RESUMO
Numerous diseases can arise as a consequence of mitochondrial malfunction. Hence, there is a significant focus on studying the role of mitochondria in cancer, ageing, neurodegenerative diseases, and the field of developmental biology. Mitochondria could exist as discrete organelles in the cell; however, they have the ability to fuse, resulting in the formation of interconnected reticular structures. The dynamic changes between these forms correlate with mitochondrial function and mitochondrial health, and consequently, there is a significant scientific interest in uncovering the specific molecular constituents that govern these transitions. Moreover, the specialized mitochondria display a wide array of variable morphologies in their cristae formations. These inner mitochondrial structures are closely associated with the specific functions performed by the mitochondria. In multiple cases, the presence of mitochondrial dysfunction has been linked to male sterility, as it has been observed to cause a range of abnormal spermatogenesis and sperm phenotypes in different species. This review aims to elucidate the dynamic alterations and functions of mitochondria in germ cell development during the spermatogenesis of Drosophila melanogaster.
Assuntos
Drosophila melanogaster , Sêmen , Masculino , Animais , Mitocôndrias , Espermatogênese , EspermatozoidesRESUMO
Nuclear Piwi/Piwi-interacting RNA complexes mediate co-transcriptional silencing of transposable elements by inducing local heterochromatin formation. In Drosophila, sumoylation plays an essential role in the assembly of the silencing complex; however, the molecular mechanism by which the sumoylation machinery is recruited to the transposon loci is poorly understood. Here, we show that the Drosophila E3 SUMO-ligase Su(var)2-10 directly binds to the Piwi protein. This interaction is mediated by the SUMO-interacting motif-like (SIM-like) structure in the C-terminal domain of Su(var)2-10. We demonstrated that the SIM-like structure binds to a special region found in the MID domain of the Piwi protein, the structure of which is highly similar to the SIM-binding pocket of SUMO proteins. Abrogation of the Su(var)2-10-binding surface of the Piwi protein resulted in transposon derepression in the ovary of adult flies. Based on our results, we propose a model in which the Piwi protein initiates local sumoylation in the silencing complex by recruiting Su(var)2-10 to the transposon loci.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Feminino , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sítios de Ligação , Elementos de DNA Transponíveis/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMO
The zinc restriction and zinc toxicity are part of host defence, called nutritional immunity. The crucial role of zinc homeostasis in microbial survival within a host is established, but little is known about these processes in the opportunistic human fungal pathogen Candida parapsilosis. Our in silico predictions suggested the presence of at least six potential zinc transporters (ZnTs) in C. parapsilosis-orthologues of ZRC1, ZRT3 and ZRT101-but an orthologue of PRA1 zincophore was not found. In addition, we detected a species-specific gene expansion of the novel ZnT ZRT2, as we identified three orthologue genes in the genome of C. parapsilosis. Based on predictions, we created homozygous mutant strains of the potential ZnTs and characterized them. Despite the apparent gene expansion of ZRT2 in C. parapsilosis, only CpZRT21 was essential for growth in a zinc-depleted acidic environment, in addition we found that CpZrc1 is essential for zinc detoxification and also protects the fungi against the elimination of murine macrophages. Significantly, we demonstrated that C. parapsilosis forms zincosomes in a Zrc1-independent manner and zinc detoxification is mediated by the vacuolar importer CpZrc1. Our study defines the functions of C. parapsilosis ZnTs, including a species-specific survival and zinc detoxification system.
Assuntos
Candida parapsilosis , Zinco , Animais , Candida parapsilosis/genética , Humanos , Macrófagos/microbiologia , Camundongos , VacúolosRESUMO
Ovarian germline stem cells (GSCs) of Drosophila melanogaster provide a valuable in vivo model to investigate how the adult stem cell identity is maintained and the differentiation of the daughter cells is regulated. GSCs are embedded into a specialized cellular microenvironment, the so-called stem cell niche. Besides the complex signaling interactions between the germ cells and the niche cells, the germ cell intrinsic mechanisms, such as chromatin regulation and transcriptional control, are also crucial in the decision about self-renewal and differentiation. The key differentiation regulator gene is the bag of marbles (bam), which is transcriptionally repressed in the GSCs and de-repressed in the differentiating daughter cell. Here, we show that the transcription factor MESR4 functions in the germline to promote GSC daughter differentiation. We find that the loss of MESR4 results in the accumulation of GSC daughter cells which fail to transit from the pre-cystoblast (pre-CB) to the differentiated cystoblast (CB) stage. The forced expression of bam can rescue this differentiation defect. By a series of epistasis experiments and a transcriptional analysis, we demonstrate that MESR4 positively regulates the transcription of bam. Our results suggest that lack of repression alone is not sufficient, but MESR4-mediated transcriptional activation is also required for bam expression.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Carbonato de Cálcio/metabolismo , Diferenciação Celular/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Células Germinativas/metabolismo , Ovário , Células-TroncoRESUMO
Сhromatin is critical for genome compaction and gene expression. On a coarse scale, the genome is divided into euchromatin, which harbors the majority of genes and is enriched in active chromatin marks, and heterochromatin, which is gene-poor but repeat-rich. The conserved molecular hallmark of heterochromatin is the H3K9me3 modification, which is associated with gene silencing. We found that in Drosophila, deposition of most of the H3K9me3 mark depends on SUMO and the SUMO ligase Su(var)2-10, which recruits the histone methyltransferase complex SetDB1/Wde. In addition to repressing repeats, H3K9me3 influences expression of both hetero- and euchromatic host genes. High H3K9me3 levels in heterochromatin are required to suppress spurious transcription and ensure proper gene expression. In euchromatin, a set of conserved genes is repressed by Su(var)2-10/SetDB1-induced H3K9 trimethylation, ensuring tissue-specific gene expression. Several components of heterochromatin are themselves repressed by this pathway, providing a negative feedback mechanism to ensure chromatin homeostasis.
Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eucromatina/metabolismo , Retroalimentação Fisiológica , Expressão Gênica/genética , Inativação Gênica/fisiologia , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Ligases/genética , Metiltransferases/genética , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
Due to the evolutionary conservation of the regulation of hematopoiesis, Drosophila provides an excellent model organism to study blood cell differentiation and hematopoietic stem cell (HSC) maintenance. The larvae of Drosophila melanogaster respond to immune induction with the production of special effector blood cells, the lamellocytes, which encapsulate and subsequently kill the invader. Lamellocytes differentiate as a result of a concerted action of all three hematopoietic compartments of the larva: the lymph gland, the circulating hemocytes, and the sessile tissue. Within the lymph gland, the communication of the functional zones, the maintenance of HSC fate, and the differentiation of effector blood cells are regulated by a complex network of signaling pathways. Applying gene conversion, mutational analysis, and a candidate based genetic interaction screen, we investigated the role of Headcase (Hdc), the homolog of the tumor suppressor HECA in the hematopoiesis of Drosophila. We found that naive loss-of-function hdc mutant larvae produce lamellocytes, showing that Hdc has a repressive role in effector blood cell differentiation. We demonstrate that hdc genetically interacts with the Hedgehog and the Decapentaplegic pathways in the hematopoietic niche of the lymph gland. By adding further details to the model of blood cell fate regulation in the lymph gland of the larva, our findings contribute to the better understanding of HSC maintenance.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Hemolinfa/citologia , Transdução de Sinais , Animais , Diferenciação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Hemolinfa/metabolismo , Modelos AnimaisRESUMO
Self-renewal and differentiation of stem cells is one of the fundamental biological phenomena relying on proper chromatin organization. In our study, we describe a novel chromatin regulator encoded by the Drosophila small ovary (sov) gene. We demonstrate that sov is required in both the germline stem cells (GSCs) and the surrounding somatic niche cells to ensure GSC survival and differentiation. sov maintains niche integrity and function by repressing transposon mobility, not only in the germline, but also in the soma. Protein interactome analysis of Sov revealed an interaction between Sov and HP1a. In the germ cell nuclei, Sov colocalizes with HP1a, suggesting that Sov affects transposon repression as a component of the heterochromatin. In a position-effect variegation assay, we found a dominant genetic interaction between sov and HP1a, indicating their functional cooperation in promoting the spread of heterochromatin. An in vivo tethering assay and FRAP analysis revealed that Sov enhances heterochromatin formation by supporting the recruitment of HP1a to the chromatin. We propose a model in which sov maintains GSC niche integrity by regulating transposon silencing and heterochromatin formation.
Assuntos
Diferenciação Celular , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Inativação Gênica , Células Germinativas/citologia , Heterocromatina/metabolismo , Células-Tronco/citologia , Animais , Apoptose , Sobrevivência Celular , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Genoma , Células Germinativas/metabolismo , Mutação/genética , Transdução de Sinais , Nicho de Células-Tronco , Células-Tronco/metabolismo , Transcrição GênicaRESUMO
Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export.
