RESUMEN
The polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia are both known to contain long cytoplasmic processes, while localized transcripts have only been studied extensively in neurons, not glia, especially in intact nervous systems. Here, we predict 1,740 localized Drosophila glial transcripts by extrapolating from our meta-analysis of seven existing studies characterizing the localized transcriptomes and translatomes of synaptically associated mammalian glia. We demonstrate that the localization of mRNAs in mammalian glial projections strongly predicts the localization of their high-confidence Drosophila homologs in larval motor neuron-associated glial projections and are highly statistically enriched for genes associated with neurological diseases. We further show that some of these localized glial transcripts are specifically required in glia for structural plasticity at the nearby neuromuscular junction synapses. We conclude that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important in disease.
Asunto(s)
Neuroglía , Unión Neuromuscular , Plasticidad Neuronal , ARN Mensajero , Animales , Neuroglía/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Plasticidad Neuronal/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/genética , Ratones , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Neuronas Motoras/metabolismo , Transcriptoma/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila/metabolismo , Drosophila/genéticaRESUMEN
While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap lines across the intact Drosophila nervous system. 97.5% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia, and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualization tools for post-transcriptional regulation.
Asunto(s)
Proteínas de Drosophila , ARN Mensajero , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , ARN Mensajero/genética , Procesamiento Postranscripcional del ARNRESUMEN
The epidermis is equipped with specialized mechanosensory organs that enable the detection of tactile stimuli. Here, by examining the differentiation of the tactile bristles, mechanosensory organs decorating the Drosophila adult epidermis, we show that neighbouring epidermal cells are essential for touch perception. Each mechanosensory bristle signals to the surrounding epidermis to co-opt a single epidermal cell, which we named the F-Cell. Once specified, the F-Cell adopts a specialized morphology to ensheath each bristle. Functional assays reveal that adult mechanosensory bristles require association with the epidermal F-Cell for touch sensing. Our findings underscore the importance of resident epidermal cells in the assembly of functional touch-sensitive organs.
Asunto(s)
Percepción del Tacto , Tacto , Animales , Tacto/fisiología , Células Epidérmicas , Epidermis , DrosophilaRESUMEN
Neurons and glia are highly polarized cells, whose distal cytoplasmic functional subdomains require specific proteins. Neurons have axonal and dendritic cytoplasmic extensions containing synapses whose plasticity is regulated efficiently by mRNA transport and localized translation. The principles behind these mechanisms are equally attractive for explaining rapid local regulation of distal glial cytoplasmic projections, independent of their cell nucleus. However, in contrast to neurons, mRNA localization has received little experimental attention in glia. Nevertheless, there are many functionally diverse glial subtypes containing extensive networks of long cytoplasmic projections with likely localized regulation that influence neurons and their synapses. Moreover, glia have many other neuron-like properties, including electrical activity, secretion of gliotransmitters and calcium signaling, influencing, for example, synaptic transmission, plasticity and axon pruning. Here, we review previous studies concerning glial transcripts with important roles in influencing synaptic plasticity, focusing on a few cases involving localized translation. We discuss a variety of important questions about mRNA transport and localized translation in glia that remain to be addressed, using cutting-edge tools already available for neurons.
Asunto(s)
Neuroglía , Neuronas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Axones/metabolismo , Sinapsis/genética , Sinapsis/metabolismo , Plasticidad Neuronal/genéticaRESUMEN
Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.
Asunto(s)
COVID-19/virología , ARN Viral/metabolismo , SARS-CoV-2/patogenicidad , Animales , Chlorocebus aethiops/genética , ARN/metabolismo , ARN Viral/genética , SARS-CoV-2/genética , Células Vero , Proteínas Virales/metabolismo , Replicación Viral/fisiologíaRESUMEN
Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Unión Neuromuscular/metabolismo , Plasticidad Neuronal , Proteínas de Unión al ARN/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Músculo Esquelético/embriología , Unión Neuromuscular/embriología , Unión Neuromuscular/genética , Proteínas de Unión al ARN/genética , Factores de TiempoRESUMEN
The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the Drosophila larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody colabeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells. These attributes make the method particularly amenable to super-resolution imaging. With 3D Structured Illumination Microscopy (3D-SIM), which increases spatial resolution by a factor of 2 in X, Y, and Z, we acquire super-resolution information about the distribution of single molecules of mRNA in relation to covisualized synaptic and cellular structures. Finally, we demonstrate the use of commercial and open source software for the quality control of single transcript expression analysis, 3D-SIM data acquisition and reconstruction as well as image archiving management and presentation. Our methods now allow the detailed mechanistic and functional analysis of sparse as well as abundant mRNAs at the NMJ in their appropriate cellular context.
Asunto(s)
Drosophila melanogaster/metabolismo , Hibridación Fluorescente in Situ/métodos , Unión Neuromuscular/metabolismo , Animales , Imagenología Tridimensional , Larva/metabolismo , Microscopía Confocal , ARN Mensajero/genética , ARN Mensajero/metabolismo , Coloración y Etiquetado , Fijación del TejidoRESUMEN
Cilia are conserved organelles that have important motility, sensory and signalling roles. The transition zone (TZ) at the base of the cilium is crucial for cilia function, and defects in several TZ proteins are associated with human congenital ciliopathies such as nephronophthisis (NPHP) and Meckel-Gruber syndrome (MKS). In several species, MKS and NPHP proteins form separate complexes that cooperate with Cep290 to assemble the TZ, but flies seem to lack core components of the NPHP module. We show that MKS proteins in flies are spatially separated from Cep290 at the TZ, and that flies mutant for individual MKS genes fail to recruit other MKS proteins to the TZ, whereas Cep290 seems to be recruited normally. Although there are abnormalities in microtubule and membrane organisation in developing MKS mutant cilia, these defects are less apparent in adults, where sensory cilia and sperm flagella seem to function quite normally. Thus, localising MKS proteins to the cilium or flagellum is not essential for viability or fertility in flies.