized feeder-free culture, and optimized conditions for clonal growth and efficient gene transfer without loss of pluripotency. Stably transfected lines retained differentiation potential, and most lines displayed normal karyotypes.

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A metric for odorant comparison.

Publication Date: 2008 May PMID: 18376403
Authors: Haddad, R. - Khan, R. - Takahashi, Y. K. - Mori, K. - Harel, D. - Sobel, N.
Journal: Nat Methods

In studies of vision and audition, stimuli can be systematically varied by wavelength and frequency, respectively, but there is no equivalent metric for olfaction. Restricted odorant-feature metrics such as number of carbons and functional group do not account for response patterns to odorants varying along other structural dimensions. We generated a multidimensional odor metric, in which each odorant molecule was represented as a vector of 1,664 molecular descriptor values. Revisiting many studies, we found that this metric and a second optimized metric were always better at accounting for neural responses than the specific metric used in each study. These metrics were applicable across studies that differed in the animals studied, the type of olfactory neurons tested, the odorants applied and the recording methods used. We use this new metric to recommend sets of odorants that span the physicochemical space for use in olfaction experiments.

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