RESUMO
Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilin's SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.
Assuntos
Caenorhabditis elegans/citologia , Endocitose , Exocitose , Vesículas Sinápticas/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Mutação , Estrutura Terciária de Proteína , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. We investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, we found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. We also found evidence that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. Our results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. We discuss how counter-repression may influence eve stripe 2 regulation and evolution.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero , Elementos Facilitadores Genéticos/genética , Feminino , Proteínas de Homeodomínio/metabolismo , MasculinoRESUMO
In a large-scale screening effort, we identified the gene gooseberry (gsb) as being necessary for synaptic homeostasis at the Drosophila neuromuscular junction. The gsb gene encodes a pair-rule transcription factor that participates in embryonic neuronal cell fate specification. Here, we define a new postembryonic role for gooseberry. We show that gsb becomes widely expressed in the postembryonic CNS, including within mature motoneurons. Loss of gsb does not alter neuromuscular growth, morphology, or the distribution of essential synaptic proteins. However, gsb function is required postembryonically for the sustained expression of synaptic homeostasis. In GluRIIA mutant animals, miniature EPSP (mEPSP) amplitudes are significantly decreased, and there is a compensatory homeostatic increase in presynaptic release that restores normal muscle excitation. Loss of gsb significantly impairs the homeostatic increase in presynaptic release in the GluRIIA mutant. Interestingly, gsb is not required for the rapid induction of synaptic homeostasis. Furthermore, gsb seems to be specifically involved in the mechanisms responsible for a homeostatic increase in presynaptic release, since it is not required for the homeostatic decrease in presynaptic release observed following an increase in mEPSP amplitude. Finally, Gsb has been shown to antagonize Wingless signaling during embryonic fate specification, and we present initial evidence that this activity is conserved during synaptic homeostasis. Thus, we have identified a gene (gsb) that distinguishes between rapid induction versus sustained expression of synaptic homeostasis and distinguishes between the mechanisms responsible for homeostatic increase versus decrease in synaptic vesicle release.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Homeostase/genética , Neurônios Motores/fisiologia , Junção Neuromuscular/fisiologia , Ribes/genética , Animais , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Dinaminas/metabolismo , Estimulação Elétrica/métodos , Embrião não Mamífero , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Proteínas de Fluorescência Verde/genética , Modelos Biológicos , Mutação/genética , Junção Neuromuscular/metabolismo , Técnicas de Patch-Clamp/métodos , Regiões Promotoras Genéticas/genética , Interferência de RNA/fisiologia , Receptores de AMPA/fisiologia , Sinapsinas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , beta-Galactosidase/metabolismoRESUMO
Enhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene's endogenous pattern, are often used to study features of enhancer function. However, evidence suggests that some enhancers require sequences outside the minimal enhancer to maintain function under environmental perturbations. We hypothesized that these additional sequences also prevent misexpression caused by a transcription factor binding site mutation within a minimal enhancer. Using the Drosophila melanogastereven-skipped stripe 2 enhancer as a case study, we tested the effect of a Giant binding site mutation (gt-2) on the expression patterns driven by minimal and extended enhancer reporter constructs. We found that, in contrast to the misexpression caused by the gt-2 binding site deletion in the minimal enhancer, the same gt-2 binding site deletion in the extended enhancer did not have an effect on expression. The buffering of expression levels, but not expression pattern, is partially explained by an additional Giant binding site outside the minimal enhancer. Deleting the gt-2 binding site in the endogenous locus had no significant effect on stripe 2 expression. Our results indicate that rules derived from mutating enhancer reporter constructs may not represent what occurs in the endogenous context.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mutação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Shank is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. Here we show that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans , Músculos/fisiologiaRESUMO
