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1.
PLoS Genet ; 18(8): e1010335, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35951645

RESUMO

Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Proteínas Correpressoras , DNA , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Displasia Ectodérmica , Humanos , Deformidades Congênitas dos Membros , Mamíferos/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Couro Cabeludo/metabolismo , Dermatoses do Couro Cabeludo/congênito , Crânio/metabolismo
2.
J Neurosci ; 39(36): 7132-7154, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31350259

RESUMO

Ca2+-activated K+ channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modeling, we discover how visual information transfer in mutants lacking the BK channel (dSlo- ), SK channel (dSK- ), or both (dSK- ;; dSlo- ) is shaped in the female fruit fly (Drosophila melanogaster) R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K+ conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and promote the mutants' distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca2+-activated K+ channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information.SIGNIFICANCE STATEMENT In this study, we directly link in vivo and ex vivo experiments with detailed stochastically operating biophysical models to extract new mechanistic knowledge of how Drosophila photoreceptor-interneuron-photoreceptor (R-LMC-R) circuitry homeostatically retains its information sampling and transmission capacity against chronic perturbations in its ion-channel composition, and what is the cost of this compensation and its impact on optomotor behavior. We anticipate that this novel approach will provide a useful template to other model organisms and computational neuroscience, in general, in dissecting fundamental mechanisms of homeostatic compensation and deepening our understanding of how biological neural networks work.


Assuntos
Retroalimentação Fisiológica , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Potenciais Sinápticos , Percepção Visual , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Interneurônios/metabolismo , Interneurônios/fisiologia , Modelos Neurológicos , Células Fotorreceptoras de Invertebrados/fisiologia , Canais de Potássio Shab/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Vias Visuais/metabolismo , Vias Visuais/fisiologia
3.
G3 (Bethesda) ; 6(10): 3139-3148, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27489208

RESUMO

Transvection-pairing-dependent interallelic regulation resulting from enhancer action in trans-occurs throughout the Drosophila melanogaster genome, likely as a result of the extensive somatic homolog pairing seen in Dipteran species. Recent studies of transvection in Drosophila have demonstrated important qualitative differences between enhancer action in cis vs. in trans, as well as a modest synergistic effect of cis- and trans-acting enhancers on total tissue transcript levels at a given locus. In the present study, we identify a system in which cis- and trans-acting GAL4-UAS enhancer synergism has an unexpectedly large quantitative influence on gene expression, boosting total tissue transcript levels at least fourfold relative to those seen in the absence of transvection. We exploit this strong quantitative effect by using publicly available UAS-shRNA constructs from the TRiP library to assay candidate genes for transvection activity in vivo The results of the present study, which demonstrate that in trans activation by simple UAS enhancers can have large quantitative effects on gene expression in Drosophila, have important new implications for experimental design utilizing the GAL4-UAS system.


Assuntos
Alelos , Drosophila melanogaster/genética , Epistasia Genética , Regulação da Expressão Gênica , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Elementos Facilitadores Genéticos , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , RNA Interferente Pequeno/genética , Transgenes
4.
J Cell Biol ; 206(1): 79-95, 2014 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-25002678

RESUMO

The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Fosfolipases A1/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Proteínas de Drosophila/química , Feminino , Masculino , Dados de Sequência Molecular , Fosfolipases A1/química , Transporte Proteico
5.
PLoS One ; 7(4): e34665, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22509342

RESUMO

A role for SK channels in synaptic plasticity has been very well-characterized. However, in the absence of simple genetic animal models, their role in behavioral memory remains elusive. Here, we take advantage of Drosophila melanogaster with its single SK gene (dSK) and well-established courtship memory assay to investigate the contribution of this channel to memory. Using two independent dSK alleles, a null mutation and a dominant negative subunit, we show that while dSK negatively regulates the acquisition of short-term memory 30 min after a short training session, it is required for normal long-term memory 24 h after extended training. These findings highlight important functions for dSK in courtship memory and suggest that SK channels can mediate multiple forms of behavioral plasticity.


