RESUMO
Mutations in Drosophila Swiss cheese (SWS) gene or its vertebrate orthologue neuropathy target esterase (NTE) lead to progressive neuronal degeneration in flies and humans. Despite its enzymatic function as a phospholipase is well established, the molecular mechanism responsible for maintaining nervous system integrity remains unclear. In this study, we found that NTE/SWS is present in surface glia that forms the blood-brain barrier (BBB) and that NTE/SWS is important to maintain its structure and permeability. Importantly, BBB glia-specific expression of Drosophila NTE/SWS or human NTE in the sws mutant background fully rescues surface glial organization and partially restores BBB integrity, suggesting a conserved function of NTE/SWS. Interestingly, sws mutant glia showed abnormal organization of plasma membrane domains and tight junction rafts accompanied by the accumulation of lipid droplets, lysosomes, and multilamellar bodies. Since the observed cellular phenotypes closely resemble the characteristics described in a group of metabolic disorders known as lysosomal storage diseases (LSDs), our data established a novel connection between NTE/SWS and these conditions. We found that mutants with defective BBB exhibit elevated levels of fatty acids, which are precursors of eicosanoids and are involved in the inflammatory response. Also, as a consequence of a permeable BBB, several innate immunity factors are upregulated in an age-dependent manner, while BBB glia-specific expression of NTE/SWS normalizes inflammatory response. Treatment with anti-inflammatory agents prevents the abnormal architecture of the BBB, suggesting that inflammation contributes to the maintenance of a healthy brain barrier. Considering the link between a malfunctioning BBB and various neurodegenerative diseases, gaining a deeper understanding of the molecular mechanisms causing inflammation due to a defective BBB could help to promote the use of anti-inflammatory therapies for age-related neurodegeneration.
Assuntos
Barreira Hematoencefálica , Hidrolases de Éster Carboxílico , Ácidos Graxos , Inflamação , Neuroglia , Animais , Barreira Hematoencefálica/metabolismo , Ácidos Graxos/metabolismo , Inflamação/metabolismo , Neuroglia/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/patologia , Humanos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genéticaRESUMO
Translational readthrough (TR) occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR has been identified in several eukaryotic organisms; however, its biological significance and mechanism remain unclear. Here, we quantify TR of several candidate genes in Drosophila melanogaster and characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj). Using CRISPR/Cas9-generated mutant flies, we show that the TR-generated Tj isoform is expressed in a subset of neural cells of the central nervous system and is excluded from the somatic cells of gonads. Control of TR in Tj is critical for preservation of neuronal integrity and maintenance of reproductive health. The tissue-specific distribution of a release factor splice variant, eRF1H, plays a critical role in modulating differential TR of leaky stop codon contexts. Fine-tuning of gene regulatory functions of transcription factors by TR provides a potential mechanism for cell-specific regulation of gene expression.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Fatores de Transcrição Maf Maior/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição , Animais , Códon de Terminação/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Adult stem cells depend on their niches for regulatory signaling that controls their maintenance, division, and their progeny differentiation. While communication between various types of stem cells and their niches is becoming clearer, the process of stem cell niche establishment is still not very well understood. Model genetic organisms provide simplified systems to address various complex questions, for example, how is a stem cell niche formed? What signaling cascades induce the stem cell niche formation? Are the mechanisms of stem cell niche formation conserved? Notch signaling is an evolutionarily conserved pathway first identified in fruit flies, crucial in fate acquisition and spatiotemporal patterning. While the core logic behind its activity is fairly simple and requires direct cell-cell interaction, it reaches an astonishing complexity and versatility by combining its different modes of action. Subtleties such as equivalency between communicating cells, their physical distance, receptor and ligand processing, and endocytosis can have an effect on the way the events unfold, and this review explores some important general mechanisms of action, later on focusing on its involvement in stem cell niche formation. First, looking at invertebrates, we will examine how Notch signaling induces the formation of germline stem cell niche in male and female Drosophila. In the developing testis, a group of somatic gonadal precursor cells receive Delta signals from the gut, activating Notch signaling and sealing their fate as niche cells even before larval hatching. Meanwhile, the ovarian germline stem cell niche is built later during late larval stages and requires a two-step process that involves terminal filament formation and cap cell specification. Intriguingly, double security mechanisms of Notch signaling activation coordinated by the soma or the germline control both steps to ensure the robustness of niche assembly. Second, in the vast domains of mammalian cellular signaling, there is an emerging picture of Notch being an active player in a variety of tissues in health and disease. Notch involvement has been shown in stem cell niche establishment in multiple organs, including the brain, muscle, and intestine, where the stem cell niches are essential for the maintenance of adult stem cells. But adult stem cells are not the only cells looking for a home. Cancer stem cells use Notch signaling at specific stages to gain an advantage over endogenous tissue and overpower it, at the same time acquiring migratory and invasive abilities to claim new tissues (e.g., bone) as their territory. Moreover, in vitro models such as organoids reveal similar Notch employment when it comes to the developing stem cell niches. Therefore, a better understanding of the processes regulating stem cell niche assembly is key for the fields of stem cell biology and regenerative medicines.
