RESUMEN
A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism.
Asunto(s)
Metabolismo Energético/genética , Epigénesis Genética , Histona Acetiltransferasas/metabolismo , Mitocondrias Musculares/enzimología , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Cardiomiopatía Hipertrófica/genética , Respiración de la Célula/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Células HeLa , Insuficiencia Cardíaca/genética , Histona Acetiltransferasas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/genética , Mitocondrias Musculares/genética , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación Oxidativa , Factores de Transcripción/genéticaRESUMEN
Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.
Asunto(s)
Lista de Verificación , Edición , Reproducibilidad de los Resultados , Procesamiento de Imagen Asistido por Computador , MicroscopíaRESUMEN
A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.
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Microscopía , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Studies on the intracellular trafficking of influenza virus ribonucleoproteins are currently limited by the lack of a method enabling their visualization during infection in single cells. This is largely due to the difficulty of encoding fluorescent fusion proteins within the viral genome. To circumvent this limitation, we used the split-green fluorescent protein (split-GFP) system (S. Cabantous, T. C. Terwilliger, and G. S. Waldo, Nat. Biotechnol. 23:102-107, 2005) to produce a quasi-wild-type recombinant A/WSN/33/influenza virus which allows expression of individually fluorescent PB2 polymerase subunits in infected cells. The viral PB2 proteins were fused to the 16 C-terminal amino acids of the GFP, whereas the large transcomplementing GFP fragment was supplied by transient or stable expression in cultured cells that were permissive to infection. This system was used to characterize the intranuclear dynamics of PB2 by fluorescence correlation spectroscopy and to visualize the trafficking of viral ribonucleoproteins (vRNPs) by dynamic light microscopy in live infected cells. Following nuclear export, vRNPs showed a transient pericentriolar accumulation and intermittent rapid (â¼1 µm/s), directional movements in the cytoplasm, dependent on both microtubules and actin filaments. Our data establish the potential of split-GFP-based recombinant viruses for the tracking of viral proteins during a quasi-wild-type infection. This new virus, or adaptations of it, will be of use in elucidating many aspects of influenza virus host cell interactions as well as in screening for new antiviral compounds. Furthermore, the existence of cell lines stably expressing the complementing GFP fragment will facilitate applications to many other viral and nonviral systems.
Asunto(s)
Proteínas Fluorescentes Verdes/genética , Virus de la Influenza A/fisiología , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética , Replicación Viral , Línea Celular , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Virus de la Influenza A/genéticaRESUMEN
Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses.
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Proteína HMGB1/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas del Núcleo Viral/metabolismo , Replicación Viral , Secuencia de Aminoácidos , Línea Celular , Proteína HMGB1/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/genética , Gripe Humana/virología , Datos de Secuencia Molecular , Proteínas de la Nucleocápside , Unión Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genéticaRESUMEN
Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality and explanatory power of microscopy data is in publications.
RESUMEN
Since currently available therapies against HIV/AIDS still show important drawbacks, the development of novel anti-HIV treatments is a key issue. We recently characterized methylated oligoribonucleotides (mONs) that extensively inhibit HIV-1 replication in primary T cells at nanomolar concentrations. The mONs were shown to target both HIV-1 reverse transcriptase (RT) and the nucleocapsid protein (NC), which is an essential partner of RT during viral DNA synthesis. To further understand the mechanism of such mONs, we studied by isothermal titration calorimetry and fluorescence-based techniques their NC binding properties and ability to inhibit the nucleic acid chaperone properties of NC. Notably, we investigated the ability of mONs to inhibit the NC-induced destabilization of the HIV-1 cTAR (complementary DNA sequence to TAR [transactivation response element]) stem-loop and the NC-promoted cTAR annealing to its complementary sequence, required at the early stage of HIV-1 viral DNA synthesis. Moreover, we compared the activity of the mONs to that of a number of modified and nonmodified oligonucleotides. Results show that the mONs inhibit NC by a competitive mechanism whereby the mONs tightly bind the NC peptide, mainly through nonelectrostatic interactions with the hydrophobic platform at the top of the NC zinc fingers. Taken together, these results favor the notion that the mONs impair the process of the RT-directed viral DNA synthesis by sequestering NC molecules, thus preventing the chaperoning of viral DNA synthesis by NC. These findings contribute to the understanding of the molecular basis for NC inhibition by mONs, which could be used for the rational design of antiretroviral compounds targeting HIV-1 NC protein.
