RESUMO
The Drosophila fruitless (fru) gene is regarded as a master regulator of the formation of male courtship circuitry, yet little is known about its molecular basis of action. We show that roundabout 1 (robo1) knockdown in females promotes formation of the male-specific neurite in sexually dimorphic mAL interneurons and that overexpression of the male-specific Fru(BM) diminishes the expression of Robo1 in the fly brain. Our electrophoretic mobility shift and reporter assays identify the 42-bp segment encompassing the palindrome sequence T T C G C T G C G C C G T G A A in the 5' UTR of robo1 exon1 as the Fru(BM)-responsive element. We find that â¼10-bp deletions in the palindrome sequence induce a loss of the male-specific neurite and disrupt male courtship patterns. This study paves the way for a thorough understanding of the mechanism whereby Fru proteins orchestrate transcription for the formation of courtship circuitry.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/crescimento & desenvolvimento , Receptores Imunológicos/metabolismo , Caracteres Sexuais , Fatores de Transcrição/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Feminino , Masculino , Proteínas RoundaboutRESUMO
Stem cell self-renewal and differentiation are coordinated to maintain tissue homeostasis and prevent cancer. Mutations causing stem cell proliferation are traditionally the focus of cancer studies. However, the contribution of the differentiating stem cell progenies in tumorigenesis is poorly characterized. Here we report that loss of the SOX transcription factor, Sox21a, blocks the differentiation programme of enteroblast (EB), the intestinal stem cell progeny in the adult Drosophila midgut. This results in EB accumulation and formation of tumours. Sox21a tumour initiation and growth involve stem cell proliferation induced by the unpaired 2 mitogen released from accumulating EBs generating a feed-forward loop. EBs found in the tumours are heterogeneous and grow towards the intestinal lumen. Sox21a tumours modulate their environment by secreting matrix metalloproteinase and reactive oxygen species. Enterocytes surrounding the tumours are eliminated through delamination allowing tumour progression, a process requiring JNK activation. Our data highlight the tumorigenic properties of transit differentiating cells.
Assuntos
Carcinogênese/metabolismo , Proteínas de Drosophila/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Intestinos/citologia , Fatores de Transcrição SOXB2/metabolismo , Células-Tronco/fisiologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Janus Quinases/genética , Janus Quinases/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Mutação , Fatores de Transcrição SOXB2/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: The diversification of immune systems during evolution involves the expansion of particular gene families in given phyla. A better understanding of the metazoan immune system requires an analysis of the logic underlying such immune gene amplification. This analysis is now within reach due to the ease with which we can generate multiple mutations in an organism. In this paper, we analyze the contribution of the three Drosophila prophenoloxidases (PPOs) to host defense by generating single, double and triple mutants. PPOs are enzymes that catalyze the production of melanin at the site of infection and around parasites. They are the rate-limiting enzymes that contribute to the melanization reaction, a major immune mechanism of arthropods. The number of PPO-encoding genes is variable among insects, ranging from one in the bee to ten in the mosquito. RESULTS: By analyzing mutations alone and in combination, we ascribe a specific function to each of the three PPOs of Drosophila. Our study confirms that two PPOs produced by crystal cells, PPO1 and PPO2, contribute to the bulk of melanization in the hemolymph, upon septic or clean injury. In contrast, PPO3, a PPO restricted to the D. melanogaster group, is expressed in lamellocytes and contributes to melanization during the encapsulation process. Interestingly, another overlapping set of PPOs, PPO2 and PPO3, achieve melanization of the capsule upon parasitoid wasp infection. CONCLUSIONS: The use of single or combined mutations allowed us to show that each PPO mutant has a specific phenotype, and that knocking out two of three genes is required to abolish fully a particular function. Thus, Drosophila PPOs have partially overlapping functions to optimize melanization in at least two conditions: following injury or during encapsulation. Since PPO3 is restricted to the D. melanogaster group, this suggests that production of PPO by lamellocytes emerged as a recent defense mechanism against parasitoid wasps. We conclude that differences in spatial localization, immediate or late availability, and mode of activation underlie the functional diversification of the three Drosophila PPOs, with each of them having non-redundant but overlapping functions.
Assuntos
Catecol Oxidase/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/imunologia , Precursores Enzimáticos/genética , Imunidade Inata , Animais , Catecol Oxidase/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/parasitologia , Precursores Enzimáticos/metabolismo , Feminino , Imunidade Inata/genética , Vespas/fisiologiaRESUMO
To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).
Assuntos
Drosophila/genética , Interferência de RNA , Acesso à Informação , Animais , Animais Geneticamente Modificados , Pesquisa Biomédica , Boston , Genes de Insetos , Vetores Genéticos , Faculdades de MedicinaRESUMO
Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, ß1,4-galactosyltransferase I, ß1,3-galactosyltransferase II, and ß1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.
Assuntos
Inativação Gênica , Família Multigênica , Fenótipo , Interferência de RNA , Animais , Drosophila , Glicosilação , Glicosiltransferases/metabolismo , Dados de Sequência Molecular , Polissacarídeos/genéticaRESUMO
The innate immune system is the first line of defense encountered by invading pathogens. Delayed and/or inadequate innate immune responses can result in failure to combat pathogens, whereas excessive and/or inappropriate responses cause runaway inflammation. Therefore, immune responses are tightly regulated from initiation to resolution and are repressed during the steady state. It is well known that glycans presented on pathogens play important roles in pathogen recognition and the interactions between host molecules and microbes; however, the function of glycans of host organisms in innate immune responses is less well known. Here, we show that innate immune quiescence and strength of the immune response are controlled by host glycosylation involving a novel UDP-galactose transporter called Senju. In senju mutants, reduced expression of galactose-containing glycans resulted in hyperactivation of the Toll signaling pathway in the absence of immune challenges. Genetic epistasis and biochemical analyses revealed that Senju regulates the Toll signaling pathway at a step that converts Toll ligand Spatzle to its active form. Interestingly, Toll activation in immune-challenged wild type (WT) flies reduced the expression of galactose-containing glycans. Suppression of the degalactosylation by senju overexpression resulted in reduced induction of Toll-dependent expression of an antimicrobial peptide, Drosomycin, and increased susceptibility to infection with Gram-positive bacteria. These data suggest that Senju-mediated galactosylation suppresses undesirable Toll signaling activation during the steady state; however, Toll activation in response to infection leads to degalactosylation, which raises the immune response to an adequate level and contributes to the prompt elimination of pathogens.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/imunologia , Glicosilação , Imunidade Inata , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Transporte de Monossacarídeos/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epistasia Genética , Galactose/química , Sistema Imunitário , Lectinas/química , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Polissacarídeos/química , Recombinação Genética , Receptores Toll-Like/metabolismoRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5-10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions. We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci - TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression. The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease.
RESUMO
We report a simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila. The system comprises two transgenic strains: one expressing Cas9 protein from the germline-specific nanos promoter and the other ubiquitously expressing a custom guide RNA (gRNA) that targets a unique site in the genome. The two strains are crossed to form an active Cas9-gRNA complex specifically in germ cells, which cleaves and mutates the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described. Founder animals stably expressing Cas9-gRNA transmitted germline mutations to an average of 60% of their progeny, a dramatic improvement in efficiency over the previous methods based on transient Cas9 expression. Simultaneous cleavage of two sites by co-expression of two gRNAs efficiently induced internal deletion with frequencies of 4.3-23%. Our method is readily scalable to high-throughput gene targeting, thereby accelerating comprehensive functional annotation of the Drosophila genome.
Assuntos
Proteínas Associadas a CRISPR/genética , Drosophila melanogaster/genética , Marcação de Genes/métodos , Mutação em Linhagem Germinativa , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Feminino , Expressão Gênica , Genes de Insetos , Masculino , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Pequeno RNA não TraduzidoRESUMO
Notch signaling is an evolutionarily conserved mechanism that controls many cell-fate specifications through local cell-cell interactions. The core mechanisms of Notch activation and its subsequent intracellular signaling are well understood. Various cellular functions are required for the activation and regulation of Notch signaling. Among them, the endocytosis of Notch and its ligands is important for the activation and suppression of Notch signaling. The endosomal sorting complex required for transport (ESCRT) proteins are required to sort ubiquitinated membrane proteins, such as Notch, into early endosomes. A loss-of-function allele of vacuolar protein sorting 2 (vps2), which encodes a component of ESCRT-III, has been reported. However, this vps2 mutant still produces the N-terminal half of the protein, and its phenotypes were studied in only a few organs. Here, we generated the first null mutant allele of Drosophila vps2, designated vps2², to better understand the function of this gene. In Drosophila wing imaginal discs homozygous for the vps2² allele, early endosomes and multivesicular bodies (MVBs) were enlarged, and Notch and Delta accumulated inside them. As reported for the previous vps2 mutant, the epithelium grew excessively under this condition. We further studied the roles of vps2 by RNA interference-knockdown. These experiments revealed that a partial reduction of vps2 attenuated Notch signaling; in contrast, the loss-of-function vps2 mutant is reported to up-regulate the Notch signaling in eye imaginal disc cells. These results suggest that Notch signaling can be up- or down-regulated, depending on the level of vps2 expression. Finally, we found that vps2 overexpression also resulted in early-endosome enlargement and the accumulation of Notch and Delta. In these cells, a portion of the Vps2 protein was detected in MVBs and colocalized with Notch. These data indicate that the expression of vps2 must be precisely regulated to maintain the normal structure of early endosomes.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Receptores Notch/metabolismo , Transdução de Sinais , Alelos , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/genética , Interferência de RNA , Receptores Notch/genéticaRESUMO
The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial-and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complexo de Golgi/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/biossíntese , Sialoglicoproteínas/biossíntese , Animais , Anticorpos Monoclonais , Transporte Biológico , Biomarcadores , Células Cultivadas , Drosophila melanogaster/citologia , Glicosilação , Glicosiltransferases/metabolismo , Complexo de Golgi/ultraestrutura , Imuno-Histoquímica , Processamento de Proteína Pós-Traducional , Ratos , Receptores de Fatores de Crescimento de Fibroblastos/imunologia , Sialoglicoproteínas/imunologiaRESUMO
Recent studies have demonstrated protective roles for autophagy in various neurodegenerative disorders, including the polyglutamine diseases; however, the role of autophagy in retinal degeneration has remained unclear. Accumulation of activated rhodopsin in some Drosophila mutants leads to retinal degeneration, and although it is known that activated rhodopsin is degraded in endosomal pathways in normal photoreceptor cells, the contribution of autophagy to rhodopsin regulation has remained elusive. This study reveals that activated rhodopsin is degraded by autophagy in collaboration with endosomal pathways to prevent retinal degeneration. Light-dependent retinal degeneration in the Drosophila visual system is caused by the knockdown or mutation of autophagy-essential components, such as autophagy-related protein 7 and 8 (atg-7/atg-8), or genes essential for PE (phosphatidylethanolamine) biogenesis and autophagosome formation, including Phosphatidylserine decarboxylase (Psd) and CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (Ept). The knockdown of atg-7/8 or Psd/Ept produced an increase in the amount of rhodopsin localized to Rab7-positive late endosomes. This rhodopsin accumulation, followed by retinal degeneration, was suppressed by overexpression of Rab7, which accelerated the endosomal degradation pathway. These results indicate a degree of cross talk between the autophagic and endosomal/lysosomal pathways. Importantly, a reduction in rhodopsin levels rescued Psd knockdown-induced retinal degeneration. Additionally, the Psd knockdown-induced retinal degeneration phenotype was enhanced by Ppt1 inactivation, which causes infantile neuronal ceroid lipofuscinosis, implying that autophagy plays a significant role in its pathogenesis. Collectively, the current data reveal that autophagy suppresses light-dependent retinal degeneration in collaboration with the endosomal degradation pathway and that rhodopsin is a key substrate for autophagic degradation in this context.