RESUMO
Various neurodegenerative disorders are associated with human NTE/PNPLA6 dysfunction. Mechanisms of neuropathogenesis in these diseases are far from clearly elucidated. Hereditary spastic paraplegia belongs to a type of neurodegeneration associated with NTE/PNLPLA6 and is implicated in neuron death. In this study, we used Drosophila melanogaster to investigate the consequences of neuronal knockdown of swiss cheese (sws)-the evolutionarily conserved ortholog of human NTE/PNPLA6-in vivo. Adult flies with the knockdown show longevity decline, locomotor and memory deficits, severe neurodegeneration progression in the brain, reactive oxygen species level acceleration, mitochondria abnormalities and lipid droplet accumulation. Our results suggest that SWS/NTE/PNPLA6 dysfunction in neurons induces oxidative stress and lipid metabolism alterations, involving mitochondria dynamics and lipid droplet turnover in neurodegeneration pathogenesis. We propose that there is a complex mechanism in neurological diseases such as hereditary spastic paraplegia, which includes a stress reaction, engaging mitochondria, lipid droplets and endoplasmic reticulum interplay.
Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Encéfalo/citologia , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster , Metabolismo dos Lipídeos , Mitocôndrias/ultraestrutura , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Estresse OxidativoRESUMO
Here we show that multiple modes of Notch signaling activation specify the complexity of spatial cellular interactions necessary for stem cell niche assembly. In particular, we studied the formation of the germline stem cell niche in Drosophila ovaries, which is a two-step process whereby terminal filaments are formed first. Then, terminal filaments signal to the adjacent cap cell precursors, resulting in Notch signaling activation, which is necessary for the lifelong acquisition of stem cell niche cell fate. The genetic data suggest that in order to initiate the process of stem cell niche assembly, Notch signaling is activated among non-equipotent cells via distant induction, where germline Delta is delivered to somatic cells located several diameters away via cellular projections generated by primordial germ cells. At the same time, to ensure the robustness of niche formation, terminal filament cell fate can also be induced by somatic Delta via cis- or trans-inhibition. This exemplifies a double security mechanism that guarantees that the germline stem cell niche is formed, since it is indispensable for the adjacent germline precursor cells to acquire and maintain stemness necessary for successful reproduction. These findings contribute to our understanding of the formation of stem cell niches in their natural environment, which is important for stem cell biology and regenerative medicine.
