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1.
PLoS Genet ; 9(9): e1003814, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24068974

RESUMO

Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.


Assuntos
Cílios/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Proteínas do Olho/genética , Audição/genética , Som , Estimulação Acústica , Animais , Animais Geneticamente Modificados , Cílios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Mecanotransdução Celular/genética , Neurônios/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo
2.
Development ; 138(17): 3835-45, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21828100

RESUMO

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. Here, we isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, our study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Animais , Western Blotting , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Imunoprecipitação , Hibridização In Situ , Masculino , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Nature ; 424(6944): 81-4, 2003 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-12819662

RESUMO

The many types of insect ear share a common sensory element, the chordotonal organ, in which sound-induced antennal or tympanal vibrations are transmitted to ciliated sensory neurons and transduced to receptor potentials. However, the molecular identity of the transducing ion channels in chordotonal neurons, or in any auditory system, is still unknown. Drosophila that are mutant for NOMPC, a transient receptor potential (TRP) superfamily ion channel, lack receptor potentials and currents in tactile bristles but retain most of the antennal sound-evoked response, suggesting that a different channel is the primary transducer in chordotonal organs. Here we describe the Drosophila Nanchung (Nan) protein, an ion channel subunit similar to vanilloid-receptor-related (TRPV) channels of the TRP superfamily. Nan mediates hypo-osmotically activated calcium influx and cation currents in cultured cells. It is expressed in vivo exclusively in chordotonal neurons and is localized to their sensory cilia. Antennal sound-evoked potentials are completely absent in mutants lacking Nan, showing that it is an essential component of the chordotonal mechanotransducer.


Assuntos
Canais de Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Audição/fisiologia , Sequência de Aminoácidos , Animais , Células CHO , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Sinalização do Cálcio , Cricetinae , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/genética , Evolução Molecular , Deleção de Genes , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Neurônios/metabolismo , Pressão Osmótica , Técnicas de Patch-Clamp , Canais de Potencial de Receptor Transitório
4.
Genes Genomics ; 42(6): 609-617, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32297146

RESUMO

It is now evident that transcriptional gene regulation usually requires the re-organization of chromatin architecture. Increasing evidence suggested various kinds of RNAs are involved in this process. Especially the nascent RNAs retained at their site of transcription can serve as a scaffold for organizing transcriptionally either favorable or unfavorable chromatin structures. An emerging concept of phase separation explains how these chromatin structures can be maintained as physically discrete subcompartments within membrane-less nucleoplasm. Evidences that support the crucial role of nascent RNAs in the formation of phase-separated condensates are now rapidly growing.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , RNA/genética , Animais , Cromatina/química , Cromatina/metabolismo , Humanos , RNA/metabolismo , Sítio de Iniciação de Transcrição
5.
Mol Cells ; 43(12): 1002-1010, 2020 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-33323559

RESUMO

Cilia are important eukaryotic cellular compartments required for diverse biological functions. Recent studies have revealed that protein targeting into the proper ciliary subcompartments is essential for ciliary function. In Drosophila chordotonal cilium, where mechano-electric transduction occurs, two transient receptor potential (TRP) superfamily ion channels, TRPV and TRPN, are restricted to the proximal and distal subcompartments, respectively. To understand the mechanisms underlying the sub-ciliary segregation of the two TRPs, we analyzed their localization under various conditions. In developing chordotonal cilia, TRPN was directly targeted to the ciliary tip from the beginning of its appearance and was retained in the distal subcompartment throughout development, whereas the ciliary localization of TRPV was considerably delayed. Lack of intraflagella transport-related proteins affected TRPV from the initial stage of its pre-ciliary trafficking, whereas it affected TRPN from the ciliary entry stage. The ectopic expression of the two TRP channels in both ciliated and nonciliated cells revealed their intrinsic properties related to their localization. Taken together, our results suggest that subciliary segregation of the two TRP channels relies on their distinct intrinsic properties, and begins at the initial stage of their pre-ciliary trafficking.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Neurônios/metabolismo , Transporte Proteico
6.
Sci Rep ; 7: 46473, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28621308

RESUMO

We conducted an inhalation toxicity test on the alternative animal model, Drosophila melanogaster, to investigate potential hazards of indoor air pollution. The inhalation toxicity of toluene and formaldehyde was investigated using comprehensive transcriptomics and computational behavior analyses. The ingenuity pathway analysis (IPA) based on microarray data suggests the involvement of pathways related to immune response, stress response, and metabolism in formaldehyde and toluene exposure based on hub molecules. We conducted a toxicity test using mutants of the representative genes in these pathways to explore the toxicological consequences of alterations of these pathways. Furthermore, extensive computational behavior analysis showed that exposure to either toluene or formaldehyde reduced most of the behavioral parameters of both wild-type and mutants. Interestingly, behavioral alteration caused by toluene or formaldehyde exposure was most severe in the p38b mutant, suggesting that the defects in the p38 pathway underlie behavioral alteration. Overall, the results indicate that exposure to toluene and formaldehyde via inhalation causes severe toxicity in Drosophila, by inducing significant alterations in gene expression and behavior, suggesting that Drosophila can be used as a potential alternative model in inhalation toxicity screening.