Assuntos
Proteínas de Drosophila/genética , Proteínas dos Microfilamentos/genética , Proteínas Associadas à Matriz Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , RNA Mensageiro/genética , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosforilação , Ligação Proteica , Transporte de RNA/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/genéticaRESUMO
Inducible protein degradation techniques have considerable advantages over classical genetic approaches, which generate loss-of-function phenotypes at the gene or mRNA level. The plant-derived auxin-inducible degradation system (AID) is a promising technique which enables the degradation of target proteins tagged with the AID motif in nonplant cells. Here, we present a detailed characterization of this method employed during the adult oogenesis of Drosophila. Furthermore, with the help of CRISPR/Cas9-based genome editing, we improve the utility of the AID system in the conditional elimination of endogenously expressed proteins. We demonstrate that the AID system induces efficient and reversible protein depletion of maternally provided proteins both in the ovary and the early embryo. Moreover, the AID system provides a fine spatiotemporal control of protein degradation and allows for the generation of different levels of protein knockdown in a well-regulated manner. These features of the AID system enable the unraveling of the discrete phenotypes of genes with highly complex functions. We utilized this system to generate a conditional loss-of-function allele which allows for the specific degradation of the Vasa protein without affecting its alternative splice variant (solo) and the vasa intronic gene (vig). With the help of this special allele, we demonstrate that dramatic decrease of Vasa protein in the vitellarium does not influence the completion of oogenesis as well as the establishment of proper anteroposterior and dorsoventral polarity in the developing oocyte. Our study suggests that both the localization and the translation of gurken mRNA in the vitellarium is independent from Vasa.
Assuntos
Sistemas CRISPR-Cas , RNA Helicases DEAD-box/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/efeitos dos fármacos , Edição de Genes/métodos , Ácidos Indolacéticos/farmacologia , Fator de Crescimento Transformador alfa/genética , Alelos , Animais , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , RNA Helicases DEAD-box/deficiência , Relação Dose-Resposta a Droga , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Herança Materna , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Oogênese/genética , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Fenótipo , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Dorsal closure of the Drosophila embryonic epithelium provides an excellent model system for the in vivo analysis of molecular mechanisms regulating cytoskeletal rearrangements. In this study, we investigated the function of the Drosophila spectraplakin Short stop (Shot), a conserved cytoskeletal structural protein, during closure of the dorsal embryonic epithelium. We show that Shot is essential for the efficient final zippering of the opposing epithelial margins. By using isoform-specific mutant alleles and genetic rescue experiments with truncated Shot variants, we demonstrate that Shot functions as an actin-microtubule cross-linker in mediating zippering. At the leading edge of epithelial cells, Shot regulates protrusion dynamics by promoting filopodia formation. Fluorescence recovery after photobleaching (FRAP) analysis and in vivo imaging of microtubule growth revealed that Shot stabilizes dynamic microtubules. The actin- and microtubule-binding activities of Shot are simultaneously required in the same molecule, indicating that Shot is engaged as a physical crosslinker in this process. We propose that Shot-mediated interactions between microtubules and actin filaments facilitate filopodia formation, which promotes zippering by initiating contact between opposing epithelial cells.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Morfogênese , Actinas/metabolismo , Animais , Proteínas de Drosophila/química , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Células Epiteliais/citologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas dos Microfilamentos/química , Mutação/genética , Domínios Proteicos , Pseudópodes/metabolismoRESUMO
During catabolism of tryptophan through the kynurenine (KYN) pathway, several endogenous metabolites with neuromodulatory properties are produced, of which kynurenic acid (KYNA) is one of the highest significance. The causal role of altered KYNA production has been described in several neurodegenerative and neuropsychiatric disorders (e.g., Parkinson's disease, Huntington's disease, schizophrenia) and therefore kynurenergic manipulation with the aim of therapy has recently been proposed. Conventionally, KYNA is produced from its precursor L-KYN with the aid of the astrocytic kynurenine aminotransferase-2 (KAT-2) in the murine brain. Although the mouse is a standard therapeutic research organism, the presence of KAT-2 in mice has not been described in detail. This study demonstrates the presence of kat-2 mRNA and protein throughout the adult C57Bl6 mouse brain. In addition to the former expression data from the rat, we found prominent KAT-2 expression not only in the astrocyte, but also in neurons in several brain regions (e.g., hippocampus, substantia nigra, striatum, and prefrontal cortex). A significant number of the KAT-2 positive neurons were positive for GAD67; the presence of the KAT-2 enzyme we could also demonstrate in mice brain homogenate and in cells overexpressing recombinant mouse KAT-2 protein. This new finding attributes a new role to interneuron-derived KYNA in neuronal network operation. Furthermore, our results suggest that the thorough investigation of the spatio-temporal expression pattern of the relevant enzymes of the KYN pathway is a prerequisite for developing and understanding the pharmacological and transgenic murine models of kynurenergic manipulation.
Assuntos
Astrócitos/enzimologia , Encéfalo/enzimologia , Transaminases/análise , Animais , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/análiseRESUMO
Ezrin-Radixin-Moesin proteins are highly conserved, actin-binding cytoskeletal proteins that play an essential role in microvilli formation, T-cell activation, and tumor metastasis by linking actin filaments to the plasma membrane. Recent studies demonstrated that the only Ezrin-Radixin-Moesin protein of Drosophila melanogaster, