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PAR-CLIP analysis uncovers AUF1 impact on target RNA fate and genome integrity
AUF1 is an RNA-binding protein believed to function mostly by regulating the decay of its target transcripts. Here, Yoon et al.systematically identify the targets of AUF1 and provide insights into how AUF1 functions to regulate various cellular processes by enhancing the decay, stability or translation of specific RNAs.
- Je-Hyun Yoon
- , Supriyo De
- & Myriam Gorospe
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A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging
Current calcium-sensitive probes based on red fluorescent proteins are unsuitable for two-photon excitation at the near-infrared wavelengths commonly used for green fluorescent probes. Wu et al. use a structure-guided approach to engineer a red fluorescent probe with optimal two-photon excitation at these wavelengths.
- Jiahui Wu
- , Ahmed S. Abdelfattah
- & Robert E. Campbell
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A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations
Phage-assisted continuous evolution (PACE) has the potential to rapidly evolve drug-resistant mutations. Here, Dickinson et al.present a protease PACE system that identifies clinically relevant mutations conferring resistance to protease inhibitors in only a few days of continuous evolution.
- Bryan C. Dickinson
- , Michael S. Packer
- & David R. Liu
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Implementation of the CRISPR-Cas9 system in fission yeast
The fission yeast, Schizosaccharomyces pombe, is a valuable model organism, but the lack of a portable RNA Pol III promoter has prevented the implementation of the CRISPR/Cas9 system. Here the authors develop a CRISPR/Cas9 system that achieves selection-free specific mutagenesis with very high efficiencies in S. pombe.
- Jake Z. Jacobs
- , Keith M. Ciccaglione
- & Mikel Zaratiegui
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RNAi-based functional selection identifies novel cell migration determinants dependent on PI3K and AKT pathways
Large-scale genetic screens combined with phenotype assays can identify novel regulators of specific cellular functions. Here, Seo et al. use shRNA-based genetic selection to identify novel genes regulating cell migration, functioning within the PI 3-kinase/PTEN/AKT signalling pathway.
- Minchul Seo
- , Shinrye Lee
- & Kyoungho Suk
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Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide
Current genetically encoded sensors for hydrogen peroxide, such as HyPer, emit in the green region giving potential for overlap with other probes. Here, the authors report HyPerRed, a red fluorescent redox probe capable of monitoring intracellular hydrogen peroxide.
- Yulia G. Ermakova
- , Dmitry S. Bilan
- & Vsevolod V. Belousov
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| Open AccessGraphene-based carbon-layered electrode array technology for neural imaging and optogenetic applications
Monitoring neuronal activity in the rodent in vivobrain is commonly done using micro-electrode arrays but these devices are not normally compatible with optical technologies. Here the authors design a transparent and flexible electrode array based on graphene that allows them to combine electrophysiological recordings with optogenetic and imaging experiments.
- Dong-Wook Park
- , Amelia A. Schendel
- & Justin C. Williams
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Transparent and flexible low noise graphene electrodes for simultaneous electrophysiology and neuroimaging
Monitoring neuronal activity of large populations of neurons at high-temporal and spatial resolution is important to understand neurophysiology but requires improved tools and methods. Here the authors develop a transparent and flexible electrode based on graphene that allows them to combine electrophysiological recordings with calcium imaging.
- Duygu Kuzum
- , Hajime Takano
- & Brian Litt
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Magneto-fluorescent core-shell supernanoparticles
Magneto-fluorescent nanoparticles hold promise for bioimaging applications, but synthesizing uniform particles with tunable sizes remains challenging. Chen et al. propose an approach for co-assembling magnetic particles with fluorescent quantum dots, leading to well-defined core-shell structures.
- Ou Chen
- , Lars Riedemann
- & Moungi G. Bawendi
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| Open AccessCombining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice
Next-generation sequencing technology has made the generation of huge amounts of genetic data possible, but phenotype characterization remains slow and difficult. Here the authors develop a high-throughput phenotyping facility for rice that is able to accurately identify and characterize traits related to morphology, biomass and yield.