Assuntos
Receptores Notch/metabolismo , Transdução de Sinais , Nicho de Células-Tronco , Células-Tronco/metabolismo , Animais , Diferenciação Celular/genética , Drosophila , Feminino , Células Germinativas/metabolismo , Modelos Biológicos , Mutação , Organogênese/genética , Ovário/embriologia , Ovário/metabolismo , Receptores Notch/genética , Nicho de Células-Tronco/genéticaRESUMO
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with a very poor prognosis and constantly growing incidence. Among other primary risks of HCC, metabolic disorders and obesity have been extensively investigated over recent decades. The latter can promote nonalcoholic fatty liver disease (NAFLD) leading to the inflammatory form of nonalcoholic steatohepatitis (NASH), that, in turn, promotes HCC. Molecular determinants of this pathogenic progression, however, remain largely undefined. In this study, we have focussed on the investigation of α-dicarbonyl compounds (α-dC), highly reactive and tightly associated with overweight-induced metabolic disorders, and studied their potential role in NAFLD and progression toward HCC using murine models. NAFLD was induced using high-fat diet (HFD). Autochthonous HCC was induced using transposon-based stable intrahepatic overexpression of oncogenic NRASG12V in mice lacking p19Arf tumor suppressor. Our study demonstrates that the HFD regimen and HCC resulted in strong upregulation of α-dC in the liver, heart, and muscles. In addition, an increase in α-dC was confirmed in sera of NAFLD and NASH patients. Furthermore, higher expression of the receptor for advanced glycation products (RAGE) was detected exclusively on immune cells and not on stroma cells in livers of mice with liver cancer progression. Our work confirms astable interplay of liver inflammation, carbonyl stress mediated by α-dC, and upregulated RAGE expression on CD8+ Tand natural killer (NK) cells in situ in NAFLD and HCC, as key factors/determinants in liver disease progression. The obtained findings underline the role of α-dC and RAGE+CD8+ Tand RAGE+ NK cells as biomarkers and candidates for a local therapeutic intervention in NAFLD and malignant liver disease.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Carcinoma Hepatocelular/etiologia , Progressão da Doença , Produtos Finais de Glicação Avançada , Humanos , Camundongos , Receptor para Produtos Finais de Glicação Avançada/genéticaRESUMO
To assemble a brain, differentiating neurons must make proper connections and establish specialized brain compartments. Abnormal levels of cell adhesion molecules disrupt these processes. Dystroglycan (Dg) is a major non-integrin cell adhesion receptor, deregulation of which is associated with dramatic neuroanatomical defects such as lissencephaly type II or cobblestone brain. The previously established Drosophila model for cobblestone lissencephaly was used to understand how Dg is regulated in the brain. During development, Dg has a spatiotemporally dynamic expression pattern, fine-tuning of which is crucial for accurate brain assembly. In addition, mass spectrometry analyses identified numerous components associated with Dg in neurons, including several proteins of the exocyst complex. Data show that exocyst-based membrane trafficking of Dg allows its distinct expression pattern, essential for proper brain morphogenesis. Further studies of the Dg neuronal interactome will allow identification of new factors involved in the development of dystroglycanopathies and advance disease diagnostics in humans.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Distroglicanas/genética , Lisencefalia/genética , Animais , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Distroglicanas/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Neurônios/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
BACKGROUND: Dystroglycanopathies are a group of inherited disorders characterized by vast clinical and genetic heterogeneity and caused by abnormal functioning of the ECM receptor dystroglycan (Dg). Remarkably, among many cases of diagnosed dystroglycanopathies, only a small fraction can be linked directly to mutations in Dg or its regulatory enzymes, implying the involvement of other, not-yet-characterized, Dg-regulating factors. To advance disease diagnostics and develop new treatment strategies, new approaches to find dystroglycanopathy-related factors should be considered. The Dg complex is highly evolutionarily conserved; therefore, model genetic organisms provide excellent systems to address this challenge. In particular, Drosophila is amenable to experiments not feasible in any other system, allowing original insights about the functional interactors of the Dg complex. METHODS: To identify new players contributing to dystroglycanopathies, we used Drosophila as a genetic muscular dystrophy model. Using mass spectrometry, we searched for muscle-specific Dg interactors. Next, in silico analyses allowed us to determine their association with diseases and pathological conditions in humans. Using immunohistochemical, biochemical, and genetic interaction approaches followed by the detailed analysis of the muscle tissue architecture, we verified Dg interaction with some of the discovered factors. Analyses of mouse muscles and myocytes were used to test if interactions are conserved in vertebrates. RESULTS: The muscle-specific Dg complexome revealed novel components that influence the efficiency of Dg function in the muscles. We identified the closest human homologs for Dg-interacting partners, determined their significant enrichment in disease-associations, and verified some of the newly identified Dg interactions. We found that Dg associates with two components of the mechanosignaling Hippo pathway: the WW domain-containing proteins Kibra and Yorkie. Importantly, this conserved interaction manages adult muscle size and integrity. CONCLUSIONS: The results presented in this study provide a new list of muscle-specific Dg interactors, further analysis of which could aid not only in the diagnosis of muscular dystrophies, but also in the development of new therapeutics. To regulate muscle fitness during aging and disease, Dg associates with Kibra and Yorkie and acts as a transmembrane Hippo signaling receptor that transmits extracellular information to intracellular signaling cascades, regulating muscle gene expression.
