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Neurexins are key adhesion proteins that coordinate extracellular and intracellular synaptic components. Nonetheless, the low abundance of these multidomain proteins has complicated any localization and structure-function studies. Here we combine an ALFA tag (AT)/nanobody (NbALFA) tool with classic genetics, cell biology and electrophysiology to examine the distribution and function of the Drosophila Nrx-1 in vivo. We generate full-length and ΔPDZ ALFA-tagged Nrx-1 variants and find that the PDZ binding motif is key to Nrx-1 surface expression. A PDZ binding motif provided in trans, via genetically encoded cytosolic NbALFA-PDZ chimera, fully restores the synaptic localization and function of NrxΔPDZ-AT. Using cytosolic NbALFA-mScarlet intrabody, we achieve compartment-specific detection of endogenous Nrx-1, track live Nrx-1 transport along the motor neuron axons, and demonstrate that Nrx-1 co-migrates with Rab2-positive vesicles. Our findings illustrate the versatility of the ALFA system and pave the way towards dissecting functional domains of complex proteins in vivo.
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Proteínas de Drosophila , Anticorpos de Domínio Único , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Anticorpos de Domínio Único/metabolismo , Drosophila melanogaster/metabolismo , Neurônios Motores/metabolismo , Domínios PDZ , Axônios/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Transporte Proteico , Moléculas de Adesão Celular NeuronaisRESUMO
The formation of functional synapses requires co-assembly of ion channels with their accessory proteins which controls where, when, and how neurotransmitter receptors function. The auxiliary protein Neto modulates the function of kainate-type glutamate receptors in vertebrates as well as at the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse widely used for genetic studies on synapse development. We previously reported that Neto is essential for the synaptic recruitment and function of glutamate receptors. Here, using outside-out patch-clamp recordings and fast ligand application, we examine for the first time the biophysical properties of recombinant Drosophila NMJ receptors expressed in HEK293T cells and compare them with native receptor complexes of genetically controlled composition. The two Neto isoforms, Neto-α and Neto-ß, differentially modulate the gating properties of NMJ receptors. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of iGluR desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals.
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The Drosophila larval ventral nerve cord (VNC) shares many similarities with the spinal cord of vertebrates and has emerged as a major model for understanding the development and function of motor systems. Here, we use high-quality scRNA-seq, validated by anatomical identification, to create a comprehensive census of larval VNC cell types. We show that the neural lineages that comprise the adult VNC are already defined, but quiescent, at the larval stage. Using fluorescence-activated cell sorting (FACS)-enriched populations, we separate all motor neuron bundles and link individual neuron clusters to morphologically characterized known subtypes. We discovered a glutamate receptor subunit required for basal neurotransmission and homeostasis at the larval neuromuscular junction. We describe larval glia and endorse the general view that glia perform consistent activities throughout development. This census represents an extensive resource and a powerful platform for future discoveries of cellular and molecular mechanisms in repair, regeneration, plasticity, homeostasis, and behavioral coordination.
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Drosophila melanogaster , Larva , Neurônios Motores , Animais , Larva/genética , Larva/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Neuroglia/metabolismo , Neuroglia/citologia , Junção Neuromuscular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , RNA-Seq/métodos , Análise da Expressão Gênica de Célula ÚnicaRESUMO
Synapse development is coordinated by intercellular communication between the pre- and postsynaptic compartments, and by neuronal activity itself. In flies as in vertebrates, neuronal activity induces input-specific changes in the synaptic strength so that the entire circuit maintains stable function in the face of many challenges, including changes in synapse number and strength. But how do neurons sense synapse activity? In several studies carried out using the Drosophila neuromuscular junction (NMJ), we demonstrated that local BMP signaling provides an exquisite sensor for synapse activity. Here we review the main features of this exquisite sensor and discuss its functioning beyond monitoring the synapse activity but rather as a key controller that operates in coordination with other BMP signaling pathways to balance synapse growth, maturation and function.
