RESUMO
In vertebrates, mutations in Protein O-mannosyltransferase1 (POMT1) or POMT2 are associated with muscular dystrophy due to a requirement for O-linked mannose glycans on the Dystroglycan (Dg) protein. In this study we examine larval body wall muscles of Drosophila mutant for Dg, or RNA interference knockdown for Dg and find defects in muscle attachment, altered muscle contraction, and a change in muscle membrane resistance. To determine if POMTs are required for Dg function in Drosophila, we examine larvae mutant for genes encoding POMT1 or POMT2. Larvae mutant for either POMT, or doubly mutant for both, show muscle attachment and muscle contraction phenotypes identical to those associated with reduced Dg function, consistent with a requirement for O-linked mannose on Drosophila Dg. Together these data establish a central role for Dg in maintaining integrity in Drosophila larval muscles and demonstrate the importance of glycosylation to Dg function in Drosophila. This study opens the possibility of using Drosophila to investigate muscular dystrophy.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Distroglicanas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Manosiltransferases/metabolismo , Músculos/metabolismo , Animais , Drosophila melanogaster/genética , Distroglicanas/genética , Eletrofisiologia , Laminina/metabolismo , Larva/genética , Larva/metabolismo , Manosiltransferases/genética , Músculos/anormalidades , Mutação/genética , Interferência de RNA , Sarcômeros/metabolismoRESUMO
N-ethylmaleimide sensitive factor (NSF) can dissociate the soluble NSF attachment receptor (SNARE) complex, but NSF also participates in other intracellular trafficking functions by virtue of SNARE-independent activity. Drosophila that express a neural transgene encoding a dominant-negative form of NSF2 show an 80% reduction in the size of releasable synaptic vesicle pool, but no change in the number of vesicles in nerve terminal boutons. Here we tested the hypothesis that vesicles in the NSF2 mutant terminal are less mobile. Using a combination of genetics, pharmacology, and imaging we find a substantial reduction in vesicle mobility within the nerve terminal boutons of Drosophila NSF2 mutant larvae. Subsequent analysis revealed a decrease of filamentous actin in both NSF2 dominant-negative and loss-of-function mutants. Lastly, actin-filament disrupting drugs also decrease vesicle movement. We conclude that a factor contributing to the NSF mutant phenotype is a reduction in vesicle mobility, which is associated with decreased presynaptic F-actin. Our data are consistent with a model in which actin filaments promote vesicle mobility and suggest that NSF participates in establishing or maintaining this population of actin.
Assuntos
Actinas/metabolismo , Alelos , Drosophila/metabolismo , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Drosophila/citologia , Drosophila/efeitos dos fármacos , Recuperação de Fluorescência Após Fotodegradação , Larva/citologia , Larva/efeitos dos fármacos , Toxinas Marinhas/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Tiazolidinas/farmacologia , Fatores de TempoRESUMO
Adult Drosophila mutant for the glycosyltransferase beta1,4-N-acetlygalactosaminyltransferase-A (beta4GalNAcTA) display an abnormal locomotion phenotype, indicating a role for this enzyme, and the glycan structures that it generates, in the neuromuscular system. To investigate the functional role of this enzyme in more detail, we turned to the accessible larval neuromuscular system and report here that larvae mutant for beta4GalNAcTA display distinct nerve and muscle phenotypes. Mutant larvae exhibit abnormal backward crawling, reductions in nerve terminal bouton number, decreased spontaneous transmitter-release frequency, and short, wide muscles. This muscle shape change appears to result from hypercontraction since the individual sarcomeres are shorter in mutant muscles. Analysis of muscle calcium signals showed altered calcium handling in the mutant, suggesting a mechanism by which hypercontraction could occur. All of these phenotypes can be rescued by a transgene carrying the beta4GalNAcTA genomic region. Tissue-specific expression, using the Gal4-UAS system, reveals that neural expression rescues the mutant crawling phenotype, while muscle expression rescues the muscle defect. Tissue-specific expression did not appear to rescue the decrease in neuromuscular junction bouton number, suggesting that this defect arises from cooperation between nerve and muscle. Altogether, these results suggest that beta4GalNAcTA has at least three distinct functional roles.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Músculos/enzimologia , N-Acetilgalactosaminiltransferases/metabolismo , Neurônios/enzimologia , Animais , Comportamento Animal , Cálcio/metabolismo , Tamanho Celular , Potenciais Pós-Sinápticos Excitadores , Larva/enzimologia , Contração Muscular , Mutação/genética , Junção Neuromuscular/citologia , Fenótipo , Terminações Pré-Sinápticas , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Mutations that diminish the function of the extracellular matrix receptor Dystroglycan (DG) result in muscular dystrophies, with associated neuronal migration defects in the brain and mental retardation e.g. Muscle Eye Brain Disease. To gain insight into the function of DG in the nervous system we initiated a study to examine its contribution to development of the eye of Drosophila melanogaster. Immuno-histochemistry showed that DG is concentrated on the apical surface of photoreceptors (R) cells during specification of cell-fate in the third instar larva and is maintained at this location through early pupal stages. In point mutations that are null for DG we see abortive R cell elongation during differentiation that first appears in the pupa and results in stunted R cells in the adult. Overexpression of DG in R cells results in a small but significant increase in their size. R cell differentiation defects appear at the same stage in a deficiency line Df(2R)Dg(248) that affects Dg and the neighboring mitochondrial ribosomal gene, mRpL34. In the adult, these flies have severely disrupted R cells as well as defects in the lens and ommatidia. Expression of an mRpL34 transgene rescues much of this phenotype. We conclude that DG does not affect neuronal commitment but functions R cell autonomously to regulate neuronal elongation during differentiation in the pupa. We discuss these findings in view of recent work implicating DG as a regulator of cell metabolism and its genetic interaction with mRpL34, a member of a class of mitochondrial genes essential for normal metabolic function.