- Wanneng Yang
- , Zilong Guo
- & Lizhong Xiong
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Visualizing the replication of respiratory syncytial virus in cells and in living mice
Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Here, Rameix-Welti et al. create fluorescent and bioluminescent RSV strains that allow real-time analysis of viral replication and screening of antiviral compounds in cultured cells and in live animals.
- Marie-Anne Rameix-Welti
- , Ronan Le Goffic
- & Jean-François Eléouët
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Assessing technical performance in differential gene expression experiments with external spike-in RNA control ratio mixtures
Differential gene expression experiments yield quantitative insight into biological activity and may be important in disease classification and treatment. Here, the authors analyse external spike-in RNA controls to provide a standard method to assess and compare experiment performance.
- Sarah A. Munro
- , Steven P. Lund
- & Marc Salit
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Phenotypic characterization of missense polymerase-δ mutations using an inducible protein-replacement system
The essential nature of replicative polymerases has hampered the study of polymerase-δ mutations found in colorectal cancer cells. Here, using polymerase-δ mutations as a proof of principle, the authors present an inducible single vector system that replaces any endogenous gene with an RNAi-resistant mutant version.
- Medini Manohar Ghodgaonkar
- , Patrick Kehl
- & Josef Jiricny
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An optimized optogenetic clustering tool for probing protein interaction and function
Protein–protein interactions are fundamental to nearly all molecular and cellular processes. Here Taslimi et al.describe a versatile new optogenetic module that can be used to visualize protein–protein interactions, as well as reversibly control them with light with spatiotemporal resolution.
- Amir Taslimi
- , Justin D. Vrana
- & Chandra L. Tucker
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| Open AccessIn vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans
Single molecule fluorescence microscopy is a powerful technique to study protein dynamics in cells, but it has not been applied to adult animals. The authors use complementation-activated light microscopy in C. elegansto discover that dystrophin regulates the diffusion properties of voltage-dependent calcium ion channels at the surface of body-wall muscle cells.
- Hong Zhan
- , Ramunas Stanciauskas
- & Fabien Pinaud
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Metabolic properties in stroked rats revealed by relaxation-enhanced magnetic resonance spectroscopy at ultrahigh fields
1H magnetic resonance can detect endogenous metabolites at physiological concentrations but its application in animal models is challenged by low sensitivity. Here the authors focus ultrahigh magnetic fields on metabolites whose spectral signatures are selectively excited, and achieve microarchitectural insight of rat brains after a stroke.
- Noam Shemesh
- , Jens T. Rosenberg
- & Lucio Frydman
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Carbon nanotubes as in vivo bacterial probes
The non-invasive detection and imaging of bacterial infection in living organisms is increasingly important. Here, the authors demonstrate the use of labelled carbon nanotubes as bacterial probes in living hosts, and are able to image deep tissue infections with higher signal amplification than dye imaging.
- Neelkanth M. Bardhan
- , Debadyuti Ghosh
- & Angela M. Belcher
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Determination of collagen fibril size via absolute measurements of second-harmonic generation signals
Quantitative determination of collagen fibril size is important for investigating pathological disorders of the extracellular matrix. Here, the authors show that SHG microscopy is capable of measuring fibril diameter after calibration of SHG radiation using correlation to electron microscopy.
- Stéphane Bancelin
- , Carole Aimé
- & Marie-Claire Schanne-Klein
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| Open AccessAn assay to image neuronal microtubule dynamics in mice
Microtubule dynamics in neurons play critical roles in physiology, injury and disease. Here the authors develop a transgenic mouse line expressing a fluorescently tagged version of the microtubule binding protein EB3, and using a range of imaging techniques, study microtubule dynamics under normal and injury conditions in living mice.