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Proteínas de Drosophila , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Junção Neuromuscular , Transdução de Sinais/fisiologia , SinapsesRESUMO
Single-cell RNA sequencing provides a new approach to an old problem: how to study cellular diversity in complex biological systems. This powerful tool has been instrumental in profiling different cell types and investigating, at the single-cell level, cell states, functions, and responses. However, mining these data requires new analytical and statistical methods for high-dimensional analyses that must be customized and adapted to specific goals. Here we present a custom multistage analysis pipeline which integrates modules contained in different R packages to ensure flexible, high-quality RNA-seq data analysis. We describe this workflow step by step, providing the codes, explaining the rationale for each function, and discussing the results and the limitations. We apply this pipeline to analyze different datasets of Drosophila larval ventral cords, identifying and describing rare cell types, such as astrocytes and neuroendocrine cells. This multistage analysis pipeline can be easily implemented by both novice and experienced scientists interested in neuronal and/or cellular diversity beyond the Drosophila model system. © 2021 US Government.
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Análise de Célula Única , Software , Animais , Drosophila/genética , Perfilação da Expressão Gênica , Larva/genéticaRESUMO
Drosophila provides a powerful genetic system and an excellent model to study the development and function of the nervous system. The fly's small brain and complex behavior has been instrumental in mapping neuronal circuits and elucidating the neural basis of behavior. The fast pace of fly development and the wealth of genetic tools has enabled systematic studies on cell differentiation and fate specification, and has uncovered strategies for axon guidance and targeting. The accessibility of neuronal structures and the ability to edit and manipulate gene expression in selective cells and/or synaptic compartments has revealed mechanisms for synapse assembly and neuronal connectivity. Recent advances in single-cell RNA sequencing (scRNA-seq) have further enhanced our appreciation and understanding of neuronal diversity in a fly brain. However, due to the small size of the fly brain and its constituent cells, scRNA-seq methodologies require a few adaptations. Here, we describe a set of protocols optimized for scRNA-seq analysis of the Drosophila larval ventral nerve cord, starting from tissue dissection and cell dissociation to cDNA library preparation, sequencing, and data analysis. We apply this workflow to three separate samples and detail the technical challenges associated with successful application of scRNA-seq to studies on neuronal diversity. An accompanying article (Vicidomini, Nguyen, Choudhury, Brody, & Serpe, 2021) presents a custom multistage analysis pipeline that integrates modules contained in different R packages to ensure high-flexibility, high-quality RNA-seq data analysis. These protocols are developed for Drosophila larval ventral nerve cord, but could easily be adapted to other tissues and model organisms. © 2021 U.S. Government. Basic Protocol 1: Dissection of larval ventral nerve cords and preparation of single-cell suspensions Basic Protocol 2: Preparation and sequencing of single-cell transcriptome libraries Basic Protocol 3: Alignment of raw sequencing data to indexed genome and generation of count matrices.
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Drosophila , Análise de Célula Única , Animais , Drosophila/genética , Larva/genética , Análise de Sequência de RNA , SoftwareRESUMO
Bone morphogenetic proteins (BMPs) shape normal development and function via canonical and noncanonical signaling pathways. BMPs initiate canonical signaling by binding to transmembrane receptors that phosphorylate Smad proteins and induce their translocation into the nucleus and regulation of target genes. Phosphorylated Smads also accumulate at cellular junctions, but this noncanonical, local BMP signaling modality remains less defined. We have recently reported that phosphorylated Smad (pMad in Drosophila) accumulates at synaptic junctions in protein complexes with genetically distinct composition and regulation. Here, we examined a wide collection of DrosophilaMad alleles and searched for molecular features relevant to pMad accumulation at synaptic junctions. We found that strong Mad alleles generally disrupt both synaptic and nuclear pMad, whereas moderate Mad alleles have a wider range of phenotypes and can selectively impact different BMP signaling pathways. Interestingly, regulatory Mad mutations reveal that synaptic pMad appears to be more sensitive to a net reduction in Mad levels than nuclear pMad. Importantly, a previously uncharacterized allele, Mad8 , showed markedly reduced synaptic pMad but only moderately diminished nuclear pMad. The postsynaptic composition and electrophysiological properties of Mad8 neuromuscular junctions (NMJs) were also altered. Using biochemical approaches, we examined how a single point mutation in Mad8 could influence the Mad-receptor interface and identified a key motif, the H2 helix. Our study highlights the biological relevance of Smad-dependent, synaptic BMP signaling and uncovers a highly conserved structural feature of Smads, critical for normal development and function.
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Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Junção Neuromuscular/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Animais , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Mutação , Junção Neuromuscular/fisiologia , Transdução de Sinais , Potenciais Sinápticos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Homeostase , Proteínas de Membrana/metabolismo , Junção Neuromuscular/metabolismo , Sinapses/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Drosophila/química , Drosophila melanogaster/ultraestrutura , Proteínas de Membrana/química , Junção Neuromuscular/ultraestrutura , Densidade Pós-Sináptica/ultraestrutura , Domínios Proteicos , Receptores de Glutamato/metabolismoRESUMO
The maintenance of the intestinal epithelium is ensured by the controlled proliferation of intestinal stem cells (ISCs) and differentiation of their progeny into various cell types, including enterocytes (ECs) that both mediate nutrient absorption and provide a barrier against pathogens. The signals that regulate transition of proliferative ISCs into differentiated ECs are not fully understood. IRBIT is an evolutionarily conserved protein that regulates ribonucleotide reductase (RNR), an enzyme critical for the generation of DNA precursors. Here, we show that IRBIT expression in ISC progeny within the Drosophila midgut epithelium cells regulates their differentiation via suppression of RNR activity. Disruption of this IRBIT-RNR regulatory circuit causes a premature loss of intestinal tissue integrity. Furthermore, age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny. Collectively, our findings demonstrate a role of the IRBIT-RNR pathway in gut homeostasis.
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Assembly, maintenance and function of synaptic junctions depend on extracellular matrix (ECM) proteins and their receptors. Here we report that Tenectin (Tnc), a Mucin-type protein with RGD motifs, is an ECM component required for the structural and functional integrity of synaptic specializations at the neuromuscular junction (NMJ) in Drosophila. Using genetics, biochemistry, electrophysiology, histology and electron microscopy, we show that Tnc is secreted from motor neurons and striated muscles and accumulates in the synaptic cleft. Tnc selectively recruits αPS2/ßPS integrin at synaptic terminals, but only the cis Tnc/integrin complexes appear to be biologically active. These complexes have distinct pre- and postsynaptic functions, mediated at least in part through the local engagement of the spectrin-based membrane skeleton: the presynaptic complexes control neurotransmitter release, while postsynaptic complexes ensure the size and architectural integrity of synaptic boutons. Our study reveals an unprecedented role for integrin in the synaptic recruitment of spectrin-based membrane skeleton.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Cadeias alfa de Integrinas/metabolismo , Junção Neuromuscular/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas da Matriz Extracelular/genética , Cadeias alfa de Integrinas/genética , Microscopia Confocal , Neurônios Motores/metabolismo , Complexos Multiproteicos/metabolismo , Músculo Estriado/metabolismo , Espectrina/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Sinaptossomos/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1005810.].
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Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation. VIDEO ABSTRACT.
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Sistema Nervoso Central/metabolismo , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Animais , Canais de Cálcio , Cristalografia , Drosophila melanogaster , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Células HEK293 , Humanos , Ligantes , N-Metilaspartato/farmacologia , Ácido Quisquálico/farmacologia , Receptores de AMPA/agonistas , Receptores de AMPA/antagonistas & inibidores , Receptores de Glutamato/metabolismo , Receptores de Ácido Caínico/agonistas , Receptores de Ácido Caínico/antagonistas & inibidores , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Glutamate receptors are ligand-gated tetrameric ion channels that mediate synaptic transmission in the central nervous system. They are instrumental in vertebrate cognition and their dysfunction underlies diverse diseases. In both the resting and desensitized states of AMPA and kainate receptor subtypes, the ion channels are closed, whereas the ligand-binding domains, which are physically coupled to the channels, adopt markedly different conformations. Without an atomic model for the desensitized state, it is not possible to address a central problem in receptor gating: how the resting and desensitized receptor states both display closed ion channels, although they have major differences in the quaternary structure of the ligand-binding domain. Here, by determining the structure of the kainate receptor GluK2 subtype in its desensitized state by cryo-electron microscopy (cryo-EM) at 3.8 Å resolution, we show that desensitization is characterized by the establishment of a ring-like structure in the ligand-binding domain layer of the receptor. Formation of this 'desensitization ring' is mediated by staggered helix contacts between adjacent subunits, which leads to a pseudo-four-fold symmetric arrangement of the ligand-binding domains, illustrating subtle changes in symmetry that are important for the gating mechanism. Disruption of the desensitization ring is probably the key switch that enables restoration of the receptor to its resting state, thereby completing the gating cycle.