Assuntos
Autofagia/fisiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Degeneração Retiniana/prevenção & controle , Rodopsina/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Endossomos/ultraestrutura , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Marcação In Situ das Extremidades Cortadas/métodos , Larva , Luz/efeitos adversos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Microscopia Imunoeletrônica/métodos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras de Invertebrados/ultraestrutura , Interferência de RNA/fisiologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/genética , Rodopsina/genética , Estatísticas não Paramétricas , Tioléster Hidrolases , Fatores de Tempo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Walker-Warburg syndrome, a progressive muscular dystrophy, is a severe disease with various kinds of symptoms such as muscle weakness and occasional seizures. The genes of protein O-mannosyltransferases 1 and 2 (POMT1 and POMT2), fukutin, and fukutin-related protein are responsible for this syndrome. In our previous study, we cloned Drosophila orthologs of human POMT1 and POMT2 and identified their activity. However, the mechanism of onset of this syndrome is not well understood. Furthermore, little is known about the behavioral properties of the Drosophila POMT1 and POMT2 mutants, which are called rotated abdomen (rt) and twisted (tw), respectively. First, we performed various kinds of behavioral tests and described in detail the muscle structures by using these mutants. The mutant flies exhibited abnormalities in heavy exercises such as climbing or flight but not in light movements such as locomotion. Defective motor function in mutants appeared immediately after eclosion and was exaggerated with aging. Along with motor function, muscle ultrastructure in the tw mutant was altered, as seen in human patients. We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively. Flies expressing RNAi had reduced lifespans. These findings clearly demonstrate that Drosophila POMT mutants are models for human muscular dystrophy. We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity. In this paper, we propose a novel mechanism for the development of muscular dystrophy: POMT mutation causes high myoblast density and position derangement, which result in apoptosis, muscle disorganization, and muscle cell defects.
Assuntos
Apoptose/fisiologia , Distrofias Musculares/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Animais , Apoptose/genética , Western Blotting , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Imuno-Histoquímica , Locomoção/genética , Locomoção/fisiologia , Longevidade/genética , Longevidade/fisiologia , Manosiltransferases/genética , Manosiltransferases/metabolismo , Microscopia Eletrônica de Transmissão , Distrofias Musculares/genética , Reação em Cadeia da Polimerase , Interferência de RNARESUMO
Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA-binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Genômica , Animais , Drosophila melanogaster/genética , Glicosilação , Sistema Nervoso/metabolismo , Especificidade de ÓrgãosRESUMO
The class III phosphatidylinositol-3 kinase (PI3K (III)) regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate (PI(3)P). While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes.
Assuntos
Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Asas de Animais/metabolismo , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Drosophila , Drosophila melanogaster/metabolismo , Endocitose , Lisossomos/metabolismo , Mutação , Fenótipo , Estrutura Terciária de ProteínaRESUMO
The genetically amenable organism Drosophila melanogaster has been estimated to have 14,076 protein coding genes in the genome, according to the flybase release note R5.13 (http://flybase.bio.indiana.edu/static_pages/docs/release_notes.html). Recent application of RNA interference (RNAi) to the study of developmental biology in Drosophila has enabled us to carry out a systematic investigation of genes affecting various specific phenotypes. In order to search for genes supporting cell survival, we conducted an immunohistochemical examination in which the RNAi of 2,497 genes was independently induced within the dorsal compartment of the wing imaginal disc. Under these conditions, the activities of a stress-activated protein kinase JNK (c-Jun N-terminal kinase) and apoptosis-executing factor Caspase-3 were monitored. Approximately half of the genes displayed a strong JNK or Caspase-3 activation when their RNAi was induced. Most of the JNK activation accompanied Caspase-3 activation, while the opposite did not hold true. Interestingly, the area activating Caspase-3 was more broadly seen than that activating JNK, suggesting that JNK is crucial for induction of non-autonomous apoptosis in many cases. Furthermore, the RNAi of essential factors commonly regulating transcription and translation showed a severe and cell-autonomous apoptosis but also elicited another apoptosis at an adjacent area in a non-autonomous way. We also found that the frequency of apoptosis varies depending on the tissues.
RESUMO
Epidermal cell migration is critical for restoration of tissue structure and function after damage. However, the mechanisms by which differentiated cells neighboring the wound sense the wound and assume a motile phenotype remain unclear. Here, we show that Pvr, a receptor tyrosine kinase (RTK) related to platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptors, and one of its ligands, Pvf1, are required for epidermal wound closure. Morphological comparison of wound-edge cells lacking Pvr or the Jun N-terminal kinase (JNK) signaling pathway previously implicated in larval wound closure suggests that Pvr signaling leads wound-margin epidermal cells to extend actin-based cell processes into the wound gap while JNK mediates transient dedifferentiation of cells at the wound margin. Genetic epistasis experiments reinforce the conclusion that the JNK and Pvr signaling pathways act in parallel. Tissue-specific knockdown and rescue experiments suggest that epidermally derived Pvf1 may be sequestered in the blood and that tissue damage exposes blood-borne Pvf1 to Pvr receptors on wound-edge epidermal cells and initiates the extension of cell processes into the wound gap. These results uncover a novel mechanism of sensing tissue damage and suggest that PDGF/VEGF ligands and receptors may play a conserved autocrine role in epidermal wound closure.
