Search

CRISPR/Cas9–based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40–300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation.

Malfunctions in the basic epigenetic mechanisms such as histone modifications, DNA methylation, and chromatin remodeling are implicated in a number of cancers and immunological and neurodegenerative conditions. Within GlaxoSmithKline (GSK) we have utilized a number of variations of the NanoBRET technology for the direct measurement of compound-target engagement within native cellular environments to drive high-throughput, routine structure-activity relationship (SAR) profiling across differing epigenetic targets. NanoBRET is a variation of the bioluminescence resonance energy transfer (BRET) methodology utilizing proteins of interest fused to either NanoLuc, a small, high-emission-intensity luciferase, or HaloTag, a modified dehalogenase enzyme that can be selectively labeled with a fluorophore. The combination of these two technologies has enabled the application of NanoBRET to biological systems such as epigenetic protein-protein interactions, which have previously been challenging. By synergizing target engagement assays with more complex primary cell phenotypic assays, we have been able to demonstrate compound-target selectivity profiles to enhance cellular potency and offset potential liability risks. Additionally, we have shown that in the absence of a robust, cell phenotypic assay, it is possible to utilize NanoBRET target engagement assays to aid chemistry in progressing at a higher scale than would have otherwise been achievable. The NanoBRET target engagement assays utilized have further shown an excellent correlation with more reductionist biochemical and biophysical assay systems, clearly demonstrating the possibility of using such assay systems at scale, in tandem with, or in preference to, lower-throughput cell phenotypic approaches.

Dambournet et al. generate genome-edited human embryonic stem cells (hESCs) labeled with endocytic markers. They comparatively and quantitatively analyze the dynamics of clathrin-mediated endocytosis during differentiation through live-cell imaging and platinum replica EM in hESCs and their isogenic progeny., We developed a general approach for investigation of how cellular processes become adapted for specific cell types during differentiation. Previous studies reported substantial differences in the morphology and dynamics of clathrin-mediated endocytosis (CME) sites. However, associating specific CME properties with distinct differentiated cell types and determining how these properties are developmentally specified during differentiation have been elusive. Using genome-edited human embryonic stem cells, and isogenic fibroblasts and neuronal progenitor cells derived from them, we established by live-cell imaging and platinum replica transmission electron microscopy that CME site dynamics and ultrastructure on the plasma membrane are precisely reprogrammed during differentiation. Expression levels for the endocytic adaptor protein AP2μ2 were found to underlie dramatic changes in CME dynamics and structure. Additionally, CME dependency on actin assembly and phosphoinositide-3 kinase activity are distinct for each cell type. Collectively, our results demonstrate that key CME properties are reprogrammed during differentiation at least in part through AP2μ2 expression regulation.

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.

Recent studies have revealed the detailed timing of protein recruitment to endocytic sites in budding yeast. However, little is understood about the early stages of their formation. Here we identify the septin-associated protein Syp1p as a component of the machinery that drives clathrin-mediated endocytosis in budding yeast. Syp1p arrives at endocytic sites early in their formation and shares unique dynamics with the EH-domain protein Ede1p. We find that Syp1p is related in amino acid sequence to several mammalian proteins one of which, SGIP1-α, is an endocytic component that binds the Ede1p homolog Eps15. Like Syp1p, SGIP1-α arrives early at sites of clathrin-mediated endocytosis, suggesting that Syp1p/Ede1p and SGIP1-α/Eps15 may have a conserved function. In yeast, both Syp1p and Ede1p play important roles in the rate of endocytic site turnover. Additionally, Ede1p is important for endocytic site formation, whereas Syp1p acts as a polarized factor that recruits both Ede1p and endocytic sites to the necks of emerging buds. Thus Ede1p and Syp1p are conserved, early-arriving endocytic proteins with roles in the formation and placement of endocytic sites, respectively.

Actin assembly influences the precise temporal and quantitative recruitment of dynamin2 to sites of clathrin-mediated endocytosis., Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2–4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization.

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3' end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.DOI:http://dx.doi.org/10.7554/eLife.00471.001.

The Caenorhabditis elegans SH3 domain interactome was mapped and compared with the yeast SH3 interactome. Orthologous SH3 domain-mediated interactions are highly rewired, but the general function of the SH3 domain network is conserved between the two species, C. elegans Src homology 3 (SH3) domain interactome was mapped using stringent yeast two-hybrid, resulting in a total of 1070 interactions among 79 out of 84 worm SH3 domains and 475 proteins. SH3 domain binding specificities were profiled for 36 worm SH3 domains using peptide phage display. The yeast and worm SH3 domain interactomes are significantly enriched in endocytosis proteins, but the specific interactions mediated by orthologous SH3 domains are highly rewired. Using the worm SH3 interactome, we identified new endocytosis proteins in worm and human., Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.

Our understanding of clathrin-mediated endocytosis is derived from analysis of live-cell protein recruitment kinetics in combination with static ultrastructure images of coated pit progression. These analyses however, cannot directly correlate membrane curvature dynamics with the arrival and activities of the endocytic machinery. Polarized Total Internal Reflection (pol-TIR) fluorescence microscopy can visualize membrane topology in cells labeled with lipophilic fluorophores whose dipoles align relative to the plasma membrane. We describe a new approach for creating s-pol and p-pol TIR fields in a commercial microscope utilizing a 2-dimensional scan head to position polarized lasers at orthogonal azimuthal positions in the back focal plane of a high numerical aperture TIR objective lens. This configuration reduces interference fringing of the coherent laser light. Ratio imaging of the two excitation polarizations permits visualization of subresolution membrane curvature with subsecond temporal resolution. By monitoring membrane topology relative to the arrival of chromosomally-tagged GFP-dynamin in skin melanoma cells, we were able to define the kinetics of membrane curvature relative to vesicle scission. Blocking the catalytic GTPase cycle of dynamin with the drug, Dynasore permitted visualization of striking curved invaginations, consistent with expected “frozen” clathrin coated pits. Tracking of single dynamin recruitment events provided a region in which to quantify membrane topology. Surprisingly, regions of high curvature were often observed adjacent to clathrin pits. This observation is consistent with a model for actin-mediated membrane protrusions that could drive pit closure. The role for actin was assessed using CFP-LifeAct, and perturbed with the drug, Latrunculin. These studies provide insight into the sequential topological changes during clathrin-mediated endocytosis and move toward a comprehensive understanding of this mechanism.

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals., Author Summary Severe acute respiratory syndrome (SARS) is a severe, sometimes fatal respiratory disease caused by a coronavirus (SARS-CoV). In order to study the disease and evaluate vaccines and antiviral drugs, animal models that mimic the disease are necessary. However, no single animal model for SARS reproduces all aspects of the disease as it affects humans. SARS-CoV replicates in the lungs of young mice, but they do not show signs of illness. Adaptation of SARS-CoV by serial passage in the lungs of mice resulted in a virus (MA15) that is lethal for young mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by hematological changes and pathological changes in the lungs. Mice infected with MA15 virus die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes, and ciliated epithelial cells. The MA15 virus has six coding mutations in its genome, which, when introduced into a recombinant SARS-CoV, confer lethality. The MA15 virus will enhance the use of the mouse model for SARS because infection with this virus in mice reproduces many aspects of severe human disease, including morbidity, mortality, and pulmonary pathology.