Assuntos
Proteínas de Drosophila/metabolismo , Distroglicanas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Atrofia Muscular/metabolismo , Distrofias Musculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Envelhecimento/metabolismo , Animais , Modelos Animais de Doenças , Drosophila , Distroglicanas/genética , Feminino , Masculino , Espectrometria de Massas , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Mutação , Mapas de Interação de ProteínasRESUMO
Stem cell niches act as signaling platforms that regulate stem cell self-renewal and sustain stem cells throughout life; however, the specific developmental events controlling their assembly are not well understood. Here, we show that during Drosophila ovarian germline stem cell niche formation, the status of Notch signaling in the cell can be reprogrammed. This is controlled via steroid-induced miR-125, which targets a negative regulator of Notch signaling, Tom. Thus, miR-125 acts as a spatiotemporal coordinator between paracrine Notch and endocrine steroid signaling. Moreover, a dual security mechanism for Notch signaling activation exists to ensure the robustness of niche assembly. Particularly, stem cell niche cells can be specified either via lateral inhibition, in which a niche cell precursor acquires Notch signal-sending status randomly, or via peripheral induction, whereby Delta is produced by a specific cell. When one mechanism is perturbed due to mutations, developmental defects or environmental stress, the remaining mechanism ensures that the niche is formed, perhaps abnormally, but still functional. This guarantees that the germline stem cells will have their residence, thereby securing progressive oogenesis and, thus, organism reproduction.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , MicroRNAs/genética , Receptores Notch/genética , Nicho de Células-Tronco/genética , Animais , Animais Geneticamente Modificados , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ecdisona/metabolismo , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Ovário/citologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Nicho de Células-Tronco/fisiologia , Esteroides/metabolismoRESUMO
Many proteins are expressed dynamically during different stages of cellular life and the accuracy of protein amounts is critical for cell endurance. Therefore, cells should have a perceptive system that notifies about fluctuations in the amounts of certain components and an executive system that efficiently restores their precise levels. At least one mechanism that evolution has employed for this task is regulation of 3'-UTR length for microRNA targeting. Here we show that in Drosophila the microRNA complex miR-310s acts as an executive mechanism to buffer levels of the muscular dystrophy-associated extracellular matrix receptor dystroglycan via its alternative 3'-UTR. miR-310s gene expression fluctuates depending on dystroglycan amounts and nitric oxide signalling, which perceives dystroglycan levels and regulates microRNA gene expression. Aberrant levels of dystroglycan or deficiencies in miR-310s and nitric oxide signalling result in cobblestone brain appearance, resembling human lissencephaly type II phenotype.
Assuntos
Regiões 3' não Traduzidas , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Distroglicanas/química , Distroglicanas/genética , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Animais , Axônios/metabolismo , Sequência de Bases , Encéfalo/patologia , Soluções Tampão , Adesão Celular , Cruzamentos Genéticos , Drosophila , Matriz Extracelular/metabolismo , Feminino , Genótipo , Masculino , MicroRNAs/genética , MicroRNAs/fisiologia , Mitose , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Óxido Nítrico/química , Fenótipo , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , Transdução de SinaisRESUMO
Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.
