RESUMO
Neurotransmitters are released at presynaptic active zones (AZs). In the fly Drosophila, monoclonal antibody (MAB) nc82 specifically labels AZs. We employ nc82 to identify Bruchpilot protein (BRP) as a previously unknown AZ component. BRP shows homology to human AZ protein ELKS/CAST/ERC, which binds RIM1 in a complex with Bassoon and Munc13-1. The C terminus of BRP displays structural similarities to multifunctional cytoskeletal proteins. During development, transcription of the bruchpilot locus (brp) coincides with neuronal differentiation. Panneural reduction of BRP expression by RNAi constructs permits a first functional characterization of this large AZ protein: larvae show reduced evoked but normal spontaneous transmission at neuromuscular junctions. In adults, we observe loss of T bars at active zones, absence of synaptic components in electroretinogram, locomotor inactivity, and unstable flight (hence "bruchpilot"-crash pilot). We propose that BRP is critical for intact AZ structure and normal-evoked neurotransmitter release at chemical synapses of Drosophila.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/fisiologia , Homologia Estrutural de Proteína , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Animais Geneticamente Modificados , Comportamento Animal , Northern Blotting/métodos , Western Blotting/métodos , Clonagem Molecular , Drosophila , Proteínas de Drosophila/genética , Dinaminas/metabolismo , Eletroforese em Gel Bidimensional/métodos , Proteínas de Fluorescência Verde/biossíntese , Humanos , Imunoquímica/métodos , Hibridização In Situ/métodos , Peptídeos e Proteínas de Sinalização Intracelular/química , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Microscopia Eletrônica de Transmissão/métodos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Junção Neuromuscular/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , RNA Polimerase I , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Caminhada/fisiologiaRESUMO
The hangover gene defines a cellular stress pathway that is required for rapid ethanol tolerance in Drosophila melanogaster. To understand how cellular stress changes neuronal function, we analyzed Hangover function on a cellular and neuronal level. We provide evidence that Hangover acts as a nuclear RNA binding protein and we identified the phosphodiesterase 4d ortholog dunce as a target RNA. We generated a transcript-specific dunce mutant that is impaired not only in ethanol tolerance but also in the cellular stress response. At the neuronal level, Dunce and Hangover are required in the same neuron pair to regulate experience-dependent motor output. Within these neurons, two cyclic AMP (cAMP)-dependent mechanisms balance the degree of tolerance. The balance is achieved by feedback regulation of Hangover and dunce transcript levels. This study provides insight into how nuclear Hangover/RNA signaling is linked to the cytoplasmic regulation of cAMP levels and results in neuronal adaptation and behavioral changes.
Assuntos
AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Proteínas de Drosophila/metabolismo , RNA Nuclear/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Adaptação Fisiológica/efeitos dos fármacos , Animais , Comportamento Animal , Citoplasma/metabolismo , Etanol/farmacologia , Isoenzimas/metabolismo , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Synapsins are evolutionarily conserved, highly abundant vesicular phosphoproteins in presynaptic terminals. They are thought to regulate the recruitment of synaptic vesicles from the reserve pool to the readily-releasable pool, in particular when vesicle release is to be maintained at high spiking rates. As regulation of transmitter release is a prerequisite for synaptic plasticity, we use the fruit fly Drosophila to ask whether Synapsin has a role in behavioral plasticity as well; in fruit flies, Synapsin is encoded by a single gene (syn). We tackled this question for associative olfactory learning in larval Drosophila by using the deletion mutant syn(97CS), which had been backcrossed to the Canton-S wild-type strain (CS) for 13 generations. We provide a molecular account of the genomic status of syn(97CS) by PCR and show the absence of gene product on Western blots and nerve-muscle preparations. We found that olfactory associative learning in syn(97CS) larvae is reduced to approximately 50% of wild-type CS levels; however, responsiveness to the to-be-associated stimuli and motor performance in untrained animals are normal. In addition, we introduce two novel behavioral control procedures to test stimulus responsiveness and motor performance after "sham training." Wild-type CS and syn(97CS) perform indistinguishably also in these tests. Thus, larval Drosophila can be used as a case study for a role of Synapsin in associative learning.