RESUMEN
Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.
Asunto(s)
Drosophila melanogaster/metabolismo , Larva/metabolismo , Neuronas Motoras/metabolismo , Péptidos/genética , Transgenes , Proteínas Virales/genética , Animales , Drosophila melanogaster/citología , Expresión Génica , Ingeniería Genética , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Larva/citología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Neuronas Motoras/citología , Péptidos/química , Péptidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Teschovirus/genética , Teschovirus/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Increases in vesicular glutamate transporter (VGLUT) levels are observed after a variety of insults including hypoxic injury, stress, methamphetamine treatment, and in genetic seizure models. Such overexpression can cause an increase in the amount of glutamate released from each vesicle, but it is unknown whether this is sufficient to induce excitotoxic neurodegeneration. Here we show that overexpression of the Drosophila vesicular glutamate transporter (DVGLUT) leads to excess glutamate release, with some vesicles releasing several times the normal amount of glutamate. Increased DVGLUT expression also leads to an age-dependent loss of motor function and shortened lifespan, accompanied by a progressive neurodegeneration in the postsynaptic targets of the DVGLUT-overexpressing neurons. The early onset lethality, behavioral deficits, and neuronal pathology require overexpression of a functional DVGLUT transgene. Thus overexpression of DVGLUT is sufficient to generate excitotoxic neuropathological phenotypes and therefore reducing VGLUT levels after nervous system injury or stress may mitigate further damage.
Asunto(s)
Drosophila melanogaster/metabolismo , Degeneración Nerviosa/metabolismo , Regulación hacia Arriba/genética , Proteínas de Transporte Vesicular de Glutamato/biosíntesis , Animales , Animales Modificados Genéticamente , Senescencia Celular/genética , Modelos Animales de Enfermedad , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Longevidad/genética , Masculino , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neurotoxinas/biosíntesis , Neurotoxinas/genética , Fenotipo , Terminales Presinápticos/metabolismo , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/patología , Proteínas de Transporte Vesicular de Glutamato/genéticaRESUMEN
During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific vesicular transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study vesicular transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT may undergo different modes of trafficking at the synapse. Further in vivo studies of DVMAT trafficking mutants will allow us to determine how changes in the localization of vesicular transporters affect the nervous system as a whole and complex behaviors mediated by aminergic circuits.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Transmisión Sináptica/fisiología , Proteínas Transportadoras Vesiculares de Neurotransmisores/fisiología , Animales , Transporte Biológico , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Neurotransmisores/metabolismo , Proteínas Transportadoras Vesiculares de Neurotransmisores/genética , Proteínas Transportadoras Vesiculares de Neurotransmisores/metabolismoRESUMEN
Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps-HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps-HOPS complex function.
Asunto(s)
Proteínas de Drosophila/metabolismo , Endosomas/metabolismo , Membranas Intracelulares/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endosomas/ultraestructura , Humanos , Lectinas Tipo C/genética , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/genética , Unión Neuromuscular/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sinapsis/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteínas de Transporte Vesicular/genéticaRESUMEN
