RESUMEN
The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.
Asunto(s)
Hiperfagia/complicaciones , Hipotálamo/metabolismo , MicroARNs/metabolismo , Obesidad/etiología , Obesidad/patología , Absorciometría de Fotón , Proteína Relacionada con Agouti/genética , Proteína Relacionada con Agouti/metabolismo , Animales , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Proteína Oncogénica v-akt/metabolismo , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Ribonucleasa III/deficiencia , Ribonucleasa III/genética , Serina-Treonina Quinasas TOR/metabolismo , Transducción GenéticaRESUMEN
We have developed a method to perform microscopic temporal and spacial multi-scale experiments by imaging cellular phenotypes of interest on complementary fluorescence microscopy systems. In a low-resolution fast data acquisition screen for phenotypic cellular responses induced by small interfering RNA (siRNA), cells in spots of siRNA cell arrays showing characteristic alterations have been selected automatically by feature space analysis. These objects were imaged on a second super-resolution dSTORM microscope (direct stochastic optical reconstruction microscopy). The coordinate transfer was based on fixed cells as reference points without the use of additional fiducial markers. This procedure is suitable to combine any kind of fluorescence microscopy technique, in order to gain further insights on the observed specimen at multiple temporal or special scales.
Asunto(s)
Microscopía Fluorescente/métodos , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Células HeLa , Humanos , Microscopía Fluorescente/instrumentación , ARN Interferente Pequeño/genética , Células Tumorales CultivadasRESUMEN
The activity of the SMN complex in promoting the assembly of pre-mRNA processing UsnRNPs correlates with condensation of the complex in nuclear Cajal bodies. While mechanistic details of its activity have been elucidated, the molecular basis for condensation remains unclear. High SMN complex phosphorylation suggests extensive regulation. Here, we report on systematic siRNA-based screening for modulators of the capacity of SMN to condense in Cajal bodies and identify mTOR and ribosomal protein S6 kinase ß-1 as key regulators. Proteomic analysis reveals TOR-dependent phosphorylations in SMN complex subunits. Using stably expressed or optogenetically controlled phospho mutants, we demonstrate that serine 49 and 63 phosphorylation of human SMN controls the capacity of the complex to condense in Cajal bodies via liquid-liquid phase separation. Our findings link SMN complex condensation and UsnRNP biogenesis to cellular energy levels and suggest modulation of TOR signaling as a rational concept for therapy of the SMN-linked neuromuscular disorder spinal muscular atrophy.
Asunto(s)
Ribonucleoproteínas Nucleares Pequeñas/biosíntesis , Proteínas del Complejo SMN/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Núcleo Celular/metabolismo , Células HeLa , Humanos , Mutación/genética , Fosforilación , Fosfoserina/metabolismo , Multimerización de Proteína , Proteómica , Reproducibilidad de los Resultados , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismoRESUMEN
Macrophage-derived foam cells are key regulators of atherogenesis. They accumulate in atherosclerotic plaques and support inflammatory processes by producing cytokines and chemokines. Identifying factors that regulate macrophage lipid uptake may reveal therapeutic targets for coronary artery disease (CAD). Here, we establish a high-throughput screening workflow to systematically identify genes that impact the uptake of DiI-labeled low-density lipoprotein (LDL) into monocyte-derived primary human macrophages. For this, monocytes isolated from peripheral blood were seeded onto 384-well plates, solid-phase transfected with siRNAs, differentiated in vitro into macrophages, and LDL-uptake per cell was measured by automated microscopy and quantitative image analysis. We applied this workflow to study how silencing of 89 genes impacts LDL-uptake into cells from 16 patients with CAD and 16 age-matched controls. Silencing of four novel genes (APOC1, CMTM6, FABP4, WBP5) reduced macrophage LDL-uptake. Additionally, knockdown of the chemokine receptor CXCR4 reduced LDL-uptake, most likely through a G-protein coupled mechanism that involves the CXCR4 ligand macrophage-induced factor (MIF), but is independent of CXCL12. We introduce a high-throughput strategy to systematically study gene function directly in primary CAD-patient cells. Our results propose a function for the MIF/CXCR4 signaling pathway, as well as several novel candidate genes impacting lipid uptake into human macrophages.