Assuntos
Diferenciação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Distroglicanas/genética , Olho/patologia , Proteínas Mitocondriais/genética , Proteínas Ribossômicas/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Linhagem da Célula , Sobrevivência Celular , Proteínas de Drosophila/metabolismo , Distroglicanas/metabolismo , Olho/metabolismo , Olho/ultraestrutura , Larva , Proteínas Mitocondriais/metabolismo , Mutação/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/patologia , Células Fotorreceptoras de Invertebrados/ultraestrutura , Proteínas Ribossômicas/metabolismo , Propriedades de Superfície , Transgenes/genéticaRESUMO
Drosophila melanogaster has two beta4-N-acetylgalactosaminyltransferases, beta4GalNAcTA and beta4GalNAcTB, that are able to catalyse the formation of lacdiNAc (GalNAcbeta,4GlcNAc). LacdiNAc is found as a structural element of Drosophila glycosphingolipids (GSLs) suggesting that beta4GalNAcTs contribute to the generation of GSL structures in vivo. Mutations in Egghead and Brainaic, enzymes that generate the beta4GalNAcT trisaccharide acceptor structure GlcNAcbeta,3Manbeta,4GlcbetaCer, are lethal. In contrast, flies doubly mutant for the beta4GalNAcTs are viable and fertile. Here, we describe the structural analysis of the GSLs in beta4GalNAcT mutants and find that in double mutant flies no lacdiNAc structure is generated and the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer accumulates. We also find that phosphoethanolamine transfer to GlcNAc in the trisaccharide does not occur, demonstrating that this step is dependent on prior or simultaneous transfer of GalNAc. By comparing GSL structures generated in the beta4GalNAcT single mutants we show that beta4GalNAcTB is the major enzyme for the overall GSL biosynthesis in adult flies. In beta4GalNAcTA mutants, composition of GSL structures is indistinguishable from wild-type animals. However, in beta4GalNAcTB mutants precursor structures are accumulating in different steps of GSL biosynthesis, without the complete loss of lacdiNAc, indicating that beta4GalNAcTA plays a minor role in generating GSL structures. Together our results demonstrate that both beta4GalNAcTs are able to generate lacdiNAc structures in Drosophila GSL, although with different contributions in vivo, and that the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer is sufficient to avoid the major phenotypic consequences associated with the GSL biosynthetic defects in Brainiac or Egghead.