Vesicular neurotransmitter transporters must localize to synaptic vesicles (SVs) to allow regulated neurotransmitter release at the synapse. However, the signals required to localize vesicular proteins to SVs in vivo remain unclear. To address this question we have tested the effects of mutating proposed trafficking domains in Drosophila orthologs of the vesicular monoamine and glutamate transporters, DVMAT-A and DVGLUT. We show that a tyrosine-based motif (YXXY) is important both for DVMAT-A internalization from the cell surface in vitro, and localization to SVs in vivo. In contrast, DVGLUT deletion mutants that lack a putative C-terminal trafficking domain show more modest defects in both internalization in vitro and trafficking to SVs in vivo. Our data show for the first time that mutation of a specific trafficking motif can disrupt localization to SVs in vivo and suggest possible differences in the sorting of VMATs versus VGLUTs to SVs at the synapse.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Vesículas Sinápticas/metabolismo , Tirosina/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Endocitosis/fisiología , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular de Glutamato/genética , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
Biogenesis of lysosome-related organelles complex 1 (BLOC-1) is a protein complex formed by the products of eight distinct genes. Loss-of-function mutations in two of these genes, DTNBP1 and BLOC1S3, cause Hermansky-Pudlak syndrome, a human disorder characterized by defective biogenesis of lysosome-related organelles. In addition, haplotype variants within the same two genes have been postulated to increase the risk of developing schizophrenia. However, the molecular function of BLOC-1 remains unknown. Here, we have generated a fly model of BLOC-1 deficiency. Mutant flies lacking the conserved Blos1 subunit displayed eye pigmentation defects due to abnormal pigment granules, which are lysosome-related organelles, as well as abnormal glutamatergic transmission and behavior. Epistatic analyses revealed that BLOC-1 function in pigment granule biogenesis requires the activities of BLOC-2 and a putative Rab guanine-nucleotide-exchange factor named Claret. The eye pigmentation phenotype was modified by misexpression of proteins involved in intracellular protein trafficking; in particular, the phenotype was partially ameliorated by Rab11 and strongly enhanced by the clathrin-disassembly factor, Auxilin. These observations validate Drosophila melanogaster as a powerful model for the study of BLOC-1 function and its interactions with modifier genes.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , Animales , Animales Modificados Genéticamente , Drosophila melanogaster/metabolismo , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/metabolismo , Humanos , Modelos Animales , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Orgánulos/metabolismo , FenotipoRESUMEN
Synaptic transmission requires the localization of presynaptic release machinery to active zones. Mechanisms regulating the abundance of such synaptic proteins at individual release sites are likely determinants of site-specific synaptic efficacy. We now identify a role for the small GTPase Rab3 in regulating the distribution of presynaptic components to active zones. At Drosophila rab3 mutant NMJs, the presynaptic protein Bruchpilot, calcium channels, and electron-dense T bars are concentrated at a fraction of available active zones, leaving the majority of sites devoid of these key presynaptic release components. Late addition of Rab3 to mutant NMJs rapidly reverses this phenotype by recruiting Brp to sites previously lacking the protein, demonstrating that Rab3 can dynamically control the composition of the presynaptic release machinery. While previous studies of Rab3 have focused on its role in the synaptic vesicle cycle, these findings demonstrate an additional and unexpected function for Rab3 in the localization of presynaptic proteins to active zones.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Unión Neuromuscular/citología , Dinámicas no Lineales , Terminales Presinápticos/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Animales , Animales Modificados Genéticamente , Calcio/farmacología , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Microscopía Electrónica de Transmisión/métodos , Placa Motora/metabolismo , Placa Motora/ultraestructura , Mutación/genética , Unión Neuromuscular/efectos de los fármacos , Terminales Presinápticos/ultraestructura , Receptores de Glutamato/genética , Proteínas de Unión al GTP rab3/genéticaRESUMEN