Asunto(s)
Diferenciación Celular/genética , Enfermedad de la Arteria Coronaria/patología , Células Espumosas/metabolismo , Monocitos/metabolismo , Transducción de Señal/genética , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/prevención & control , Técnicas de Silenciamiento del Gen , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/metabolismo , Metabolismo de los Lípidos/genética , Lipoproteínas LDL/metabolismo , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Cultivo Primario de Células , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM-CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM-CF operations elaborated by the workgroups of the German network of ALM-CFs, German Bio-Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM-CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech. 79:463-479, 2016. © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC.
Asunto(s)
Instituciones de Salud , Laboratorios , Microscopía , Investigación Biomédica , Alemania , HumanosRESUMEN
Using RNAi interference (RNAi), it is possible to study the effect of specific gene knockdowns in mammalian cells. In this protocol we present the automated preparation of "ready to transfect" multiwell plates and cell arrays, on which cells can be grown which are then reversely transfected with one type of siRNA in every individual well or spot. Additionally, different microscope types for screening approaches are compared and considerations about the information workflow are made.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Microscopía Fluorescente/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , Transfección/métodos , Minería de Datos/métodosRESUMEN
The survival motor neuron (SMN) complex fulfils essential functions in the assembly of snRNPs, which are key components in the splicing of pre-mRNAs. Little is known about the regulation of SMN complex activity by posttranslational modification despite its complicated phosphorylation pattern. Several phosphatases had been implicated in the regulation of SMN, including the nuclear phosphatases PPM1G and PP1γ. Here we systematically screened all human phosphatase gene products for a regulatory role in the SMN complex. We used the accumulation of SMN in Cajal bodies of intact proliferating cells, which actively assemble snRNPs, as a readout for unperturbed SMN complex function. Knockdown of 29 protein phosphatases interfered with SMN accumulation in Cajal bodies, suggesting impaired SMN complex function, among those the catalytically inactive, non-receptor-type tyrosine phosphatase PTPN23/HD-PTP. Knockdown of PTPN23 also led to changes in the phosphorylation pattern of SMN without affecting the assembly of the SMN complex. We further show interaction between SMN and PTPN23 and document that PTPN23, like SMN, shuttles between nucleus and cytoplasm. Our data provide the first comprehensive screen for SMN complex regulators and establish a novel regulatory function of PTPN23 in maintaining a highly phosphorylated state of SMN, which is important for its proper function in snRNP assembly.
Asunto(s)
Cuerpos Enrollados/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN/metabolismo , Biocatálisis , Western Blotting , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Electroforesis en Gel Bidimensional , Células HeLa , Humanos , Microscopía Confocal , Fosforilación , Transporte de Proteínas , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Interferencia de ARNRESUMEN
High-throughput microscopy is an effective tool for rapidly collecting data on a large scale. However, high throughput comes at the cost of low spatial resolution. Here we introduce correlative light microscopy by combining fast automated widefield imaging, confocal microscopy and super-resolution microscopy. We demonstrate the potential of this approach for scalable experiments. The workflow consists of a robust approach for selecting cells of interest on a wide-field screening microscope at low resolution and subsequently re-localizing those cells with micrometer precision for confocal and super-resolution imaging. As a case study, we visualized and quantified cis- and trans-Golgi markers at increasing resolution.