Assuntos
Dióxido de Carbono/metabolismo , Drosophila melanogaster/metabolismo , Animais , Dióxido de Carbono/análise , Drosophila melanogaster/genética , Metabolismo Energético , Masculino , Modelos Animais , Oxirredução , Mecânica Respiratória/fisiologiaRESUMO
Establishment of intercellular interactions between various cell types of different origin is vital for organism development and tissue maintenance. Therefore, precise timing, expression pattern, and amounts of extracellular matrix (ECM) proteins must be tightly regulated. Particularly, the ECM is important for the development and function of myotendinous junctions (MTJs). We find that precise levels of the ECM receptor Dystroglycan (Dg) are required for MTJ formation in Drosophila and that Dg levels in this process are controlled by miR-9a. In the embryo, Dg is enriched at the termini of the growing muscles facing the tendon matrix and absent from miR-9a-expressing tendons. This gradient of Dg expression is crucial for proper muscle-tendon attachments and is adjusted by miR-9a. In addition to Dg, miR-9a regulates the expression of several other critical muscle genes, and we therefore propose that during embryogenesis, miR-9a specifically controls the expression of mesodermal genes to canalize MTJ morphogenesis.
Assuntos
Drosophila melanogaster/genética , Distroglicanas/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Músculo Esquelético/metabolismo , Tendões/metabolismo , Animais , Western Blotting , Proliferação de Células , Drosophila melanogaster/crescimento & desenvolvimento , Distroglicanas/genética , Embrião não Mamífero/citologia , Imunofluorescência , Técnicas Imunoenzimáticas , Morfogênese , Músculo Esquelético/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tendões/citologiaRESUMO
Previously, it has been shown that in Drosophila steroid hormones are required for progression of oogenesis during late stages of egg maturation. Here, we show that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay to switch on a differentiation programme. This differentiation impediment is associated with reduced TGF-ß signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Ecdisteroides/farmacologia , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Nicho de Células-Tronco , Animais , Western Blotting , Adesão Celular , Linhagem da Célula , Proliferação de Células , Proteínas de Drosophila , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Oogênese/efeitos dos fármacos , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles (1) and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies (2). Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.
Assuntos
Drosophila/anatomia & histologia , Músculo Esquelético/anatomia & histologia , Inclusão em Parafina/métodos , Animais , Drosophila/fisiologia , Músculo Esquelético/fisiologia , Coloração e Rotulagem/métodos , Tórax/anatomia & histologiaRESUMO
The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-beta and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought.
Assuntos
Drosophila melanogaster/genética , Distroglicanas/metabolismo , Distrofina/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Receptores ErbB/metabolismo , Mutação , Receptores Notch/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
The conserved dystroglycan-dystrophin (Dg.Dys) complex connects the extracellular matrix to the cytoskeleton. In humans as well as Drosophila, perturbation of this complex results in muscular dystrophies and brain malformations and in some cases cellular polarity defects. However, the regulation of the Dg.Dys complex is poorly understood in any cell type. We now find that in loss-of-function and overexpression studies more than half (34 residues) of the Dg proline-rich conserved C-terminal regions can be truncated without significantly compromising its function in regulating cellular polarity in Drosophila. Notably, the truncation eliminates the WW domain binding motif at the very C terminus of the protein thought to mediate interactions with dystrophin, suggesting that a second, internal WW binding motif can also mediate this interaction. We confirm this hypothesis by using a sensitive fluorescence polarization assay to show that both WW domain binding sites of Dg bind to Dys in humans (K(d) = 7.6 and 81 microM, respectively) and Drosophila (K(d) = 16 and 46 microM, respectively). In contrast to the large deletion mentioned above, a single proline to an alanine point mutation within a predicted Src homology 3 domain (SH3) binding site abolishes Dg function in cellular polarity. This suggests that an SH3-containing protein, which has yet to be identified, functionally interacts with Dg.
Assuntos
Polaridade Celular/fisiologia , Proteínas de Drosophila/metabolismo , Distroglicanas/metabolismo , Distrofina/metabolismo , Domínios de Homologia de src , Motivos de Aminoácidos , Sequência de Aminoácidos/genética , Substituição de Aminoácidos , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Distroglicanas/genética , Distrofina/genética , Humanos , Mutação Puntual , Ligação Proteica/genética , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Domínios de Homologia de src/genéticaRESUMO
Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.