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Microscopia Crioeletrônica , Receptores de Ácido Caínico/metabolismo , Receptores de Ácido Caínico/ultraestrutura , Animais , Sítios de Ligação , Regulação para Baixo , Ativação do Canal Iônico , Ligantes , Modelos Moleculares , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores de Ácido Caínico/química , Receptor de GluK2 CainatoRESUMO
At the Drosophila NMJ, BMP signaling is critical for synapse growth and homeostasis. Signaling by the BMP7 homolog, Gbb, in motor neurons triggers a canonical pathway-which modulates transcription of BMP target genes, and a noncanonical pathway-which connects local BMP/BMP receptor complexes with the cytoskeleton. Here we describe a novel noncanonical BMP pathway characterized by the accumulation of the pathway effector, the phosphorylated Smad (pMad), at synaptic sites. Using genetic epistasis, histology, super resolution microscopy, and electrophysiology approaches we demonstrate that this novel pathway is genetically distinguishable from all other known BMP signaling cascades. This novel pathway does not require Gbb, but depends on presynaptic BMP receptors and specific postsynaptic glutamate receptor subtypes, the type-A receptors. Synaptic pMad is coordinated to BMP's role in the transcriptional control of target genes by shared pathway components, but it has no role in the regulation of NMJ growth. Instead, selective disruption of presynaptic pMad accumulation reduces the postsynaptic levels of type-A receptors, revealing a positive feedback loop which appears to function to stabilize active type-A receptors at synaptic sites. Thus, BMP pathway may monitor synapse activity then function to adjust synapse growth and maturation during development.
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Junção Neuromuscular/genética , Organogênese , Sinapses/genética , Transmissão Sináptica/genética , Animais , Animais Geneticamente Modificados , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Neurônios Motores/metabolismo , Junção Neuromuscular/crescimento & desenvolvimento , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
I am extremely honored to be the recipient of the 2015 Women in Cell Biology Junior Award. When I reflect on my journey in science, many great people and memorable experiences come to mind. Some of these encounters were truly career-defining moments. Others provided priceless lessons. In this essay, I recount some of the moments and experiences that influenced my scientific trajectory with the hope that they may inspire others.
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Distinções e Prêmios , Biologia Celular , Logro , Feminino , HumanosRESUMO
The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-ß, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-ß or its intracellular domain were generated. When Neto-ß is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-ß mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-ß rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-ß restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes.
