Protein synthesis is a dynamic process to tune the cellular proteome to internal and external demands. Metabolic labeling approaches identify the general proteomic response but missing is a tool to visualize within cells specific newly synthesized proteins. Here we describe a technique that couples non-canonical amino acid tagging or puromycylation with the proximity-ligation assay to visualize identified newly synthesized proteins and monitor their origin, redistribution and turnover in situ.
DEAP-3600 is a single-phase liquid argon (LAr) direct-detection dark matter experiment, operating 2 km underground at SNOLAB (Sudbury, Canada). The detector consists of 3279 kg of LAr contained in a spherical acrylic vessel. This paper reports on the analysis of a 758 tonne · day exposure taken over a period of 231 live-days during the first year of operation. No candidate signal events are observed in the WIMP-search region of interest, which results in the leading limit on the WIMP-nucleon spin-independent cross section on a LAr target of 3.9 × 10 −45 cm 2 (1.5 × 10 −44 cm 2 ) for a 100 GeV=c 2 (1 TeV=c 2 ) WIMP mass at 90% C.L. In addition to a detailed background model, this analysis demonstrates the best pulseshape discrimination in LAr at threshold, employs a Bayesian photoelectron-counting technique to improve the energy resolution and discrimination efficiency, and utilizes two position reconstruction algorithms based on the charge and photon detection time distributions observed in each photomultiplier tube.
N-glycosylation – the sequential addition of complex sugars to adhesion proteins, neurotransmitter receptors, ion channels and secreted trophic factors as they progress through the endoplasmic reticulum and the Golgi apparatus – is one of the most frequent protein modifications. In mammals, most organ-specific N-glycosylation events occur in the brain. Yet, little is known about the nature, function and regulation of N-glycosylation in neurons. Using imaging, quantitative immunoblotting and mass spectrometry, we show that hundreds of neuronal surface membrane proteins are core-glycosylated, resulting in the neuronal membrane displaying surprisingly high levels of glycosylation profiles that are classically associated with immature intracellular proteins. We report that while N-glycosylation is generally required for dendritic development and glutamate receptor surface expression, core-glycosylated proteins are sufficient to sustain these processes, and are thus functional. This atypical glycosylation of surface neuronal proteins can be attributed to a bypass or a hypo-function of the Golgi apparatus. Core-glycosylation is regulated by synaptic activity, modulates synaptic signaling and accelerates the turnover of GluA2-containing glutamate receptors, revealing a novel mechanism that controls the composition and sensing properties of the neuronal membrane.DOI: http://dx.doi.org/10.7554/eLife.20609.001
In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations based on their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells, and predicting assay outcomes using machine learning, among many others. Here we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, golgi apparatus, plasma membrane, endoplasmic reticulum, and mitochondria. The original protocol was updated in 2016 based on several years' experience running it at two sites, after optimizing it by visual stain quality. Here we describe the work of the Joint Undertaking for Morphological Profiling (JUMP) Cell Painting Consortium, aiming to improve upon the assay via quantitative optimization, based on the measured ability of the assay to detect morphological phenotypes and group similar perturbations together. We find that the assay gives very robust outputs despite a variety of changes to the protocol and that two vendors' dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1 to 2 weeks for a typically sized batch of 20 or fewer plates; feature extraction and data analysis take an additional 1 to 2 weeks.
Image-based profiling has emerged as a powerful technology for various steps in basic biological and pharmaceutical discovery, but the community has lacked a large, public reference set of data from chemical and genetic perturbations. Here we present data generated by the Joint Undertaking for Morphological Profiling (JUMP)-Cell Painting Consortium, a collaboration between 10 pharmaceutical companies, six supporting technology companies, and two non-profit partners. When completed, the dataset will contain images and profiles from the Cell Painting assay for over 116,750 unique compounds, over-expression of 12,602 genes, and knockout of 7,975 genes using CRISPR-Cas9, all in human osteosarcoma cells (U2OS). The dataset is estimated to be 115 TB in size and capturing 1.6 billion cells and their single-cell profiles. File quality control and upload is underway and will be completed over the coming months at the Cell Painting Gallery: https://registry.opendata.aws/cellpainting-gallery. A portal to visualize a subset of the data is available at https://phenaid.ardigen.com/jumpcpexplorer/.
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