Asunto(s)
VIH-1/metabolismo , Chaperonas Moleculares/antagonistas & inhibidores , Proteínas de la Nucleocápside/antagonistas & inhibidores , Oligorribonucleótidos/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral/biosíntesis , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismoRESUMEN
Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential. In this study, we systematically investigated the interactions between the polymerase subunits and their localization in living cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2. Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together, our results demonstrate which of the previously discussed influenza virus polymerase transport models operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects of different host range mutations in the future.
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Núcleo Celular/enzimología , ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Influenza A/enzimología , Espectrometría de Fluorescencia/métodos , Línea Celular , Humanos , Fracciones Subcelulares/enzimologíaRESUMEN
Optical tissue clearing refers to physico-chemical treatments which make thick biological samples transparent by removal of refractive index gradients and light absorbing substances. Although tissue clearing was first reported in 1914, it was not widely used in light microscopy until 21th century, because instrumentation of that time did not permit to acquire and handle images of thick (mm to cm) samples as whole. Rapid progress in optical instrumentation, computers and software over the last decades made micrograph acquisition of centimeter-thick samples feasible. This boosted tissue clearing use and development. Numerous diverse protocols have been developed. They use organic solvents or water-miscible substances, such as detergents and chaotropic agents; some protocols require application of electric field or perfusion with special devices. There is no 'best-for-all' tissue clearing method. Depending on the case, one or another protocol is more suitable. Most of protocols require days or even weeks to complete, thus choosing an unsuitable protocol may cause an important waste of time. Several inter-dependent parameters should be taken into account to choose a tissue clearing protocol, such as: (1) required image quality (resolution, contrast, signal to noise ratio etc), (2) nature and size of the sample, (3) type of labels, (4) characteristics of the available instrumentation, (5) budget, (6) time budget, and (7) feasibility. Present review focusses on the practical aspects of various tissue clearing techniques. It is aimed to help non-experts to choose tissue clearing techniques which are optimal for their particular cases.
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Técnicas de Preparación Histocitológica/métodos , Animales , Humanos , Microscopía/métodosRESUMEN
Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid's position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.
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Microscopía Fluorescente/métodos , Dinámicas Mitocondriales/fisiología , Proteínas Mitocondriales/metabolismo , HumanosRESUMEN
Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syndromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex-Mof, Kansl2 or Kansl3-unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)-NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work establishes that neurovascular function is determined by the neural metabolic environment.
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Núcleo Celular/patología , Cromatina/metabolismo , Histona Acetiltransferasas/fisiología , Inflamación/patología , Neovascularización Patológica/patología , Neuronas/patología , Pericitos/patología , Animales , Encéfalo/citología , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Cromatina/genética , Ácidos Grasos/metabolismo , Femenino , Feto/citología , Feto/metabolismo , Humanos , Inflamación/metabolismo , Masculino , Metaboloma , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pericitos/metabolismoRESUMEN
The nucleocapsid protein (NC) of HIV-1 is a highly conserved protein essential for the virus life cycle that constitutes an attractive target for new antiviral agents. Most NC functions rely on its binding to the HIV-1 genomic RNA and its DNA copies that contain multiple and possibly interdependent binding sites. Therefore, a detailed understanding of NC binding requires a site-specific experimental approach. We have recently shown that 2-aminopurine (2Ap), a fluorescent adenine analogue, can site-selectively probe the binding of NC. Here, we introduced 2Ap at various positions of model single-stranded dodecanucleotides containing two TG motifs which constitute putative specific binding sites. Steady-state and time-resolved fluorescence experiments indicated that NC binding strongly increased the fluorescence quantum yield of 2AP by reducing the dynamic quenching of 2Ap by its close neighbors and slowing the picosecond to nanosecond conformational fluctuations of the oligonucleotides. The dodecanucleotides were found to bind two NC molecules at physiological salt concentrations, confirming the preferential binding of NC to TG motifs and an occluded binding site size for NC of five to six bases. Using the NC-induced changes in 2Ap fluorescence, we determined the microscopic affinity constants of the individual binding sites and showed that affinities can significantly differ from one site to another within the same dodecanucleotide, depending on the position of the TG dinucleotide and the nature of its close neighbors. Moreover, our data suggest that binding of NC even to close binding sites shows no strong cooperativity.
Asunto(s)
Oligonucleótidos/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , 2-Aminopurina/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , VIH-1/química , Cinética , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
The nucleocapsid protein (NC) plays seminal roles in HIV replication, thus representing a major drug target. NC functions rely on its two zinc-fingers and flanking basic residues. Zinc ejectors inhibit NC functions, but with limited specificity. New classes of molecules competing with NC or its viral nucleic acid and enzyme partners are reviewed here.
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Fármacos Anti-VIH/química , Sistemas de Liberación de Medicamentos , VIH-1/efectos de los fármacos , Proteínas de la Nucleocápside/efectos de los fármacos , Fármacos Anti-VIH/farmacología , VIH-1/fisiología , Humanos , Modelos Biológicos , Proteínas de la Nucleocápside/química , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos , Dedos de Zinc/efectos de los fármacosRESUMEN
Mitochondrial DNA molecules coated with proteins form compact particles called mitochondrial nucleoids. They are redistributed within mitochondrial network undergoing morphological changes. The straightforward technique to characterize nucleoids' motions is fluorescence microscopy. Mitochondrial nucleoids are commonly labelled with fluorescent protein tags, which is not always feasible and was reported to cause artifacts. Organic DNA-binding dyes are free of these drawbacks, but they lack specificity to mitochondrial DNA. Here, considering physico-chemical properties of such dyes, we achieved preferential live-cell labelling of mitochondrial nucleoids by a nucleic acid staining dye SYBR Gold. It enabled time-lapse imaging of mitochondrial nucleoids by structured illumination microscopy and quantification of their motions.
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Colorantes/metabolismo , ADN Mitocondrial/ultraestructura , Mitocondrias/ultraestructura , Compuestos Orgánicos/metabolismo , Células A549 , Animales , Chlorocebus aethiops , ADN Mitocondrial/metabolismo , Células HeLa , Humanos , Microscopía/métodos , Mitocondrias/metabolismo , Imagen de Lapso de Tiempo , Células VeroRESUMEN
Conventional fragments of fluorescent proteins used in bimolecular fluorescence complementation technique (BiFC), form light-emitting species only when they are kept in close proximity by interacting proteins of interest. By contrast, certain fluorescent protein fragments complement spontaneously, namely those corresponding to the 1st to 10th beta-strands (GFP1-10) and the 11th beta-strand of superfolder GFP (GFP11). They were designed as folding reporters for high throughput expression and structure biology. Besides, for light microscopy, self-associating fluorescent protein fragments constitute a valuable and sometimes unique tool. The GFP11 tag is very advantageous when a full-length fluorescent protein cannot be fused to a protein of interest, namely for live imaging of certain pathogens. Self-associating GFP fragments enable live labelling of specific synapses, visualization of proteins topology and their exposure to particular subcellular compartments. Present review aims to attract attention of scientific community to these tools and to inspire their further development and applications.
