RESUMO
Here we identify a new role for Syndecan (Sdc), the only transmembrane heparan sulphate proteoglycan in Drosophila, in tracheal development. Sdc is required cell autonomously for efficient directed migration and fusion of dorsal branch cells, but not for dorsal branch formation per se. The cytoplasmic domain of Sdc is dispensable, indicating that Sdc does not transduce a signal by itself. Although the branch-specific phenotype of sdc mutants resembles those seen in the absence of Slit/Robo2 signalling, genetic interaction experiments indicate that Sdc also helps to suppress Slit/Robo2 signalling. We conclude that Sdc cell autonomously regulates Slit/Robo2 signalling in tracheal cells to guarantee ordered directional migration and branch fusion.
Assuntos
Movimento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Sindecanas/metabolismo , Animais , Sequência de Bases , Movimento Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica , Ordem dos Genes , Dados de Sequência Molecular , Fenótipo , Estabilidade Proteica , Alinhamento de Sequência , Transdução de Sinais , Sindecanas/genética , Traqueia/metabolismo , Proteínas RoundaboutRESUMO
Targeted gene silencing by RNA interference allows the study of gene function in plants and animals. In cell culture and small animal models, genetic screens can be performed--even tissue-specifically in Drosophila--with genome-wide RNAi libraries. However, a major problem with the use of RNAi approaches is the unavoidable false-positive error caused by off-target effects. Until now, this is minimized by computational RNAi design, comparing RNAi to the mutant phenotype if known, and rescue with a presumed ortholog. The ultimate proof of specificity would be to restore expression of the same gene product in vivo. Here, we present a simple and efficient method to rescue the RNAi-mediated knockdown of two independent genes in Drosophila. By exploiting the degenerate genetic code, we generated Drosophila RNAi Escape Strategy Construct (RESC) rescue proteins containing frequent silent mismatches in the complete RNAi target sequence. RESC products were no longer efficiently silenced by RNAi in cell culture and in vivo. As a proof of principle, we rescue the RNAi-induced loss of function phenotype of the eye color gene white and tracheal defects caused by the knockdown of the heparan sulfate proteoglycan syndecan. Our data suggest that RESC is widely applicable to rescue and validate ubiquitous or tissue-specific RNAi and to perform protein structure-function analysis.
Assuntos
Drosophila/genética , Técnicas de Silenciamento de Genes , Interferência de RNA , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , Drosophila/anatomia & histologia , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Olho/anatomia & histologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Genes de Insetos , Fenótipo , Sindecanas/antagonistas & inibidores , Sindecanas/genéticaRESUMO
The brain's impotence to utilize long-chain fatty acids as fuel, one of the dogmas in neuroscience, is surprising, since the nervous system is the tissue most energy consuming and most vulnerable to a lack of energy. Challenging this view, we here show in vivo that loss of the Drosophila carnitine palmitoyltransferase 2 (CPT2), an enzyme required for mitochondrial ß-oxidation of long-chain fatty acids as substrates for energy production, results in the accumulation of triacylglyceride-filled lipid droplets in adult Drosophila brain but not in obesity. CPT2 rescue in glial cells alone is sufficient to restore triacylglyceride homeostasis, and we suggest that this is mediated by the release of ketone bodies from the rescued glial cells. These results demonstrate that the adult brain is able to catabolize fatty acids for cellular energy production.
Assuntos
Drosophila melanogaster/metabolismo , Metabolismo Energético , Neuroglia/metabolismo , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Homeostase , Humanos , Larva/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Mutação , Obesidade/patologia , Oxirredução , Fosfolipídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sobrevida , Triglicerídeos/metabolismoAssuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antitrombinas , Isquemia Encefálica/tratamento farmacológico , Coagulantes/administração & dosagem , Dabigatrana/antagonistas & inibidores , Acidente Vascular Cerebral/tratamento farmacológico , Administração Intravenosa , Idoso , Ensaios Clínicos como Assunto , Feminino , Humanos , Terapia TrombolíticaRESUMO
Polarization of a neuron begins with the appearance of the first neurite, thus defining the ultimate growth axis. Unlike late occurring polarity events (such as axonal growth), very little is known about this fundamental process. We show here that, in Drosophila melanogaster neurons in vivo, the first membrane deformation occurred 3.6 min after precursor division. Clustering of adhesion complex components (Bazooka (Par-3), cadherin-catenin) marked this place by 2.8 min after division; the upstream phosphatidylinositol 4,5-bisphosphate, by 0.7 min after division; and the furrow components RhoA and Aurora kinase, from the time of cytokinesis. Local DE-cadherin inactivation prevented sprout formation, whereas perturbation of division orientation did not alter polarization from the cytokinesis pole. This is, to our knowledge, the first molecular study of initial neuronal polarization in vivo. The mechanisms of polarization seem to be defined at the precursor stage.
Assuntos
Polaridade Celular/fisiologia , Citocinese/fisiologia , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Receptoras Sensoriais/metabolismo , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Ciclo Celular/genética , Diferenciação Celular , Polaridade Celular/genética , Centríolos/fisiologia , Citocinese/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Peroxidase do Rábano Silvestre/metabolismo , Imageamento Tridimensional , Proteínas Luminescentes/genética , Microscopia Confocal , Mutação/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Pupa , Órgãos dos Sentidos/citologia , Órgãos dos Sentidos/embriologia , Células Receptoras Sensoriais/citologia , Fatores de Tempo , alfa Catenina/metabolismoRESUMO
The syndecans play critical roles in several signal transduction pathways. The core proteins of these heparan sulfate proteoglycans are characterized by highly conserved transmembrane and intracellular domains which are required for signaling across the membrane and for interaction with cytosolic proteins. However, regulatory mechanisms controlling these functions remain largely unknown. Here we show that, upon ligand-induced primary proteolytic cleavage within the ectodomain, the intracellular domain of syndecan 3 is released by regulated intramembrane proteolysis. The cleavage is mediated by presenilin/gamma-secretase complex and negatively regulates the plasma membrane targeting of the transcriptional cofactor CASK.
Assuntos
Citosol/metabolismo , Endopeptidases/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Hidrólise , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Sindecana-3RESUMO
To determine whether neurite outgrowth depends upon the mevalonate pathway, we blocked mevalonate synthesis in nerve growth factor-treated PC12 cells or primary cortical neurones with atorvastatin, a 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and substituted different intermediates of the mevalonate pathway. We show that HMG-CoA reductase inhibition causes a profound reduction of neurite length, neurite loss and ultimatively cell death in undifferentiated and pre-differentiated PC12 cells and also in rat primary cortical neurones. Geranylgeranylpyrophosphate, but not farnesylpyrophosphate, squalene or cholesterol, completely compensated for the lack of mevalonate. Our data indicate that, under HMG-CoA reductase inhibition, geranylgeranylpyrophosphate rather than farnesylpyrophosphate or cholesterol is critical for neurite outgrowth and/or maintenance. Loss of neurites is an early manifestation of various neurodegenerative disorders, and dysfunction of isoprenylation might play a role in their pathogenesis.