Asunto(s)
Aparato de Golgi/ultraestructura , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Animales , Línea Celular , Procesamiento de Imagen Asistido por Computador/métodos , Riñón/citología , RatasRESUMEN
Despite the critical contributions of cilia to embryonic development and human health, key regulators of cilia formation await identification. In this paper, a functional RNA interference-based screen linked 30 novel protein kinases with ciliogenesis. Of them, we have studied the role of the microtubule (MT)-associated protein/MT affinity regulating kinase 4 (MARK4) in depth. MARK4 associated with the basal body and ciliary axoneme in human and murine cell lines. Ultrastructural and functional analyses established that MARK4 kinase activity was required for initiation of axoneme extension. We identified the mother centriolar protein ODF2 as an interaction partner of MARK4 and showed that ODF2 localization to the centriole partially depended on MARK4. Our data indicated that, upon MARK4 or ODF2 knockdown, the ciliary program arrested before the complete removal of the CP110-Cep97 inhibitory complex from the mother centriole, suggesting that these proteins act at this level of axonemal extension. We propose that MARK4 is a critical positive regulator of early steps in ciliogenesis.
Asunto(s)
Axonema/metabolismo , Cilios/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Axonema/ultraestructura , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cilios/ultraestructura , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiología , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Células 3T3 NIH , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARNRESUMEN
A high-content colocalization RNA interference screen based on automatic three-color confocal fluorescence microscopy was developed to analyze the alternative lengthening of telomeres (ALT) pathway. Via this pathway telomerase-negative cancer cells can maintain their telomeres and with it their unlimited proliferative potential. A hallmark of ALT cells is the colocalization of promyelocytic leukemia (PML) nuclear bodies with telomeres to form ALT-associated PML nuclear bodies (APBs). In our screen, the presence of APBs was used as a marker to identify proteins required for the ALT mechanism. A cell-based assay and an automatic confocal image acquisition procedure were established. Using automatic image analysis based on 3D parametric intensity models to identify APBs, we conducted an unbiased and quantitative analysis of nine different candidate genes. A comparison with the literature and manual analysis of the gene knockdown demonstrates the reliability of our approach. It extends the available repertoire of high-content screening to studies of cellular colocalizations and allows the identification of candidate genes for the ALT mechanism that represent possible targets for cancer therapy.
Asunto(s)
Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Interferencia de ARN , Telómero/genética , Telómero/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen/métodos , Células HeLa , Humanos , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal/genética , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Endocytosis is one of the most essential cellular processes, which enables cells to internalise diverse -material. It is crucial for regulation of receptor activity and signalling, cell polarisation, attachment and motility, and a great number of other cellular functions. A number of diverse endocytosis pathways are described by now; however, their specificity for different cellular cargoes is poorly resolved. Only few of endocytosis regulators are well-characterised and even less are attributed to the specific cargo. That is very true for the integrin endocytosis pathway, which is a key process in cell migration, adhesion, and signalling. The recent advent of quantitative fluorescent microscopy and cell arrays opened an exciting possibility to systematically characterise molecules playing a role in this crucially important process. Here, we describe a fluorescent screening microscopy-based assay to identify regulators of integrin α2 internalisation. The experimental procedure is the best suited for a highly parallel screening format, such as cell arrays, albeit can be used in single experiments. We provide protocols for sample preparation, fabrication of cell arrays and quantification of integrin α2 internalisation. The approach can be modified to quantify endocytosis of other cargo, and can be used under the conditions of knock-down and knock-in as well as for chemical screening.
Asunto(s)
Comunicación Celular/genética , Endocitosis/genética , Integrina alfa2/metabolismo , Microscopía Fluorescente/métodos , Análisis por Matrices de Proteínas/métodos , Interferencia de ARN , Comunicación Celular/fisiología , Endocitosis/fisiologíaRESUMEN
The Golgi complex is the central organelle in the secretory membrane trafficking and its organization strongly depends upon the flow of coming and leaving material. The principles of cargo transfer to, through, and away from the Golgi complex were investigated in numerous studies. However, the knowledge of how the Golgi complex responses to changes in diverse trafficking events (e.g., ER exit block) on a molecular level is far from being complete. In order to identify regulatory molecules playing a role in the dynamic organization of the Golgi complex, we established a fluorescent microscopy-based quantitative assay to measure rates of the Golgi redistribution and assembly after addition and washout of BFA, respectively. At first, we tested our system under the condition of over-expression of GFP-tagged proteins. We measured their influence upon BFA-induced effects in a format, suitable for large-scale studies in living cell, namely cell arrays. The approach can be applied for large-scale RNA interference studies as well as for chemical screening.