The synapse is composed of an active zone apposed to a postsynaptic cluster of neurotransmitter receptors. Each Drosophila neuromuscular junction comprises hundreds of such individual release sites apposed to clusters of glutamate receptors. Here, we show that protein phosphatase 2A (PP2A) is required for the development of structurally normal active zones opposite glutamate receptors. When PP2A is inhibited presynaptically, many glutamate receptor clusters are unapposed to Bruchpilot (Brp), an active zone protein required for normal transmitter release. These unapposed receptors are not due to presynaptic retraction of synaptic boutons, since other presynaptic components are still apposed to the entire postsynaptic specialization. Instead, these data suggest that Brp localization is regulated at the level of individual release sites. Live imaging of glutamate receptors demonstrates that this disruption to active zone development is accompanied by abnormal postsynaptic development, with decreased formation of glutamate receptor clusters. Remarkably, inhibition of the serine-threonine kinase GSK-3beta completely suppresses the active zone defect, as well as other synaptic morphology phenotypes associated with inhibition of PP2A. These data suggest that PP2A and GSK-3beta function antagonistically to control active zone development, providing a potential mechanism for regulating synaptic efficacy at a single release site.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Unión Neuromuscular/fisiología , Proteína Fosfatasa 2/metabolismo , Animales , Animales Modificados Genéticamente , Citoesqueleto/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Tomografía con Microscopio Electrónico/métodos , Regulación del Desarrollo de la Expresión Génica/genética , Glucógeno Sintasa Quinasa 3 beta , Microscopía Confocal , Mutación/genética , Unión Neuromuscular/citología , Unión Neuromuscular/ultraestructura , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Transporte de Proteínas/genética , Receptores de Glutamato/metabolismo , Transmisión Sináptica/genética , Transmisión Sináptica/fisiologíaRESUMEN
Axon degeneration underlies many common neurological disorders, but the signaling pathways that orchestrate axon degeneration are unknown. We found that dual leucine kinase (DLK) [corrected to add (DLK) abbreviation] promoted degeneration of severed axons in Drosophila and mice, and that its target, c-Jun N-terminal kinase, promoted degeneration locally in axons as they committed to degenerate. This pathway also promoted degeneration after chemotherapy exposure and may be a component of a general axon self-destruction program.
Asunto(s)
Axones/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Neuronas/patología , Degeneración Walleriana/patología , Animales , Animales Modificados Genéticamente , Antracenos/farmacología , Axones/metabolismo , Axones/ultraestructura , Axotomía/métodos , Células Cultivadas , Modelos Animales de Enfermedad , Drosophila , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Proteínas Fluorescentes Verdes/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/ultraestructura , Neuronas Receptoras Olfatorias/patología , Nervio Ciático/lesiones , Nervio Ciático/patología , Degeneración Walleriana/genéticaRESUMEN
Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system (CNS) and at Drosophila neuromuscular junctions (NMJs). Although glutamate is also used as a transmitter in the Drosophila CNS, there has been no systematic description of the central glutamatergic signaling system in the fly. With the recent cloning of the Drosophila vesicular glutamate transporter (DVGLUT), it is now possible to mark many, if not all, central glutamatergic neurons and synapses. Here we present the pattern of glutamatergic synapses and cell bodies in the late larval CNS and in the adult fly brain by using an anti-DVGLUT antibody. We also introduce two new tools for studying the Drosophila glutamatergic system: a dvglut promoter fragment fused to Gal4 whose expression labels glutamatergic neurons and a green fluorescent protein (GFP)-tagged DVGLUT transgene that localizes to synapses. In the larval CNS, we find synaptic DVGLUT immunoreactivity prominent in all brain lobe neuropil compartments except for the mushroom body. Likewise in the adult CNS, glutamatergic synapses are abundant throughout all major brain structures except the mushroom body. We also find that the larval ventral nerve cord neuropil is rich in glutamatergic synapses, which are primarily located near the dorsal surface of the neuropil, segregated from the ventrally positioned cholinergic processes. This description of the glutamatergic system in Drosophila highlights the prevalence of glutamatergic neurons in the CNS and presents tools for future study and manipulation of glutamatergic transmission.