- Tatjana Kleele
- , Petar Marinković
- & Thomas Misgeld
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Spatial-temporal model for silencing of the mitotic spindle assembly checkpoint
During cell division, a single chromosome that lacks attachment to microtubules is sufficient to delay chromosome segregation. Chen and Liu construct a model demonstrating that the transport of regulators along microtubules may explain the remarkable sensitivity and robustness of this checkpoint.
- Jing Chen
- & Jian Liu
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Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria
Research on the roles played by gut microbes in human health is hampered by our inability to isolate and study them in pure cultures. Here, the authors describe an approach that allows targeted cultivation and rapid characterization of a significant proportion of human gut bacteria.
- Elizabeth A. Rettedal
- , Heidi Gumpert
- & Morten O.A. Sommer
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| Open AccessScreening of DUB activity and specificity by MALDI-TOF mass spectrometry
Deubiquitylases (DUBs) remove ubiquitin chains from proteins. Here the authors develop a mass spectrometry-based DUB activity screen using unmodified diubiquitin isomers to characterize substrate specificity for 42 human DUBs, and assess the potency and selectivity of 11 DUB inhibitors.
- Maria Stella Ritorto
- , Richard Ewan
- & Matthias Trost
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Functional high-throughput screening identifies the miR-15 microRNA family as cellular restriction factors for Salmonella infection
The functional role of microRNAs in the interplay between bacterial pathogens and host cells is not well defined. Here, using an image-based high-throughput screening approach, the authors identify a family of microRNAs that regulates Salmonellainfection and characterize their mechanism of action.
- Claire Maudet
- , Miguel Mano
- & Ana Eulalio
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| Open AccessLive imaging of calcium spikes during double fertilization in Arabidopsis
Intracellular calcium waves are key signalling elements during the fertilization process of animals, involved in egg activation. Here the authors image calcium oscillations during the fertilization process in flowering plants, revealing specific signatures involved in the success of this process.
- Yuki Hamamura
- , Moe Nishimaki
- & Tetsuya Higashiyama
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| Open AccessIdentification of genetic variants associated with alternative splicing using sQTLseekeR
RNA sequencing has enabled the global analysis of both gene expression levels and splicing events. Here, the authors develop a multivariate approach that is able to identify SNPs that influence splicing, and investigate the overlap of these with functional domains across the genome, including previously identified GWAS signals.
- Jean Monlong
- , Miquel Calvo
- & Roderic Guigó
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Enhancement of biological reactions on cell surfaces via macromolecular crowding
Optimizing cell-surface biological reactions is an important goal of biotechnology and industrial processes. Here the authors use macromolecular crowding to enhance the enzymatic conversion of red blood cells to the universal type O blood type, using orders of magnitude less enzyme than was previously required.
- Rafi Chapanian
- , David H. Kwan
- & Jayachandran N Kizhakkedathu
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| Open AccessUnderstanding functional miRNA–target interactions in vivo by site-specific genome engineering
Identifying miRNA response elements (MREs) within target mRNAs can be done computationally but the functional validation of putative MREs remains challenging. Here, Bassett et al. describe applications of genome engineering to target and assess the functional significance of MREs in different organisms and stages of development.
- Andrew R. Bassett
- , Ghows Azzam
- & Tudor A. Fulga
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| Open AccessQuantitative super-resolution imaging of Bruchpilot distinguishes active zone states
Complex molecular interactions occur in the active zone cytomatrix (CAZ) within the presynaptic terminal to regulate synaptic plasticity. Here, the authors use imaging techniques to show that the CAZ is composed of units containing on average 137 Bruchpilot proteins, many of which are arranged into clusters.
- Nadine Ehmann
- , Sebastian van de Linde
- & Robert J. Kittel
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Bright and fast multicoloured voltage reporters via electrochromic FRET
Genetically encoded voltage sensors are useful tools for the analysis of membrane potential and its influence on cell function. Here, the authors present a range of these sensors with varying colours for rapid and sensitive neuronal voltage imaging.