Assuntos
Encéfalo/fisiologia , Corte , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Proteínas rab de Ligação ao GTP/genética , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica , Inativação Gênica , Memória de Longo Prazo/fisiologia , Memória de Curto Prazo/fisiologia , Mutação , Comportamento Sexual Animal/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia
6.
Neurobiol Dis ; 46(3): 655-62, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22426392

RESUMO

Endocytosis-mediated cell death is a form of degeneration displayed in several Drosophila mutants. This form of degeneration is displayed in several Drosophila mutant lines including flies lacking the eye-specific PLC (norpA). The cell death pathway is initiated by the stabilization of complexes between rhodopsin and arrestin which undergo massive endocytosis into the cell body. The internalized rhodopsin becomes insoluble and builds up in the late endosomal system, wherein it triggers cell death. Cathepsins are resident late endosome/lysosome proteases that have been shown to mediate apoptosis in many disease models. Therefore we sought to test the involvement of cathepsins in endocytosis-mediated retinal degeneration. Here we show that cathepsins mediate cell death in light-exposed norpA eyes. Moreover, we show that the cathepsin L-like cysteine protease, CP1, specifically mediates retinal degeneration, while the aspartyl protease, cathepsin D, does not. Furthermore, eye-specific expression of pan-cathepsin inhibitors also blocks cell death. Western blot analysis demonstrates that cathepsin L levels remain unchanged during retinal degeneration. However, whole mount immunohistochemistry performed on light-exposed retinas revealed a decrease in cathepsin L levels and a loss of rhodopsin/ CP1 colocalization, suggesting that cathepsin L translocates during the degeneration process. Lastly, we show that the retinal degeneration can be enhanced by the overexpression of cathepsin L in the sensitized norpA background. Together these data show that cathepsins play a crucial role in endocytosis-mediated retinal degeneration and are consistent with a model where rhodopsin internalization and accumulation in the endosomal/lysosomal system triggers cathepsin translocation to the cytosol.


Assuntos
Catepsinas/fisiologia , Células Fotorreceptoras de Invertebrados/patologia , Degeneração Retiniana/patologia , Animais , Western Blotting , Catepsina L/metabolismo , Morte Celular/efeitos dos fármacos , Citosol/metabolismo , Drosophila , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Imuno-Histoquímica , Lisossomos/metabolismo , Transporte Proteico , Retina/metabolismo , Retina/patologia , Rodopsina/metabolismo
7.
Proc Natl Acad Sci U S A ; 109(10): E605-12, 2012 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-22328149

RESUMO

Photoreceptor cell death accompanying many retinal degenerative disorders results in irreversible loss of vision in humans. However, the precise molecular pathway that executes cell death is not known. Our results from a Drosophila model of retinal degeneration corroborate previously reported findings that the developmental apoptotic pathway is not involved in photoreceptor cell demise. By undertaking a candidate gene approach, we find that players involved in the immune response against gram-negative bacteria are involved in retinal degeneration. Here, we report that the NF-κB transcription factor Relish regulates neuronal cell death. Retinal degeneration is prevented in genetic backgrounds that block Relish activation. We also report that the N-terminal domain of Relish encodes unique toxic functions. These data uncover a unique molecular pathway of retinal degeneration in Drosophila and identify a previously unknown function of NF-κB signaling in cell death.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Animais Geneticamente Modificados , Apoptose , Caspases/metabolismo , Morte Celular , Feminino , Sistema Imunitário , Masculino , Modelos Biológicos , Modelos Genéticos , Células Fotorreceptoras de Invertebrados/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais
8.
Mol Vis ; 17: 3224-33, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22194648

RESUMO

PURPOSE: In this study we investigated the biochemical and cell biologic characteristics of flies expressing two novel dominant alleles of the major rhodopsin encoding gene neither inactivation nor afterpotential E (ninaE) in a heterozygous background. METHODS: Presence of the deep pseudopupil in flies was assayed 5 days post eclosion. For structural analysis, 1-µm-retinal cross sections were obtained from fixed and resin-embedded Drosophila heads. Confocal microscopy was performed on dissected retinas stained with antibodies specific for rhodopsin, NinaA, and F-actin. Rhodopsin levels were determined by western and slot blot analysis. RESULTS: Dominant rhodopsin mutants showed progressive age-dependent and light-independent loss of the deep pseudopupil, without any apparent retinal degeneration at the morphological level. Expression of mutant rhodopsin caused rhodopsin to mislocalize to the cell body and the endoplasmic reticulum compartment. Mutant rhodopsin also caused loss of solubility of wild-type rhodopsin and its accumulation presumably as a high molecular mass complex in the photoreceptor cell body. CONCLUSIONS: In heterozygous mutant flies, there is loss of wild-type rhodopsin immunoreactivity on a western assay but less reduction using slot blot analysis. This suggests that mutant rhodopsin is likely inducing the misfolding and insolubility of wild-type rhodopsin. Localization of rhodopsin revealed that in mutant flies, wild-type rhodopsin is mislocalized to the cell body and the endoplasmic reticulum.