Assuntos
Movimento Celular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Proteínas do Ovo/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Drosophila/citologia , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Epistasia Genética , Hemolinfa/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Larva/citologia , Larva/fisiologia , Ligantes , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Interferência de RNA , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
BACKGROUND: A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate alpha-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. CONCLUSIONS/SIGNIFICANCE: These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.
Assuntos
Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Polissacarídeos/biossíntese , Animais , Glicosilação , Espectrometria de Massas/métodos , Redes e Vias Metabólicas , Polissacarídeos/química , Polissacarídeos/metabolismo , Conformação ProteicaRESUMO
T antigen (Galbeta1-3GalNAcalpha1-Ser/Thr), the well-known tumor-associated antigen, is a core 1 mucin-type O-glycan structure that is synthesized by core 1 beta1,3-galactosyltransferase (C1beta3GalT), which transfers Gal from UDP-Gal to Tn antigen (GalNAcalpha1-Ser/Thr). Three putative C1beta3GalTs have been identified in Drosophila. However, although all three are expressed in embryos, their roles during embryogenesis have not yet been clarified. In this study, we used P-element inserted mutants to show that CG9520, one of the three putative C1beta3GalTs, synthesizes T antigen expressed on the central nervous system (CNS) during embryogenesis. We also found that T antigen was expressed on a subset of the embryonic hemocytes. CG9520 mutant embryos showed the loss of T antigens on the CNS and on a subset of hemocytes. Then, the loss of T antigens was rescued by precise excision of the P-element inserted into the CG9520 gene. Our data demonstrate that T antigens expressed on the CNS and on a subset of hemocytes are synthesized by CG9520 in the Drosophila embryo. In addition, we found that the number of circulating hemocytes was reduced in third instar larvae of CG9520 mutant. We, therefore, named the CG9520 gene Drosophila core 1 beta1,3-galactosyltransferase 1 because it is responsible for the synthesis and function of T antigen in vivo.
Assuntos
Antígenos Glicosídicos Associados a Tumores/biossíntese , Proteínas de Drosophila/genética , Drosophila/embriologia , Drosophila/enzimologia , Galactosiltransferases/genética , Hemócitos/metabolismo , Animais , Antígenos Glicosídicos Associados a Tumores/imunologia , Sistema Nervoso Central/metabolismo , Drosophila/genética , Embrião não Mamífero/enzimologia , Histocitoquímica , Mutação , FilogeniaRESUMO
The Tol2 element is a transposon found from a genome of a vertebrate, a small teleost medaka fish. Tol2 encodes a gene for a transposase which is active in vertebrate animals so far tested; for instance, in fish, frog, chicken and mammals, and transgenesis methods using Tol2 have been developed in these model vertebrates. However, it has not been known whether Tol2 can transpose in animals other than vertebrates. Here we report transposition of Tol2 in an invertebrate Drosophila melanogaster. First, we injected a transposon donor plasmid containing a Tol2 construct and mRNA encoding the Tol2 transposase into Drosophila eggs, and found that the Tol2 construct could be excised from the plasmid. Second, we crossed the injected flies, raised the offspring, and found that the Tol2 construct was integrated into the genome of germ cells and transmitted to the next generation. Finally, we constructed a Tol2 construct containing the white gene and injected the transposon donor plasmid and the transposase mRNA into fertilized eggs from the white mutant. We analyzed their offspring, and found that G1 flies with wild type red eyes could be obtained from 35% of the injected fly. We cloned and sequenced 34 integration loci from these lines and showed that these insertions were indeed created through transposition and distributed throughout the genome. Our present study demonstrates that the medaka fish Tol2 transposable element does not require vertebrate-specific host factors for its transposition, and also provides a possibility that Tol2 may be used as a new genetic tool for transgenesis and genome analysis in Drosophila.