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Proteínas Fluorescentes Verdes/metabolismo , Sondas Moleculares/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Microscopía Fluorescente/métodos , Sondas Moleculares/química , Sondas Moleculares/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Unión Proteica , Multimerización de ProteínaRESUMEN
The numbers of thymic epithelial cells (TECs) and thymocytes steadily increase during embryogenesis. To examine this dynamic, we generated several TEC-specific transgenic mouse lines, which express fluorescent proteins in the nucleus, the cytosol and in the membranes under the control of the Foxn1 promoter. These tools enabled us to determine TEC numbers in tissue sections by confocal fluorescent microscopy, and in the intact organ by light-sheet microscopy. Compared to histological procedures, flow cytometric analysis of thymic cellularity is shown to underestimate the numbers of TECs by one order of magnitude; using enzymatic digestion of thymic tissue, the loss of cortical TECs (cTECs) is several fold greater than that of medullary TECs (mTECs), although different cTEC subsets appear to be still present in the final preparation. Novel reporter lines driven by Psmb11 and Prss16 promoters revealed the trajectory of differentiation of cTEC-like cells, and, owing to the additional facility of conditional cell ablation, allowed us to follow the recovery of such cells after their depletion during embryogenesis. Multiparametric histological analyses indicate that the new transgenic reporter lines not only reveal the unique morphologies of different TEC subsets, but are also conducive to the analysis of the complex cellular interactions in the thymus.
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Epitelio/embriología , Timo/embriología , Animales , Comunicación Celular , Microambiente Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Expresión Génica , Genes Reporteros , Ratones Transgénicos , Células del Estroma/citología , Células del Estroma/metabolismo , Timo/metabolismoRESUMEN
Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a "back-to-back" fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling.
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Inmunoglobulina G/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Dimerización , Humanos , Modelos Moleculares , Transducción de Señal/fisiología , Proteínas RoundaboutRESUMEN
Influenza viruses are a global health concern because of the permanent threat of novel emerging strains potentially capable of causing pandemics. Viral ribonucleoproteins (vRNPs) containing genomic RNA segments, nucleoprotein oligomers, and the viral polymerase, play a central role in the viral replication cycle. Our knowledge about critical events such as vRNP assembly and interactions with other viral and cellular proteins is poor and could be substantially improved by time lapse imaging of the infected cells. However, such studies are limited by the difficulty to achieve live-cell compatible labeling of active vRNPs. Previously we designed the first unimpaired recombinant influenza WSN-PB2-GFP11 virus allowing fluorescent labeling of the PB2 subunit of the viral polymerase (Avilov et al., J.Virol. 2012). Here, we simultaneously labeled the viral PB2 protein using the above-mentioned strategy, and virus-encoded progeny RNPs through spontaneous incorporation of transiently expressed NP-mCherry fusion proteins during RNP assembly in live infected cells. This dual labeling enabled us to visualize progeny vRNPs throughout the infection cycle and to characterize independently the mobility, oligomerization status and interactions of vRNP components in the nuclei of live infected cells.
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Virus de la Influenza A/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Transporte de Proteínas , Espectrometría de FluorescenciaRESUMEN
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.
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Instituciones de Salud , Laboratorios , Microscopía , Investigación Biomédica , Alemania , HumanosRESUMEN
The single Cys residue in the C-terminal domain of bovine eye lens alpha-crystallin was covalently labelled with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone. This novel SH-reactive two-band ratiometric fluorescent dye is characterized by excited state intramolecular proton transfer reaction yielding two highly emissive N* and T* bands separated by more than 100 nm. Their relative intensities are known to be highly sensitive to the H-bonding ability of the environment. Properties of the environment of the dye attached to the protein were studied under native-like conditions and at a range of elevated temperatures that are known to facilitate alpha-crystallin chaperone-like activity. We observe that on heating, the environment of the dye becomes more flexible and the H-bonding of the dye with the protein vicinity decreases. The spectroscopic properties observed on heating were partially restored after cooling, but the initial values were not reached on the time scale of our experiments (up to 3 h). This suggests that the changes of the dye microenvironment are connected with the rearrangements of alpha-crystallin quaternary structure. Since there is only one Cys residue in alphaA subunit of alpha-crystallin (whereas alphaB subunit contains no Cys), we attributed the observed temperature-induced changes of the dye's microenvironment to the particular site within alpha-crystallin molecule.