Assuntos
Poluentes Atmosféricos/toxicidade , Poluição do Ar em Ambientes Fechados/efeitos adversos , Comportamento Animal/efeitos dos fármacos , Drosophila melanogaster/efeitos dos fármacos , Animais , Exposição por Inalação/análise , Transcriptoma
7.
BMB Rep ; 49(7): 355-6, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27312604

RESUMO

The THO/TREX complex consists of several conserved subunits and is required for mRNA export. In metazoans, THO/TREX binds a subset of mRNAs during RNA splicing, and facilitates their nuclear export. How THO/TREX selects RNA targets is, however, incompletely understood. In our recent study, we reported that THO is loaded onto Piwi-interacting RNA (piRNA) precursor transcripts independent of splicing, and facilitates convergent transcription in Drosophila ovary. The precursors are later processed into mature piRNAs, small noncoding RNAs that silence transposable elements (TEs). We observed that piRNAs originating from dual-strand clusters, where precursors are transcribed from both strands, were specifically affected by THO mutation. Analysis of THO-bound RNAs showed enrichment of dual-strand cluster transcripts. Interestingly, THO loading onto piRNA precursors was dependent on Cutoff (Cuff), which comprises the Rhino-Deadlock-Cutoff (RDC) complex that is recruited to dual-strand clusters by recognizing H3K9me3 and licenses convergent transcription from the cluster. We also found that THO mutation affected transcription from dualstrand clusters. Therefore, we concluded that THO/TREX is recruited to dual-strand piRNA clusters, independent of splicing events, via multi-protein interactions with chromatin structure. Then, it facilitates transcription likely by suppressing premature termination to ensure adequate expression of piRNA precursors. [BMB Reports 2016; 49(7): 355-356].


Assuntos
Proteínas de Drosophila/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Elementos de DNA Transponíveis/genética , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Feminino , Mutação , Proteínas Nucleares/genética , Ovário/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Interferente Pequeno/genética , Transcrição Gênica
8.
J Neurosci ; 24(41): 9059-66, 2004 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-15483124

RESUMO

Hearing in Drosophila depends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that a Drosophila protein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only other Drosophila TRPV protein is mutated in the behavioral mutant inactive (iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channel in vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.


Assuntos
Canais de Cálcio/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Audição/fisiologia , Canais Iônicos/fisiologia , Receptores de Droga/fisiologia , Animais , Canais de Cálcio/biossíntese , Canais de Cálcio/genética , Linhagem Celular , Mapeamento Cromossômico , Cílios/metabolismo , Cricetinae , Cruzamentos Genéticos , Drosophila/genética , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Feminino , Audição/genética , Transtornos da Audição/genética , Canais Iônicos/biossíntese , Canais Iônicos/genética , Masculino , Mutagênese , Mutação , Neurônios Aferentes/metabolismo , Neurônios Aferentes/ultraestrutura , Técnicas de Patch-Clamp , Subunidades Proteicas/biossíntese , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Receptores de Droga/biossíntese , Receptores de Droga/genética , Canais de Potencial de Receptor Transitório
9.
BMB Rep ; 48(7): 380-7, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25936781

RESUMO

Cilia are conserved subcellular organelles with diverse sensory and developmental roles. Recently, they have emerged as crucial organelles whose dysfunction causes a wide spectrum of disorders called ciliopathies. Recent studies on the pathological mechanisms underlying ciliopathies showed that the ciliary compartment is further divided into subdomains with specific roles in the biogenesis, maintenance and function of cilia. Several conserved sets of molecules that play specific roles in each subcompartment have been discovered. Here we review recent progress on our understanding of ciliary subcompartments, especially focusing on the molecules required for their structure and/or function.