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Proteínas de Drosophila/genética , Proteínas de Membrana/genética , Junção Neuromuscular/genética , Receptores Ionotrópicos de Glutamato/genética , Quinases Ativadas por p21/genética , Animais , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Proteínas de Membrana/biossíntese , Junção Neuromuscular/crescimento & desenvolvimento , Isoformas de Proteínas/genética , Receptores Ionotrópicos de Glutamato/biossíntese , Sinapses/genética , Sinapses/metabolismo , Transmissão Sináptica/genética , Quinases Ativadas por p21/biossínteseRESUMO
The Drosophila larval neuromuscular junction (NMJ), at which glutamate acts as the excitatory neurotransmitter, is a widely used model for genetic analysis of synapse function and development. Despite decades of study, the inability to reconstitute NMJ glutamate receptor function using heterologous expression systems has complicated the analysis of receptor function, such that it is difficult to resolve the molecular basis for compound phenotypes observed in mutant flies. We find that Drosophila Neto functions as an essential component required for the function of NMJ glutamate receptors, permitting analysis of glutamate receptor responses in Xenopus oocytes. In combination with a crystallographic analysis of the GluRIIB ligand binding domain, we use this system to characterize the subunit dependence of assembly, channel block, and ligand selectivity for Drosophila NMJ glutamate receptors.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Membrana/genética , Junção Neuromuscular/fisiologia , Receptores de Glutamato/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , DNA Complementar/metabolismo , Proteínas de Drosophila/fisiologia , Íons , Ligantes , Proteínas de Membrana/fisiologia , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Oócitos/citologia , Oócitos/metabolismo , Permeabilidade , Fenótipo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Sinapses/fisiologia , Xenopus laevisRESUMO
Stabilization of neurotransmitter receptors at postsynaptic specializations is a key step in the assembly of functional synapses. Drosophila Neto (Neuropillin and Tolloid-like protein) is an essential auxiliary subunit of ionotropic glutamate receptor (iGluR) complexes required for the iGluRs clustering at the neuromuscular junction (NMJ). Here we show that optimal levels of Neto are crucial for stabilization of iGluRs at synaptic sites and proper NMJ development. Genetic manipulations of Neto levels shifted iGluRs distribution to extrajunctional locations. Perturbations in Neto levels also produced small NMJs with reduced synaptic transmission, but only Neto-depleted NMJs showed diminished postsynaptic components. Drosophila Neto contains an inhibitory prodomain that is processed by Furin1-mediated limited proteolysis. neto null mutants rescued with a Neto variant that cannot be processed have severely impaired NMJs and reduced iGluRs synaptic clusters. Unprocessed Neto retains the ability to engage iGluRs in vivo and to form complexes with normal synaptic transmission. However, Neto prodomain must be removed to enable iGluRs synaptic stabilization and proper postsynaptic differentiation.
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Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Membrana/genética , Junção Neuromuscular/genética , Receptores Ionotrópicos de Glutamato/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Larva , Proteínas de Membrana/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/metabolismo , Interferência de RNA , Receptores Ionotrópicos de Glutamato/genética , Sinapses/genética , Sinapses/metabolismo , Transmissão Sináptica/genéticaRESUMO
Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.
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Perfilação da Expressão Gênica , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Western Blotting , Códon , Biologia Computacional , Microinjeções , Dados de Sequência Molecular , Biossíntese de ProteínasRESUMO
Effective communication between pre- and postsynaptic compartments is required for proper synapse development and function. At the Drosophila neuromuscular junction (NMJ), a retrograde BMP signal functions to promote synapse growth, stability and homeostasis and coordinates the growth of synaptic structures. Retrograde BMP signaling triggers accumulation of the pathway effector pMad in motoneuron nuclei and at synaptic termini. Nuclear pMad, in conjunction with transcription factors, modulates the expression of target genes and instructs synaptic growth; a role for synaptic pMad remains to be determined. Here, we report that pMad signals are selectively lost at NMJ synapses with reduced postsynaptic sensitivities. Despite this loss of synaptic pMad, nuclear pMad persisted in motoneuron nuclei, and expression of BMP target genes was unaffected, indicating a specific impairment in pMad production/maintenance at synaptic termini. During development, synaptic pMad accumulation followed the arrival and clustering of ionotropic glutamate receptors (iGluRs) at NMJ synapses. Synaptic pMad was lost at NMJ synapses developing at suboptimal levels of iGluRs and Neto, an auxiliary subunit required for functional iGluRs. Genetic manipulations of non-essential iGluR subunits revealed that synaptic pMad signals specifically correlated with the postsynaptic type-A glutamate receptors. Altering type-A receptor activities via protein kinase A (PKA) revealed that synaptic pMad depends on the activity and not the net levels of postsynaptic type-A receptors. Thus, synaptic pMad functions as a local sensor for NMJ synapse activity and has the potential to coordinate synaptic activity with a BMP retrograde signal required for synapse growth and homeostasis.