Assuntos
Envelhecimento/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mutação , Células Fotorreceptoras de Invertebrados/metabolismo , Retina/metabolismo , Degeneração Retiniana/genética , Rodopsina/genética , Actinas/genética , Actinas/metabolismo , Envelhecimento/metabolismo , Alelos , Animais , Western Blotting , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Retículo Endoplasmático/metabolismo , Genes Dominantes , Heterozigoto , Humanos , Microscopia Confocal , Células Fotorreceptoras de Invertebrados/citologia , Dobramento de Proteína , Retina/patologia , Degeneração Retiniana/metabolismo , Rodopsina/química , Rodopsina/metabolismo
9.
J Neurosci ; 31(39): 13897-910, 2011 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-21957252

RESUMO

The contribution of the SK (small-conductance calcium-activated potassium) channel to neuronal functions in complex circuits underlying sensory processing and behavior is largely unknown in the absence of suitable animal models. Here, we generated a Drosophila line that lacks the single highly conserved SK gene in its genome (dSK). In R1-R6 photoreceptors, dSK encodes a slow Ca²âº-activated K(+) current similar to its mammalian counterparts. Compared with wild-type, dSK(-) photoreceptors and interneurons showed accelerated oscillatory responses and adaptation. These enhanced kinetics were accompanied with more depolarized dSK(-) photoreceptors axons, assigning a role for dSK in network gain control during light-to-dark transitions. However, compensatory network adaptation, through increasing activity between synaptic neighbors, overcame many detriments of missing dSK current enabling dSK(-) photoreceptors to maintain normal information transfer rates to naturalistic stimuli. While demonstrating important functional roles for dSK channel in the visual circuitry, these results also clarify how homeostatically balanced network functions can compensate missing or faulty ion channels.


Assuntos
Proteínas de Drosophila/fisiologia , Rede Nervosa/fisiologia , Células Fotorreceptoras/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Vias Visuais/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Masculino , Dados de Sequência Molecular , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética
10.
Proc Natl Acad Sci U S A ; 106(47): 20063-8, 2009 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-19892737

RESUMO

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP(2)), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP(2) and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP(2) and PLC during phototransduction.


Assuntos
Drosophila melanogaster/fisiologia , Transdução de Sinal Luminoso/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Ceramidas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Eletrorretinografia , Homeostase , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Luz , Mutação , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Células Fotorreceptoras de Invertebrados/fisiologia , Células Fotorreceptoras de Invertebrados/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/genética
11.
PLoS Genet ; 5(2): e1000377, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19214218

RESUMO

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Células Fotorreceptoras de Invertebrados/citologia , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Animais , Morte Celular , Sobrevivência Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Endocitose , Endossomos/genética , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Mutação , Células Fotorreceptoras de Invertebrados/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/fisiopatologia , Rodopsina/genética
12.
PLoS Genet ; 3(12): e206, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18069895

RESUMO

The Drosophila melanogaster photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. We also show that by feeding wild-type flies carcinine, we can duplicate components of mutant ine ERGs. Finally, we demonstrate that treatment with H(3) receptor agonists or inverse agonists rescue several components of the mutant ine ERG phenotype. Here, we provide pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. We also speculate that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H(3) receptor.


Assuntos
Carnosina/análogos & derivados , Drosophila melanogaster/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Carnosina/metabolismo , Carnosina/farmacologia , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Eletrorretinografia , Genes de Insetos , Histamina/metabolismo , Antagonistas dos Receptores Histamínicos H3/farmacologia , Modelos Neurológicos , Mutação , Neuroglia/metabolismo , Neurônios Aferentes/metabolismo , Fenótipo , Células Fotorreceptoras de Invertebrados/efeitos dos fármacos , Células Fotorreceptoras de Invertebrados/fisiologia , Piperidinas/farmacologia , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Transdução de Sinais , Sinapses/metabolismo
13.
J Neurosci ; 26(48): 12408-14, 2006 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-17135402