Assuntos
Compartimento Celular , Cílios/metabolismo , Animais , Humanos , Membranas Intracelulares/metabolismo , Modelos Biológicos
10.
Mol Cells ; 35(3): 261-8, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23475424

RESUMO

The THO complex (THO) is an evolutionary conserved protein required for the formation of export-competent mRNP. The growing evidence indicates that the metazoan THO plays important roles in cell differentiation and cellular stress response. But the underlying mechanisms are poorly understood. Herein we examined the relevance of THO to cellular signaling pathways involved in cell differentiation and cellular stress response. When we examined the endogenous p53 level in the testis, it was sustained much longer during spermatogenesis in the THO mutant compared to that of wild-type. In flies with impaired THO, overexpression of p53 by eye-specific GAL4 not only enhanced p53-mediated retinal degeneration, but p53 level was also elevated compared to the control flies. Since the body size of the THO mutant flies was significantly larger than control flies, we also examined whether the PI3K/AKT signaling is enhanced in the mutant flies. The results showed that the endogenous level of phosphorylated AKT, which is the active form, was highly elevated in the THO mutants. Taken together our results suggested that both p53 and PI3K/AKT signalings are up-regulated in the flies with impaired THO.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Tamanho Corporal/genética , Olho Composto de Artrópodes/metabolismo , Olho Composto de Artrópodes/patologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Epistasia Genética , Expressão Gênica , Masculino , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Fosforilação , Pigmentação , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Degeneração Retiniana/genética , Transdução de Sinais , Espermatogênese , Testículo/metabolismo , Testículo/patologia , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Regulação para Cima
11.
Environ Toxicol Pharmacol ; 36(2): 548-556, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23827194

RESUMO

Silver nanoparticles (AgNPs) have been widely used in commercial goods ranging from medical devices to home appliances. Their widespread application increase the risk related to their potential toxicity. Although several studies showed their acute hazardous effects on living animals, our understanding of chronic effects of AgNPs exposed by the environment we encounter in our everyday lives is still very limited. This is partly because of the lack of versatile animal model system for studying AgNPs effects on terrestrial animals including human. In this study, we used Drosophila model to study AgNPs toxicity in terrestrial animals, and found that long-term exposure of AgNPs, but not Ag ions, at low level (0.1 and 1µg/mL) significantly shortened the lifespan. By taking advantage of the power of Drosophila genetics, we also isolated a GAL4 enhancer trap line called M95, in which the expression of GAL4 is up-regulated in response to ingestion of AgNPs at concentrations as low as 0.1µg/mL. Interestingly M95 flies showed significantly increased tolerance to both AgNPs treatment and dry starvation probably due to up-regulation of JNK signaling. These findings suggest not only that M95 may be a very useful biomarker of AgNPs because of its high sensitivity and tolerance to AgNPs, but also that Drosophila may be a versatile terrestrial invertebrate model for studying the effects of AgNPs on human health.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Drosophila/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Proteínas de Saccharomyces cerevisiae/metabolismo , Prata/toxicidade , Testes de Toxicidade/métodos , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Drosophila/genética , Drosophila/metabolismo , Genes Reporter , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Longevidade/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Inanição/genética , Inanição/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima
12.
PLoS One ; 5(6): e11012, 2010 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-20543979

RESUMO

BACKGROUND: A TRPN channel protein is essential for sensory transduction in insect mechanosensory neurons and in vertebrate hair cells. The Drosophila TRPN homolog, NOMPC, is required to generate mechanoreceptor potentials and currents in tactile bristles. NOMPC is also required, together with a TRPV channel, for transduction by chordotonal neurons of the fly's antennal ear, but the TRPN or TRPV channels have distinct roles in transduction and in regulating active antennal mechanics. The evidence suggests that NOMPC is a primary mechanotransducer channel, but its subcellular location-key for understanding its exact role in transduction-has not yet been established. METHODOLOGY/PRINCIPAL FINDINGS: Here, by immunostaining, we locate NOMPC at the tips of mechanosensory cilia in both external and chordotonal sensory neurons, as predicted for a mechanotransducer channel. In chordotonal neurons, the TRPN and TRPV channels are respectively segregated into distal and proximal ciliary zones. This zonal separation is demarcated by and requires the ciliary dilation, an intraciliary assembly of intraflagellar transport (IFT) proteins. CONCLUSIONS: Our results provide a strong evidence for NOMPC as a primary transduction channel in Drosophila mechansensory organs. The data also reveals a structural basis for the model of auditory chordotonal transduction in which the TRPN and TRPV channels play sequential roles in generating and amplifying the receptor potential, but have opposing roles in regulating active ciliary motility.


Assuntos
Cílios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Animais , Animais Geneticamente Modificados , Microscopia Confocal , Canais de Potencial de Receptor Transitório
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