Assuntos
Drosophila melanogaster/enzimologia , Glicoesfingolipídeos/biossíntese , N-Acetilgalactosaminiltransferases/fisiologia , Animais , Animais Geneticamente Modificados , Sequência de Carboidratos , Drosophila melanogaster/genética , Isoenzimas/genética , Isoenzimas/fisiologia , Dados de Sequência Molecular , Mutação , N-Acetilgalactosaminiltransferases/genéticaRESUMO
Notch signaling plays critical roles in animal development and physiology. The activation of Notch receptors by their ligands is modulated by Fringe-dependent glycosylation. Fringe catalyzes the addition of N-acetylglucosamine in a beta1,3 linkage onto O-fucose on epidermal growth factor-like domains. This modification of Notch by Fringe influences the binding of Notch ligands to Notch receptors. However, prior studies have relied on in vivo glycosylation, leaving unresolved the question of whether addition of N-acetylglucosamine is sufficient to modulate Notch-ligand interactions on its own, or whether instead it serves as a precursor to subsequent post-translational modifications. Here, we describe the results of in vitro assays using purified components of the Drosophila Notch signaling pathway. In vitro glycosylation and ligand binding studies establish that the addition of N-acetylglucosamine onto O-fucose in vitro is sufficient both to enhance Notch binding to the Delta ligand and to inhibit Notch binding to the Serrate ligand. Further elongation by galactose does not detectably influence Notch-ligand binding in vitro. Consistent with these observations, carbohydrate compositional analysis and mass spectrometry on Notch isolated from cells identified only N-acetylglucosamine added onto Notch in the presence of Fringe. These observations argue against models in which Fringe-dependent glycosylation modulates Notch signaling by acting as a precursor to subsequent modifications and instead establish the simple addition of N-acetylglucosamine as a basis for the effects of Fringe on Drosophila Notch-ligand binding.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Animais , Carboidratos/química , Fucose/química , Glicosilação , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Jagged-1 , Ligantes , Modelos Biológicos , Mutação , Ligação Proteica , Receptores Notch , Proteínas Serrate-Jagged , TransgenesRESUMO
Members of the mammalian beta1,4-galactosyltransferase family are among the best studied glycosyltransferases, but the requirements for all members of this family within an animal have not previously been determined. Here, we describe analysis of two Drosophila genes, beta4GalNAcTA (CG8536) and beta4GalNAcTB (CG14517), that are homologous to mammalian beta1,4-galactosyltransferases. Like their mammalian homologs, these glycosyltransferases use N-acetylglucosamine as an acceptor substrate. However, they transfer N-acetylgalactosamine rather than galactose. This activity, together with amino acid sequence similarity, places them among a group of recently identified invertebrate beta1,4-N-acetylgalactosaminyltransferases. To investigate the biological functions of these genes, null mutations were generated by imprecise excision of a transposable element (beta4GalNAcTA) or by gene-targeted homologous recombination (beta4GalNAcTB). Flies mutant for beta4GalNAcTA are viable and fertile but display behavioral phenotypes suggestive of essential roles for GalNAc-beta1,4-GlcNAc containing glycoconjugates in neuronal and/or muscular function. beta4GalNAcTB mutants are viable and display no evident morphological or behavioral phenotypes. Flies doubly mutant for both genes display only the behavioral phenotypes associated with mutation of beta4GalNAcTA. Thus Drosophila homologs of the mammalian beta4GalT family are essential for neuromuscular physiology or development but are not otherwise required for viability, fertility, or external morphology.
Assuntos
Proteínas de Drosophila/genética , Mutação , N-Acetilgalactosaminiltransferases/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Intracellular post-translational modifications such as phosphorylation and ubiquitylation have been well studied for their roles in regulating diverse signalling pathways, but we are only just beginning to understand how differential glycosylation is used to regulate intercellular signalling. Recent studies make clear that extracellular post-translational modifications, in the form of glycosylation, are essential for the Notch signalling pathway, and that differences in the extent of glycosylation are a significant mechanism by which this pathway is regulated.
Assuntos
Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Animais , Relógios Biológicos , Padronização Corporal , Proteínas de Drosophila , Fator de Crescimento Epidérmico/metabolismo , Fucose/química , Fucose/metabolismo , Fucosiltransferases/metabolismo , Glicosilação , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Notch , Asas de Animais/fisiologiaRESUMO
In mammals, the xylosylprotein beta4-galactosyltransferase termed beta4GalT7 (XgalT-1, EC ) participates in proteoglycan biosynthesis through the transfer of galactose to the xylose that initiates each glycosaminoglycan chain. A Drosophila cDNA homologous to mammalian beta4-galactosyltransferases was identified using a human beta4GalT7 cDNA as a probe in a BLAST analysis of expressed sequence tags. The Drosophila cDNA encodes a type II membrane protein with 322 amino acids and shows 49% identity to human beta4GalT7. Extracts from L cells transfected with the cDNA exhibited marked galactosyltransferase activity specific for a xylopyranoside acceptor. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis in beta4GalT7-deficient Chinese hamster ovary cells. In transfectant lysates the properties of Drosophila and human beta4GalT7 resembled each other, except that Drosophila beta4GalT7 showed a less restricted specificity and was active at a wider range of temperatures. Drosophila beta4GalT7 is expressed throughout development, with higher expression levels in adults. Reduction of Drosophila beta4GalT7 levels using expressed RNA interference (RNAi) in imaginal discs resulted in an abnormal wing and leg morphology similar to that of flies with defective Hedgehog and Decapentaplegic signaling, which are known to depend on intact proteoglycan biosynthesis. Immunohistochemical analysis of tissues confirmed that both heparan sulfate and chondroitin sulfate biosynthesis were impaired. Our results demonstrate that Drosophila beta4GalT7 has the in vitro and in vivo properties predicted for an ortholog of human beta4GalT7 and is essential for normal animal development through its role in proteoglycan biosynthesis.