RESUMO

Prion diseases are CNS disorders that can occur in sporadic, infectious, and inherited forms. Although all forms of prion disease are associated with the accumulation of pathogenic conformers of the prion protein, collectively termed PrP(Sc), the mechanisms by which PrP(Sc) molecules form and cause neuronal degeneration are unknown. Using the bipartite galactosidase-4-upstream activating sequence expression system, we generated transgenic Drosophila melanogaster heterologously expressing either wild-type (WT) or mutant, disease-associated (P101L) mouse PrP molecules in cholinergic neurons. Transgenic flies expressing neuronal P101L PrP molecules exhibited severe locomotor dysfunction and premature death as larvae and adults. These striking clinical abnormalities were accompanied by age-dependent accumulation of misfolded PrP molecules, intracellular PrP aggregates, and neuronal vacuoles. In contrast, transgenic flies expressing comparable levels of WT PrP displayed no clinical, pathological, or biochemical abnormalities. These results indicate that transgenic Drosophila expressing neuronal P101L PrP specifically exhibit several hallmark features of human Gerstmann-Sträussler-Scheinker (GSS) syndrome. Because the rates of abnormal PrP accumulation and clinical progression are highly accelerated in Drosophila compared with the rates of these processes in rodents or humans, the P101L mutant may be used for future genetic and pharmacologic studies as a novel invertebrate model of GSS.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Príons/química , Príons/metabolismo , Dobramento de Proteína , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Doença de Gerstmann-Straussler-Scheinker/genética , Doença de Gerstmann-Straussler-Scheinker/patologia , Humanos , Camundongos , Príons/genética
14.
J Cell Sci ; 119(Pt 15): 3141-8, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16835270

RESUMO

Previously, we have identified a class of retinal degeneration mutants in Drosophila in which the normally transient interaction between arrestin2 (Arr2) and rhodopsin is stabilized and the complexes are rapidly internalized into the cell body by receptor-mediated endocytosis. The accumulation of protein complexes in the cytoplasm eventually results in photoreceptor cell death. We now show that the endocytic adapter protein AP-2 is essential for rhodopsin endocytosis through an Arr2-AP-2beta interaction, and mutations in Arr2 that disrupt its interaction with the beta subunit of AP-2 prevent endocytosis-induced retinal degeneration. We further demonstrate that if the interaction between Arr2 and AP-2 is blocked, this also results in retinal degeneration in an otherwise wild-type background. This indicates that the Arr2-AP-2 interaction is necessary for the pathology observed in a number of Drosophila visual system mutants, and suggests that regular rhodopsin turnover in wild-type photoreceptor cells by Arr2-mediated endocytosis is essential for photoreceptor cell maintenance.


Assuntos
Arrestinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endocitose/fisiologia , Rodopsina/metabolismo , Fator de Transcrição AP-2/metabolismo , Animais , Arrestinas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Eletrorretinografia , Luz , Complexos Multiproteicos , Fosfolipase C beta , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/ultraestrutura , Mutação Puntual , Estrutura Terciária de Proteína , Retina/metabolismo , Retina/patologia , Retina/ultraestrutura , Rodopsina/genética , Fator de Transcrição AP-2/genética , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
15.
J Neurochem ; 85(6): 1614-23, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12787080

RESUMO

A wealth of evidence supports the view that conformational change of the prion protein, PrPC, into a pathogenic isoform, PrPSc, is the hallmark of sporadic, infectious, and inherited forms of prion disease. Although the central role played by PrPSc in the pathogenesis of prion disease is appreciated, the cellular mechanisms that recognize PrPSc and modulate its production, clearance, and neural toxicity have not been elucidated. To address these questions, we used a tissue-specific expression system to express wild-type and disease-associated PrP molecules heterologously in Drosophila melanogaster. Our results indicate that Drosophila brain possesses a specific and saturable mechanism that suppresses the accumulation of PG14, a disease-associated insertional PrP mutant. We also found that wild-type PrP molecules are maintained in a detergent-soluble conformation throughout life in Drosophila brain neurons, whereas they become detergent-insoluble in retinal cells as flies age. PG14 protein expression in Drosophila eye did not cause retinal pathology. Our work reveals the presence of mechanisms in neurons that specifically counterbalance the production of misfolded PrP conformations, and provides an opportunity to study these processes in a model organism amenable to genetic analysis.


Assuntos
Neurônios/metabolismo , Príons/metabolismo , Processamento de Proteína Pós-Traducional , Fatores Etários , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Química Encefálica , Detergentes/química , Drosophila melanogaster , Olho/química , Olho/metabolismo , Homozigoto , Camundongos , Especificidade de Órgãos , Príons/química , Príons/genética , Conformação Proteica , Processamento de Proteína Pós-Traducional/genética , RNA Mensageiro/metabolismo , Retina/química , Retina/metabolismo , Solubilidade
16.
Gene ; 310: 169-73, 2003 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-12801644

