RESUMEN
Heart failure (HF) is the leading cause of death in the Western world. Pathophysiological processes underlying HF development, including cardiac hypertrophy, fibrosis and inflammation, are controlled by specific microRNAs (miRNAs). Whereas most studies investigate miRNA function in one particular cardiac cell type, their multicellular function is poorly investigated. The present study probed 194 miRNAs -differentially expressed in cardiac inflammatory disease - for regulating cardiomyocyte size, cardiac fibroblasts collagen content, and macrophage polarization. Of the tested miRNAs, 13%, 26%, and 41% modulated cardiomyocyte size, fibroblast collagen production, and macrophage polarization, respectively. Seventeen miRNAs affected all three cellular processes, including miRNAs with established (miR-210) and unknown roles in cardiac pathophysiology (miR-145-3p). These miRNAs with a multi-cellular function commonly target various genes. In-depth analysis in vitro of previously unstudied miRNAs revealed that the observed phenotypical alterations concurred with changes in transcript and protein levels of hypertrophy-, fibrosis- and inflammation-related genes. MiR-145-3p and miR-891a-3p were identified to regulate the fibrotic response, whereas miR-223-3p, miR-486-3p, and miR-488-5p modulated macrophage activation and polarisation. In conclusion, miRNAs are multi-cellular regulators of different cellular processes underlying cardiac disease. We identified previously undescribed roles of miRNAs in hypertrophy, fibrosis, and inflammation, and attribute new cellular effects to various well-known miRNAs.
Asunto(s)
Cardiomegalia/patología , Insuficiencia Cardíaca/genética , MicroARNs/metabolismo , Miocarditis/inmunología , Miocardio/patología , Animales , Animales Recién Nacidos , Cardiomegalia/genética , Cardiomegalia/inmunología , Células Cultivadas , Fibroblastos , Fibrosis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Insuficiencia Cardíaca/inmunología , Insuficiencia Cardíaca/patología , Humanos , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Macrófagos , Ratones , Miocarditis/genética , Miocarditis/patología , Miocardio/citología , Miocardio/inmunología , Miocitos Cardíacos , Cultivo Primario de Células , RatasRESUMEN
The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.
Asunto(s)
Membrana Celular/enzimología , Endosomas/enzimología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Interferencia de ARN , Receptor IGF Tipo 2/metabolismo , Red trans-Golgi/enzimología , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Análisis por Conglomerados , Regulación Enzimológica de la Expresión Génica , Redes Reguladoras de Genes , Células HeLa , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Mapas de Interacción de Proteínas , Transporte de Proteínas/genética , Receptor IGF Tipo 2/genética , Transducción de Señal , Transfección , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Apically enriched Rab11-positive recycling endosomes (Rab11-REs) are important for establishing and maintaining epithelial polarity. Yet, little is known about the molecules controlling trafficking of Rab11-REs in an epithelium in vivo. Here, we report a genome-wide, image-based RNA interference screen for regulators of Rab11-RE positioning and transport of an apical membrane protein (PEPT-1) in C. elegans intestine. Among the 356 screen hits was the 14-3-3 and partitioning defective protein PAR-5, which we found to be specifically required for Rab11-RE positioning and apicobasal polarity maintenance. Depletion of PAR-5 induced abnormal clustering of Rab11-REs to ectopic sites at the basolateral cortex containing F-actin and other apical domain components. This phenotype required key regulators of F-actin dynamics and polarity, such as Rho GTPases (RHO-1 and the Rac1 orthologue CED-10) and apical PAR proteins. Our data suggest that PAR-5 acts as a regulatory hub for a polarity-maintaining network required for apicobasal asymmetry of F-actin and proper Rab11-RE positioning.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Polaridad Celular , Endosomas/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Técnica del Anticuerpo Fluorescente , Genotipo , Microscopía Confocal , Microscopía Fluorescente , Microtúbulos/metabolismo , Transportador de Péptidos 1 , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Simportadores/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
BACKGROUND: Automated microscopy and image analysis have progressed tremendously over the past 10 years and opened up a new era of high-content screening. In parallel, RNA interference (RNAi) has revolutionized the functional analysis of genes. OBJECTIVE: The focus of this review is screening of RNAi libraries, and in particular in screening of short interfering RNA (siRNA) libraries for target identification and validation in mammalian cell systems. METHODS: Recent literature of high-content siRNA screening in oncology, in intracellular trafficking and infection biology, and in neurobiology is reviewed and placed in the context of a discussion on hit verification. RESULTS/CONCLUSION: Various methods have been established for the application of RNAi in mammalian cells also, which allows efficient and reproducible silencing of individual genes to gain functional information on each individual gene. Complex multi-parametric cell-based assays, combined with both technologies, provide an extraordinary valuable tool to help understand biological and pathological processes in a systematic way and discover new targets for pharmaceutical exploitation.
RESUMEN
Advanced microscopy and the corresponding image analysis have been developed in recent years into a powerful tool for studying molecular and morphological events in cells and tissues. Cell-based high-content screening (HCS) is an upcoming methodology for the investigation of cellular processes and their alteration by multiple chemical or genetic perturbations. Multiparametric characterization of responses to such changes can be analyzed using intact live cells as reporter. These disturbances are screened for effects on a variety of molecular and cellular targets, including subcellular localization and redistribution of proteins. In contrast to biochemical screening, they detect the responses within the context of the intercellular structural and functional networks of normal and diseased cells, respectively. As cell-based HCS of small-molecule libraries is applied to identify and characterize new therapeutic lead compounds, large pharmaceutical companies are major drivers of the technology and have already shown image-based screens using more than 100,000 compounds.