Dynamic changes in synaptic connectivity and strength, which occur during both embryonic development and learning, have the tendency to destabilize neural circuits. To overcome this, neurons have developed a diversity of homeostatic mechanisms to maintain firing within physiologically defined limits. In this study, we show that activity-dependent control of mRNA for a specific voltage-gated Na+ channel [encoded by paralytic (para)] contributes to the regulation of membrane excitability in Drosophila motoneurons. Quantification of para mRNA, by real-time reverse-transcription PCR, shows that levels are significantly decreased in CNSs in which synaptic excitation is elevated, whereas, conversely, they are significantly increased when synaptic vesicle release is blocked. Quantification of mRNA encoding the translational repressor pumilio (pum) reveals a reciprocal regulation to that seen for para. Pumilio is sufficient to influence para mRNA. Thus, para mRNA is significantly elevated in a loss-of-function allele of pum (pum(bemused)), whereas expression of a full-length pum transgene is sufficient to reduce para mRNA. In the absence of pum, increased synaptic excitation fails to reduce para mRNA, showing that Pum is also necessary for activity-dependent regulation of para mRNA. Analysis of voltage-gated Na+ current (I(Na)) mediated by para in two identified motoneurons (termed aCC and RP2) reveals that removal of pum is sufficient to increase one of two separable I(Na) components (persistent I(Na)), whereas overexpression of a pum transgene is sufficient to suppress both components (transient and persistent). We show, through use of anemone toxin (ATX II), that alteration in persistent I(Na) is sufficient to regulate membrane excitability in these two motoneurons.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila/genética , Drosophila/fisiologia , Regulação da Expressão Gênica , Neurônios Motores/fisiologia , Canais de Sódio/genética , Transmissão Sináptica , Potenciais de Ação , Animais , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Condutividade Elétrica , Neurônios Motores/metabolismo , Técnicas de Patch-Clamp , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Canais de Sódio/metabolismoRESUMO
An electrophysiology-based forward genetic screen has identified two genes, pickpocket11 (ppk11) and pickpocket16 (ppk16), as being necessary for the homeostatic modulation of presynaptic neurotransmitter release at the Drosophila neuromuscular junction (NMJ). Pickpocket genes encode Degenerin/Epithelial Sodium channel subunits (DEG/ENaC). We demonstrate that ppk11 and ppk16 are necessary in presynaptic motoneurons for both the acute induction and long-term maintenance of synaptic homeostasis. We show that ppk11 and ppk16 are cotranscribed as a single mRNA that is upregulated during homeostatic plasticity. Acute pharmacological inhibition of a PPK11- and PPK16-containing channel abolishes the expression of short- and long-term homeostatic plasticity without altering baseline presynaptic neurotransmitter release, indicating remarkable specificity for homeostatic plasticity rather than NMJ development. Finally, presynaptic calcium imaging experiments support a model in which a PPK11- and PPK16-containing DEG/ENaC channel modulates presynaptic membrane voltage and, thereby, controls calcium channel activity to homeostatically regulate neurotransmitter release.
Assuntos
Sistema Nervoso Central/fisiologia , Canais Epiteliais de Sódio/metabolismo , Homeostase/fisiologia , Junção Neuromuscular/fisiologia , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/fisiologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Drosophila/genética , Drosophila melanogaster , Canais Epiteliais de Sódio/genética , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Homeostase/genética , Larva , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/citologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/genética , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Fármacos Neuroprotetores/farmacologia , Antagonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , Poliaminas/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Canais de Sódio/genéticaRESUMO
Homeostatic signaling systems stabilize neural function through the modulation of neurotransmitter receptor abundance, ion channel density, and presynaptic neurotransmitter release. Molecular mechanisms that drive these changes are being unveiled. In theory, molecular mechanisms may also exist to oppose the induction or expression of homeostatic plasticity, but these mechanisms have yet to be explored. In an ongoing electrophysiology-based genetic screen, we have tested 162 new mutations for genes involved in homeostatic signaling at the Drosophila NMJ. This screen identified a mutation in the rab3-GAP gene. We show that Rab3-GAP is necessary for the induction and expression of synaptic homeostasis. We then provide evidence that Rab3-GAP relieves an opposing influence on homeostasis that is catalyzed by Rab3 and which is independent of any change in NMJ anatomy. These data define roles for Rab3-GAP and Rab3 in synaptic homeostasis and uncover a mechanism, acting at a late stage of vesicle release, that opposes the progression of homeostatic plasticity.