Asunto(s)
Orgánulos/metabolismo , Análisis de Matrices Tisulares/métodos , Transfección , Animales , Bencimidazoles/metabolismo , Transporte Biológico/efectos de los fármacos , Brefeldino A/farmacología , Línea Celular , Colorantes Fluorescentes/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Procesamiento de Imagen Asistido por Computador , Riñón/citología , Riñón/metabolismo , Microscopía Fluorescente , Microscopía por Video , Inhibidores de la Síntesis de la Proteína/farmacología , Interferencia de ARN , RatasRESUMEN
RNA interference (RNAi) has emerged as a powerful technique for studying loss-of-function phenotypes by specific down-regulation of gene expression, allowing the investigation of virus-host interactions by large-scale high-throughput RNAi screens. Here we present a robust and sensitive small interfering RNA screening platform consisting of an experimental setup, single-cell image and statistical analysis as well as bioinformatics. The workflow has been established to elucidate host gene functions exploited by viruses, monitoring both suppression and enhancement of viral replication simultaneously by fluorescence microscopy. The platform comprises a two-stage procedure in which potential host factors are first identified in a primary screen and afterwards re-tested in a validation screen to confirm true positive hits. Subsequent bioinformatics allows the identification of cellular genes participating in metabolic pathways and cellular networks utilised by viruses for efficient infection. Our workflow has been used to investigate host factor usage by the human immunodeficiency virus-1 (HIV-1), but can also be adapted to other viruses. Importantly, we expect that the description of the platform will guide further screening approaches for virus-host interactions. The ViroQuant-CellNetworks RNAi Screening core facility is an integral part of the recently founded BioQuant centre for systems biology at the University of Heidelberg and will provide service to external users in the near future.
Asunto(s)
Biología Computacional/métodos , VIH-1/genética , Interferencia de ARN , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Replicación Viral/genética , Células HeLa , HumanosRESUMEN
Reverse transfection on cell arrays is a high-throughput method for the parallel transfection of mammalian cells for use in high-content screening light microscopy. Here, we present novel 9216-microwell cell arrays which combine the advantages of multiwell plates (physically separated samples) and cell microarrays (high sample density and long-term storage).
Asunto(s)
Microscopía de Sonda de Barrido , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Materiales Biocompatibles Revestidos/química , Estudios de Factibilidad , Gelatina/química , Vidrio/química , Células HeLa , Humanos , Miniaturización , ARN Interferente Pequeño/genética , Especificidad por Sustrato , Titanio/química , TransfecciónRESUMEN
Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved its applicability to a wide range of biological questions. For the SMI live cell imaging this system was enhanced in terms of the development of a completely new upright configuration. This so called Vertico-SMI transfers the advantages of SMI nanoscaling to vital biological systems, and is shown to work consistently at different temperatures using both oil- and water-immersion objective lenses. Furthermore, we increased the speed of data acquisition to minimize errors in the detection signal resulting from cellular or object movement. By performing accurate characterization, the present Vertico-SMI now offers a fully-fledged microscope enabling a complete three-dimensional (3D) SMI data stack to be acquired in less than 2 seconds. We have performed live cell measurements of a tet-operator repeat insert in U2OS cells, which provided the first in vivo signatures of subnuclear complexes. Furthermore, we have successfully implemented an optional optical configuration allowing the generation of high-resolution localization microscopy images of a nuclear pore complex distribution.