- Peng Zou
- , Yongxin Zhao
- & Adam E. Cohen
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Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs
Current CRISPR-mediated genome-editing methods are limited by the requirement for a specific +1 nucleotide when using the U6 promoter to drive guide RNA synthesis. Now, Ranganathan et al.report a modification of the CRISPR–Cas9 system that more than doubles the number of targetable CRISPR sites within the human genome.
- Vinod Ranganathan
- , Karl Wahlin
- & Donald J. Zack
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| Open AccessSingle-molecule force spectroscopy reveals force-enhanced binding of calcium ions by gelsolin
The application of force can influence biological processes such as ligand and protein–protein binding, with mechanical stress typically hindering such interactions. Here, the authors use atomic force microscopy to show that the binding of calcium to gelsolin can be improved under stress.
- Chunmei Lv
- , Xiang Gao
- & Wei Wang
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| Open AccessAlarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity
Alarmins are locally released during inflammation and are early amplifiers of inflammation. Here Vogl et al.show that the alarmin S100A8/S100A9 can be used as a sensitive marker to detect subclinical inflammation and follow disease progression in a variety of disease models.
- Thomas Vogl
- , Michel Eisenblätter
- & Johannes Roth
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Conformable amplified lead zirconate titanate sensors with enhanced piezoelectric response for cutaneous pressure monitoring
The development of more sensitive tools for physiological monitoring presents obvious advantages in health-care and diagnostic assessment. Here, the authors present a thin, skin-like sensor that uses enhanced responses in lead zirconate titanate for monitoring arterial pressure waves.
- Canan Dagdeviren
- , Yewang Su
- & John A. Rogers
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| Open AccessAutomated monitoring and quantitative analysis of feeding behaviour in Drosophila
Feeding is an important behaviour, but its quantification remains challenging, particularly in small animal models like Drosophila melanogaster. Here the authors describe a method which uses capacitive sensing for automated high-resolution measuring of feeding behaviour in individual flies.
- Pavel M. Itskov
- , José-Maria Moreira
- & Carlos Ribeiro
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| Open AccessCreation of a gated antibody as a conditionally functional synthetic protein
The ability to control antibody binding could have important medical implications. Here, the authors present a method to engineer phosphatase-controllable antibodies that bind to a specific recognition site in the presence of two biomarker inputs.
- Smita B. Gunnoo
- , Helene M. Finney
- & Benjamin G. Davis
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Poking cells for efficient vector-free intracellular delivery
The incorporation of foreign objects into cells can be used in various avenues of biological research, although crossing the cell membrane can be challenging. Here, the authors use a diamond nanoneedle array for enhanced delivery of various particles into cells, including neurons.
- Ying Wang
- , Yang Yang
- & Peng Shi
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Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites
The development of biosensors with improved sensitivity for the detection of biological molecules presents obvious possibilities for improved diagnostic tools in healthcare. Here, the authors present a technique for the label-free detection and super-resolution imaging of single proteins.
- Marek Piliarik
- & Vahid Sandoghdar
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| Open AccessRetrieving the intracellular topology from multi-scale protein mobility mapping in living cells
Numerous obstacles posed by cellular subcompartments and structures constrain protein transport in the cell. Here, Baum et al.map the intracellular topology from a diffusing protein’s point of view by measuring the diffusive movements of fluorescently labelled reporter proteins in living cells on multiple time and length scales.
- Michael Baum
- , Fabian Erdel
- & Karsten Rippe
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A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells
PTEN is a prominent tumour suppressor and monitoring its conformation and activity are key to developing targeted therapies. Here, the authors develop a bioluminescence resonance energy transfer biosensor for PTEN conformation and identify novel G protein-coupled receptor regulation and therapeutic activation.