Asunto(s)
Sistema Nervioso Central/citología , Sistema Nervioso Central/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/fisiología , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Larva , Masculino , Cuerpos Pedunculados/crecimiento & desarrollo , Cuerpos Pedunculados/metabolismo , Neuronas/citología , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular de Glutamato/genética , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
BACKGROUND: The growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, we have identified DFsn as a binding partner of Highwire and characterized the roles of DFsn in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance. RESULTS: We identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants - there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth. CONCLUSION: The F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. We propose that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Drosophila/metabolismo , Proteínas F-Box/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/metabolismo , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Secuencia Conservada , Regulación hacia Abajo/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Drosophila melanogaster , Evolución Molecular , Proteínas F-Box/química , Proteínas F-Box/genética , Proteínas F-Box/aislamiento & purificación , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/aislamiento & purificación , Inhibidores de Crecimiento/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Sistema Nervioso/citología , Unión Neuromuscular/embriología , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
The fly eye provides an attractive substrate for genetic studies, and critical transport activities for synaptic transmission and pigment biogenesis in the insect visual system remain unknown. We therefore screened for transporters in Drosophila melanogaster that are down-regulated by genetically ablating the eye. Using a large panel of transporter specific probes on Northern blots, we identified three transcripts that are down-regulated in flies lacking eye tissue. Two of these, CG13794 and CG13795, are part of a previously unknown subfamily of putative solute carriers within the neurotransmitter transporter family. The third, CG4476, is a member of a related subfamily that includes characterized nutrient transporters expressed in the insect gut. Using imprecise excision of a nearby transposable P element, we have generated a series of deletions in the CG4476 gene. In fast phototaxis assays, CG4476 mutants show a decreased behavioral response to light, and the most severe mutant behaves as if it were blind. These data suggest an unforeseen role for the "nutrient amino acid transporter" subfamily in the nervous system, and suggest new models to study transport function using the fly eye.
Asunto(s)
Proteínas de Transporte de Neurotransmisores/genética , Visión Ocular/fisiología , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Northern Blotting , Células Cultivadas , Regulación hacia Abajo/genética , Drosophila melanogaster , Electrorretinografía , Ojo/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Estimulación Luminosa , Filogenia , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.
Asunto(s)
Mutación Puntual , Vesículas Sinápticas/fisiología , Sintaxina 1/genética , Potenciales de Acción , Animales , Drosophila , Proteínas SNARE/fisiologíaRESUMEN
The formation of synaptic connections with target cells and maintenance of axons are highly regulated and crucial for neuronal function. The atypical cadherin and G-protein-coupled receptor Flamingo and its orthologs in amphibians and mammals have been shown to regulate cell polarity, dendritic and axonal growth, and neural tube closure. However, the role of Flamingo in synapse formation and function and in axonal health remains poorly understood. Here we show that fmi mutations cause a significant increase in the number of ectopic synapses on muscles and result in the formation of novel en passant synapses along axons, and unique presynaptic varicosities, including active zones, within axons. The fmi mutations also cause defective synaptic responses in a small subset of muscles, an age-dependent loss of muscle innervation and a drastic degeneration of axons in 3rd instar larvae without an apparent loss of neurons. Neuronal expression of Flamingo rescues all of these synaptic and axonal defects and larval lethality. Based on these observations, we propose that Flamingo is required in neurons for synaptic target selection, synaptogenesis, the survival of axons and synapses, and adult viability. These findings shed new light on a possible role for Flamingo in progressive neurodegenerative diseases.
Asunto(s)
Axones/metabolismo , Cadherinas/metabolismo , Proteínas de Drosophila/metabolismo , Degeneración Nerviosa/metabolismo , Unión Neuromuscular/metabolismo , Sinapsis/metabolismo , Animales , Axones/ultraestructura , Cadherinas/genética , Sistema Nervioso Central/embriología , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Drosophila , Proteínas de Drosophila/genética , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mutación , Unión Neuromuscular/embriología , Unión Neuromuscular/crecimiento & desarrollo , Organogénesis/fisiología , Técnicas de Placa-Clamp , Sinapsis/ultraestructuraRESUMEN
Presynaptic regulation of quantal size is an appealing mechanism for changing synapse strength. In this issue of Neuron, Steinert et al. describe an activity-dependent increase in synapse strength mediated by the formation and release of large synaptic vesicles at the Drosophila neuromuscular junction.
Asunto(s)
Drosophila/fisiología , Unión Neuromuscular/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología , Animales , Locomoción/fisiología , Terminales Presinápticos/fisiologíaRESUMEN
Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.