Assuntos
Homeostase/fisiologia , Junção Neuromuscular/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Drosophila , Proteínas de Drosophila/genética , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Homeostase/genética , Modelos Biológicos , Mutação/genética , Antagonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp/métodos , Poliaminas/farmacologia , Receptores de Glutamato/genética , Transdução de Sinais/genética , Vesículas Sinápticas/genética , Sinaptotagminas/metabolismo , Proteínas rab3 de Ligação ao GTP/genéticaRESUMO
Although Caenorhabditis elegans has been utilized extensively to study synapse formation and function, relatively little is known about synaptic plasticity in C. elegans. We show that a brief treatment with the cholinesterase inhibitor aldicarb induces a form of presynaptic potentiation whereby ACh release at neuromuscular junctions (NMJs) is doubled. Aldicarb-induced potentiation was eliminated by mutations that block processing of proneuropeptides, by mutations inactivating a single proneuropeptide (NLP-12), and by those inactivating an NLP-12 receptor (CKR-2). NLP-12 expression is limited to a single stretch-activated neuron, DVA. Analysis of a YFP-tagged NLP-12 suggests that aldicarb stimulates DVA secretion of NLP-12. Mutations disrupting the DVA mechanoreceptor (TRP-4) decreased aldicarb-induced NLP-12 secretion and blocked aldicarb-induced synaptic potentiation. Mutants lacking NLP-12 or CKR-2 have decreased locomotion rates. Collectively, these results suggest that NLP-12 mediates a mechanosensory feedback loop that couples muscle contraction to changes in presynaptic release, thereby providing a mechanism for proprioceptive control of locomotion.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Mecanorreceptores/metabolismo , Contração Muscular/fisiologia , Plasticidade Neuronal/fisiologia , Neuropeptídeos/metabolismo , Transmissão Sináptica/fisiologia , Acetilcolina/metabolismo , Aldicarb/farmacologia , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Inibidores da Colinesterase/farmacologia , Potenciais Pós-Sinápticos Inibidores/genética , Potenciais Pós-Sinápticos Inibidores/fisiologia , Locomoção/fisiologia , Mecanorreceptores/fisiologia , Mutação , Plasticidade Neuronal/genética , Neuropeptídeos/genética , Pró-Proteína Convertase 2/genética , Receptores CCR2/genética , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Canais de Cátion TRPC/genéticaRESUMO
The regulatory mechanisms that orchestrate the developmental acquisition of electrical properties in embryonic neurons are poorly understood. Progress in this important area is dependent on the availability of preparations that allow electrophysiology to be married with genetics. The powerful genetics of the fruitfly Drosophila melanogaster has long been exploited to describe fundamental mechanisms associated with structural neuronal development (i.e. axon guidance). It has not, however, been fully employed to study the final stages of embryonic neural development and in particular the acquisition of electrical activity. This review focuses on the recent development of a Drosophila preparation that allows central neurons to be accessed by patch electrodes at both embryonic and larval stages. This preparation, which allows electrophysiology to be coupled with genetics, offers the prospect of making significant advances in our understanding of functional neuron development.
Assuntos
Drosophila melanogaster/citologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Animais , Membrana Celular/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Neurônios/citologia , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Canais de Sódio/metabolismo , Sinapses/fisiologiaRESUMO
BACKGROUND: While developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons. Early decisions, including motoneuron axon targeting, are orchestrated by a cohort of transcription factors that act together in a combinatorial manner. These transcription factors include Even-skipped (Eve), islet and Lim3. The perdurance of these factors in late embryonic neurons is, however, indicative that they might also regulate additional aspects of neuron development, including the acquisition of electrical properties. RESULTS: To test the hypothesis that a combinatorial code transcription factor is also able to influence the acquisition of electrical properties in embryonic neurons we utilized the molecular genetics of Drosophila to manipulate the expression of Eve in identified motoneurons. We show that increasing expression of this transcription factor, in two Eve-positive motoneurons (aCC and RP2), is indeed sufficient to affect the electrical properties of these neurons in early first instar larvae. Specifically, we observed a decrease in both the fast K+ conductance (IKfast) and amplitude of quantal cholinergic synaptic input. We used charybdotoxin to pharmacologically separate the individual components of IKfast to show that increased Eve specifically down regulates the Slowpoke (a BK Ca2+-gated potassium channel), but not Shal, component of this current. Identification of target genes for Eve, using DNA adenine methyltransferase identification, revealed strong binding sites in slowpoke and nAcRalpha-96Aa (a nicotinic acetylcholine receptor subunit). Verification using real-time PCR shows that pan-neuronal expression of eve is sufficient to repress transcripts for both slo and nAcRalpha-96Aa. CONCLUSION: Taken together, our findings demonstrate, for the first time, that Eve is sufficient to regulate both voltage- and ligand-gated currents in motoneurons, extending its known repertoire of action beyond its already characterized role in axon guidance. Our data are also consistent with a common developmental program that utilizes a defined set of transcription factors to determine both morphological and functional neuronal properties.