- Evelyne Lima-Fernandes
- , Stanislas Misticone
- & Mark G.H. Scott
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| Open Access3D niche microarrays for systems-level analyses of cell fate
3D cell culture matrices more closely resemble the natural microenvironments of stem cells than 2D systems. Here, the authors present a 3D cell culture approach to screen for the influence of environmental parameters on self-renewal and differentiation of single mouse embryonic stem cells.
- A. Ranga
- , S. Gobaa
- & M. P. Lutolf
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A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a
β-Diguetoxin-Dc1a, a component of desert bush spider venom, targets insect voltage-gated sodium channels, but not those of humans. Bende et al. find that American, but not German cockroaches are sensitive to the toxin, and identify two residues in the voltage-sensor domain that underlie this difference.
- Niraj S. Bende
- , Sławomir Dziemborowicz
- & Frank Bosmans
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Nanoscale chromatin profiling of gastric adenocarcinoma reveals cancer-associated cryptic promoters and somatically acquired regulatory elements
Epigenetic alterations alter chromatin structure and gene expression and are known contributors to cancer development. Here, Muratani et al.profile multiple epigenetic chromatin marks in primary gastric cancers and identify hundreds of altered promoters and enhancers that drive the gene expression program in these malignancies.
- Masafumi Muratani
- , Niantao Deng
- & Patrick Tan
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Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy
Photosystem II is the biosynthetic machinery that allows the conversion of water to oxygen using light. Here, the authors combine X-ray emission and diffraction data to probe the structural changes that take place during photosystem II catalysis.
- Jan Kern
- , Rosalie Tran
- & Vittal K. Yachandra
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The Pan-Cancer analysis of pseudogene expression reveals biologically and clinically relevant tumour subtypes
The contribution of expressed pseudogenes to cancer pathogenesis is unclear. In this study, Han et al.analyse RNA-seq data from The Cancer Genome Atlas and demonstrate the potential of pseudogenes for investigating cancer mechanisms and discovering prognostic biomarkers.
- Leng Han
- , Yuan Yuan
- & Han Liang
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Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis
Many tissues can be grown as 3D spheroid models in hanging drops of media. Here, Frey et al. develop a microfluidic, interconnected hanging drop network to facilitate inter-drop communication, and demonstrate that pro-drug conversion by liver spheroids can limit the growth of cancer spheroids in adjacent drops.
- Olivier Frey
- , Patrick M. Misun
- & Andreas Hierlemann
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| Open AccessAllele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR–Cas platform
The bacterial CRISPR–Cas system is increasingly used for genome editing in animal models. Here the authors utilize this system to target and edit specific coat colour alleles in rats and demonstrate the potential of this technology for the creation of genetically engineered animal models.
- K. Yoshimi
- , T. Kaneko
- & T. Mashimo
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Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery
The activity of heat shock proteins (Hsp) is modified by binding to cochaperones. Here, the authors develop a four-colour FRET system to show that cochaperone p23 binding to Hsp90 strengthens the ATP-dependent directionality, thus validating their approach for the study of other multicomponent protein machines.
- C. Ratzke
- , B. Hellenkamp
- & T. Hugel
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| Open AccessImaging intraorganellar Ca2+ at subcellular resolution using CEPIA
The use of intracellular calcium sensors provides important information about the dynamics of calcium signalling in cells. Here Suzuki et al. develop organelle-targeted sensors to simultaneously measure calcium concentrations in ER and mitochondria, and uncover novel insights into calcium flux in mitochondria.
- Junji Suzuki
- , Kazunori Kanemaru
- & Masamitsu Iino
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Integrating sequence and array data to create an improved 1000 Genomes Project haplotype reference panel
1000 Genomes imputation can increase the power of genome-wide association studies to detect genetic variants associated with human traits and diseases. Here, the authors develop a method to integrate and analyse low-coverage sequence data and SNP array data, and show that it improves imputation performance.
- Olivier Delaneau
- , Jonathan Marchini
- & Leena Peltonenz
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