RESUMO

Alleles of pawn have numerous morphological phenotypes, including dark deposits on the eye, truncated bristles, and semilethality. The gene encoding the pawn locus was cloned. The Pawn protein is predicted to be a large cell adhesion molecule with a single transmembrane domain, a short cytoplasmic tail and two extracellular epidermal growth factor (EGF)-like repeats. The identity of the pawn gene product was confirmed by sequencing the defects in four different pawn alleles. Analysis of cDNAs showed a complex genomic structure with two 5' untranslated exons and developmental-specific RNA splicing. The gene is expressed throughout development with highest expression found in the late embryonic and larval stages.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insetos/genética , Alelos , Animais , Clonagem Molecular , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Fator de Crescimento Epidérmico/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Fenótipo , Análise de Sequência de DNA
17.
Mol Vis ; 8: 455-61, 2002 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-12486400

RESUMO

PURPOSE: To determine the fate of rhodopsin during endocytosis-mediated retinal degeneration. METHODS: Drosophila stocks were raised in complete darkness and shifted to light for 24 h prior to dissection and fixation of retinas. 1 microm frozen sections were cut on an ultracryomicrotome, then stained with antibodies specific for rhodopsin or arrestin. Localization of photoreceptor cell-specific proteins was determined by confocal microscopy. RESULTS: Flies that are in the process of undergoing endocytosis-mediated retinal degeneration exhibit an apparent loss of rhabdomeric rhodopsin at early times during the degenerative process. Using different immunological agents, genetic backgrounds, and light treatments, we have found that the binding of arrestin to rhodopsin masked the C-terminal monoclonal antibody epitope and resulted in the loss of rhodopsin immunoreactivity. The loss of immunoreactive rhabdomeric rhodopsin only occurred when rhodopsin was depleted from the plasma membrane such that it was found within the rhabdomere at stoichiometric levels with arrestin. CONCLUSIONS: When rhodopsin and arrestin are found at equal levels, binding of arrestin to rhodopsin results in the masking of the antibody epitope on the C-terminus of rhodopsin. Since masking can only occur after most of the rhodopsin has been depleted from the rhabdomere, it can be concluded that during endocytosis-induced retinal degeneration, much of the rhodopsin is localized to the cell body in small puncta. These data suggest that rhodopsin is at extremely high local concentrations in the cytoplasm. The data are discussed in the context of a model for photoreceptor cell apoptosis in retinal degenerative disorders.


Assuntos
Endocitose , Epitopos/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Animais , Arrestinas/metabolismo , Drosophila melanogaster , Técnica Indireta de Fluorescência para Anticorpo , Fosfatidilinositol Diacilglicerol-Liase , Fosfoproteínas/metabolismo , Fosfolipases Tipo C/genética
18.
Neuroscientist ; 8(4): 347-55, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12194503

RESUMO

G protein-coupled receptors are a large family of signaling molecules that respond to a wide variety of extracellular stimuli. The receptors relay the information encoded by the ligand through the activation of heterotrimeric G proteins and intracellular effector molecules. To ensure the appropriate regulation of the signaling cascade, it is vital to properly inactivate the receptor. This inactivation is achieved, in part, by the binding of a soluble protein, arrestin, which uncouples the receptor from the downstream G protein. In addition to the inactivation of G protein-coupled receptors, arrestins have also been implicated in the endocytosis of receptors and cross talk with other signaling pathways. Due to its central role in cellular signaling, misregulation or misfunction of arrestin can have dramatic affects on cell viability and have direct implications in human disease.


Assuntos
Arrestina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Retina/metabolismo , Transdução de Sinais , Animais , Morte Celular , Sobrevivência Celular , Humanos , Neurônios/metabolismo , Receptores de Neurotransmissores/metabolismo , Rodopsina/metabolismo
19.
Vision Res ; 42(4): 497-505, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11853766

RESUMO

Previously we have shown that a subset of visual transduction mutants in Drosophila melanogaster induce the formation of stable complexes between rhodopsin and arrestin. One such mutant is in a visual system-specific phospholipase C (PLC). The rhodopsin/arrestin complexes generated in PLC mutants induce massive retinal degeneration. Here we demonstrate that both arrestin and rhodopsin undergo light-dependent endocytosis in a PLC mutant background. Interestingly, the internalized rhodopsin is rapidly degraded, but the arrestin is fully stable. The data are discussed with respect to mechanisms of arrestin-mediated endocytosis and human retinal disease.


Assuntos
Arrestina/metabolismo , Endocitose , Células Fotorreceptoras de Invertebrados/metabolismo , Retinose Pigmentar/metabolismo , Rodopsina/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Drosophila melanogaster , Imuno-Histoquímica , Microscopia Confocal , Modelos Animais , Fosfatidilinositol Diacilglicerol-Liase
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