Asunto(s)
Células/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Células/química , Humanos , Microscopía , Proteínas/efectos de los fármacos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
The underlying frameworks of natural product classes with multiple biological activities can be regarded as biologically selected and prevalidated starting points in vast chemical structure space in the development of compound collections for chemical biology and medicinal chemistry research. For the synthesis of natural product-derived and -inspired compound collections, the development of enantioselective transformations in a format amenable to library synthesis, e.g., on the solid support, is a major and largely unexplored goal. We report on the enantioselective solid-phase synthesis of a natural product-inspired alpha,beta-unsaturated delta-lactone collection and its investigation in cell-based screens monitoring cell cycle progression and viral entry into cells. The screens identified modulators of both biological processes at a high hit rate. The screen for inhibition of viral entry opens up avenues of research for the identification of compounds with antiviral activity.
Asunto(s)
Productos Biológicos/química , Ciclo Celular/efectos de los fármacos , Lactonas/química , Lactonas/farmacología , Internalización del Virus/efectos de los fármacos , Animales , Sitios de Unión , Productos Biológicos/metabolismo , Línea Celular , Cercopithecus , Técnicas Químicas Combinatorias , Diseño de Fármacos , Células HeLa , Humanos , Lactonas/síntesis química , Lactonas/metabolismo , Conformación Molecular , Unión Proteica , Estereoisomerismo , Virus de la Estomatitis Vesicular IndianaRESUMEN
Dual-color cross-correlation spectroscopy allows the detection and quantification of labeled biomolecules at ultra-low concentrations, whereby the sensitivity of the assay correlates with the measurement time. We now describe a parallel multifocal dual-color spectroscopic configuration employing multiple avalanche photodiodes and hardware correlators. Cross-correlation curves are obtained from several dual-color excitation foci simultaneously. Multifocal dual-color excitation is achieved by splitting each of two laser beams (488 and 633 nm) into four sub-beams with the help of two 2x2 fan-out diffractive optical elements (DOEs), and subsequent superposition of the two sets of four foci. The fluorescence emission from double-labeled biomolecules is detected by two 2x2 fiber arrays.
Asunto(s)
Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Refractometría/instrumentación , Espectrometría de Fluorescencia/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Estudios de Factibilidad , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Refractometría/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/métodosRESUMEN
The direct analysis of single biological molecules is getting increasingly important in basic as well as pharmaceutical research (e.g. for gene expression analysis). In particular single-molecule fluorescence detection provides exciting new opportunities to probe biochemical processes in unprecedented detail. Currently several academic and industrial research groups work on the development of single molecule detection based technologies in order to directly detect and analyze RNA and DNA molecules. As these developed methods are characterized as homogenous assays and obviate any amplification of the target or the signal, they provide clear advantages compared to methods like real-time PCR or DNA- arrays. In the following we describe a recently developed approach based on fluorescence correlation spectroscopy (FCS). This expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target molecule (either DNA or RNA) in solution. The subsequent dual color cross-correlation analysis allows the quantification of the bio-molecule of interest in absolute numbers. Target concentrations of less than 10(-12) M can be easily monitored, covering the direct analysis of the expression levels of high, medium and low abundant genes.
Asunto(s)
ADN Complementario/análisis , Expresión Génica , Hibridación Fluorescente in Situ , ARN Mensajero/análisis , Espectrometría de Fluorescencia/métodos , Animales , Secuencia de Bases , Sondas de ADN , ADN Complementario/genética , Colorantes Fluorescentes , Humanos , Ácidos Nucleicos/genética , ARN Mensajero/genética , Espectrometría de Fluorescencia/instrumentaciónRESUMEN
Determination of the gene expression by direct quantification of mRNA is becoming increasingly important in basic, pharmaceutical and clinical research. We present a novel approach for gene quantification based on direct hybridization of gene-specific probes to target mRNA sequences in solution at temperatures ensuring absolute specificity of the probe-target complex. No enzymatic steps like reverse transcription or amplification by PCR are involved within the quantification process. In order to increase specificity as well as sensitivity, two probes emitting fluorescence light in different colors are used for our homogeneous assay using fluorescence cross-correlation. We relate to the single molecule sensitivity of excitation and detection in confocal cavities avoiding the amplification of the detected signal. The analysis of the expression level of high, medium and low abundant genes is described in two different cell lines, whereby the genes are quantified in absolute numbers.
Asunto(s)
Proteínas de Unión al Calcio , ADN Complementario/metabolismo , Expresión Génica , ARN Mensajero/análisis , ARN Mensajero/genética , Espectrometría de Fluorescencia/métodos , Calibración , Cromatografía Líquida de Alta Presión , Proteínas del Citoesqueleto/análisis , ADN Complementario/síntesis química , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Colorantes Fluorescentes , Células HL-60 , Humanos , Células K562 , Glicoproteínas de Membrana/análisis , Proteínas del Tejido Nervioso/análisis , Fosfoglicerato Quinasa/análisis , Reproducibilidad de los Resultados , Rodaminas , Sensibilidad y Especificidad , Soluciones , Espectrofotometría Ultravioleta , Sinaptotagminas , Factores de TiempoRESUMEN
Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.