Asunto(s)
Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Animales , Drosophila , Inmunohistoquímica , Microscopía Electrónica , Mutación , Técnicas de Placa-Clamp , Sinapsis/fisiología , Sinapsis/ultraestructura , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructura , Proteínas de Transporte Vesicular de Glutamato/genéticaRESUMEN
AP180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been implicated in retrieving SV proteins. In Drosophila, deletion of its homologue, Like-AP180 (LAP), has been shown to increase the size of SVs but decrease the number of SVs and transmitter release. However, it remains elusive whether a reduction in the total vesicle pool directly affects transmitter release. Further, it is unknown whether the lap mutation also affects vesicle protein retrieval and synaptic protein localization and, if so, how it might affect exocytosis. Using a combination of electrophysiology, optical imaging, electron microscopy, and immunocytochemistry, we have further characterized the lap mutant and hereby show that LAP plays additional roles in maintaining both normal synaptic transmission and protein distribution at synapses. While increasing the rate of spontaneous vesicle fusion, the lap mutation dramatically reduces impulse-evoked transmitter release at steps downstream of calcium entry and vesicle docking. Notably, lap mutations disrupt calcium coupling to exocytosis and reduce calcium cooperativity. These results suggest a primary defect in calcium sensors on the vesicles or on the release machinery. Consistent with this hypothesis, three vesicle proteins critical for calcium-mediated exocytosis, synaptotagmin I, cysteine-string protein, and neuronal synaptobrevin, are all mislocalized to the extrasynaptic axonal regions along with Dap160, an active zone marker (nc82), and glutamate receptors in the mutant. These results suggest that AP180 is required for either recycling vesicle proteins and/or maintaining the distribution of both vesicle and synaptic proteins in the nerve terminal.
Asunto(s)
Exocitosis/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas/fisiología , Proteínas del Tejido Nervioso/metabolismo , Unión Neuroefectora/fisiología , Vesículas Sinápticas/metabolismo , Animales , Animales Modificados Genéticamente , Calcio/farmacología , Proteínas de Unión al Calcio/metabolismo , Clatrina/metabolismo , Diagnóstico por Imagen/métodos , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Drosophila , Estimulación Eléctrica/métodos , Exocitosis/efectos de los fármacos , Proteínas del Choque Térmico HSP40 , Inmunohistoquímica/métodos , Glicoproteínas de Membrana/metabolismo , Microscopía Electrónica de Transmisión/métodos , Mutación , Unión Neuroefectora/efectos de los fármacos , Unión Neuroefectora/efectos de la radiación , Unión Neuroefectora/ultraestructura , Técnicas de Placa-Clamp/métodos , Proteínas R-SNARE , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Transmisión Sináptica/efectos de la radiación , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/ultraestructura , Sinaptotagmina I , SinaptotagminasRESUMEN
Quantal size is a fundamental parameter controlling the strength of synaptic transmission. The transmitter content of synaptic vesicles is one mechanism that can affect the physiological response to the release of a single vesicle. At glutamatergic synapses, vesicular glutamate transporters (VGLUTs) are responsible for filling synaptic vesicles with glutamate. To investigate how VGLUT expression can regulate synaptic strength in vivo, we have identified the Drosophila vesicular glutamate transporter, which we name DVGLUT. DVGLUT mRNA is expressed in glutamatergic motoneurons and a large number of interneurons in the Drosophila CNS. DVGLUT protein resides on synaptic vesicles and localizes to the presynaptic terminals of all known glutamatergic neuromuscular junctions as well as to synapses throughout the CNS neuropil. Increasing the expression of DVGLUT in motoneurons leads to an increase in quantal size that is accompanied by an increase in synaptic vesicle volume. At synapses confronted with increased glutamate release from each vesicle, there is a compensatory decrease in the number of synaptic vesicles released that maintains normal levels of synaptic excitation. These results demonstrate that (1) expression of DVGLUT determines the size and glutamate content of synaptic vesicles and (2) homeostatic mechanisms exist to attenuate the excitatory effects of excess glutamate release.