RESUMEN
Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately, >70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers.
Asunto(s)
Anticuerpos/análisis , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas/análisis , ARN/análisis , Análisis de la Célula Individual/métodos , Animales , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.
Asunto(s)
Mapeo Cromosómico , Código de Barras del ADN Taxonómico , Daño del ADN , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Reparación del ADN , Epistasis Genética , Eliminación de Gen , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Metilmetanosulfonato , Modelos Teóricos , Regiones Promotoras Genéticas , Reproducibilidad de los ResultadosRESUMEN
High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods.
Asunto(s)
Centrosoma/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Cromosomas Humanos/metabolismo , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Although RNA-mediated interference (RNAi) is a widely conserved process among eukaryotes, including many fungi, it is absent from the budding yeast Saccharomyces cerevisiae. Three human proteins, Ago2, Dicer and TRBP, are sufficient for reconstituting the RISC complex in vitro. To examine whether the introduction of human RNAi genes can reconstitute RNAi in S. cerevisiae, genes encoding these three human proteins were introduced into S. cerevisiae. We observed both siRNA and siRNA- and RISC-dependent silencing of the target gene GFP. Thus, human Ago2, Dicer and TRBP can functionally reconstitute human RNAi in S. cerevisiae, in vivo, enabling the study and use of the human RNAi pathway in a facile genetic model organism.
Asunto(s)
Interferencia de ARN , Saccharomyces cerevisiae/genética , Proteínas Argonautas , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Modelos Genéticos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Most G-proteins require a guanine nucleotide exchange factor (GEF) to regulate a variety of critical cellular processes. Interestingly, a small number of G-proteins switch between the active and inactive forms without a GEF. Translation elongation factor 1A (eEF1A) normally requires the GEF eEF1Balpha to accelerate nucleotide dissociation. However, several mutant forms of eEF1A are functional independent of this essential regulator in vivo. GEF-independent eEF1A mutations localize close to the G-protein motifs that are crucial for nucleotide binding. Kinetic analysis demonstrated that reduced GDP affinity correlates with wild type growth and high translation activities of GEF-independent mutants. Furthermore, the mutant forms show an 11-22-fold increase in rates of GDP dissociation from eEF1A compared with the wild type protein. All mutant forms have dramatically enhanced stability at elevated temperatures. This, coupled with data demonstrating that eEF1A is also more stable in the presence of nucleotides, suggests that both the GEF and nucleotide have stabilizing effects on eEF1A. The biochemical properties of these eEF1A mutants provide insight into the mechanism behind GEF-independent G-protein function.
Asunto(s)
Factor 1 Eucariótico de Iniciación/metabolismo , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mutación , Saccharomyces cerevisiae/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Bioquímica/métodos , Relación Dosis-Respuesta a Droga , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of gamendazole action are currently unknown. Gamendazole was recently designed as a potent orally active antispermatogenic male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation elongation factor 1 alpha 1]) were identified as binding targets by biotinylated gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8 ovarian cancer cells; identification was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled gamendazole. However, gamendazole did not inhibit nucleotide binding by EEF1A1. Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited luciferase refolding and was not competed by the HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of the HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from gamendazole-treated rats showed a marked, rapid increase in three interleukin 1 genes and Nfkbia (NF-kappaB inhibitor alpha) 4 h after oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.
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Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Indazoles/farmacología , Interleucina-1alfa/genética , Factor 1 de Elongación Peptídica/antagonistas & inhibidores , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Bloqueadores de Espermatogénesis/farmacología , Administración Oral , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Femenino , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Indazoles/administración & dosificación , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/metabolismo , Ratas , Bloqueadores de Espermatogénesis/administración & dosificación , Testículo/efectos de los fármacos , Testículo/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
G-proteins play critical roles in many cellular processes and are regulated by accessory proteins that modulate the nucleotide-bound state. Such proteins, including eukaryotic translation elongation factor 1A (eEF1A), are frequently reactivated by guanine nucleotide exchange factors (GEFs). In the yeast Saccharomyces cerevisiae, only the catalytic subunit of the GEF complex, eEF1Balpha, is essential for viability. The requirement for the TEF5 gene encoding eEF1Balpha can be suppressed by the presence of excess substrate, eEF1A. These cells, however, have defects in growth and translation. Two independent unbiased screens performed to dissect the cause of these phenotypes yielded dominant suppressors that bypass the requirement for extra eEF1A. Surprisingly, all mutations are in the G-protein eEF1A and cluster in its GTP-binding domain. Five mutants were used to construct novel strains expressing only the eEF1A mutant at normal levels. These strains show no growth defects and little to no decreases in total translation, which raises questions as to the evolutionary expression of GEF complexity and other potential functions of this complex. The location of the mutations on the eEF1A-eEF1Balpha structure suggests that their mechanism of suppression may depend on effects on the conserved G-protein elements: the P-loop and NKXD nucleotide-binding element.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Mutagénesis , Factor 1 de Elongación Peptídica/genética , Proteínas de Saccharomyces cerevisiae/genética , Sitios de Unión/genética , Factores de Intercambio de Guanina Nucleótido/fisiología , Estructura Terciaria de Proteína/genéticaRESUMEN
Calcium signalling is involved in myriad cellular processes such as mating morphogenesis. Mating in yeast induces changes in cell morphology with a concomitant increase in calcium uptake that is dependent on the MID1 and CCH1 genes. Mid1p and Cch1p are believed to function in a capacitive calcium entry (CCE)-like process. Amiodarone alters mammalian calcium channel activity but, despite its clinical importance, its molecular mechanisms are not clearly defined. We have shown previously that amiodarone has fungicidal activity against a broad array of fungi. We show here that amiodarone causes a dramatic increase in cytoplasmic calcium ([Ca2+]cyt) in Saccharomyces cerevisiae. The majority of this increase is dependent on extracellular Ca2+ nonetheless, a significant increase in [Ca2+]cyt is still induced by amiodarone when no uptake of extracellular Ca2+ can occur. The influx of extracellular Ca2+ may be a direct effect of amiodarone on a membrane transporter or may be by a CCE mechanism. Uptake of the extracellular Ca2+ is inhibited by caffeine and reduced in strains deleted for the mid1 gene, but not in cells deleted for cch1. Our data are the first demonstrating control of yeast calcium channels by amiodarone and caffeine.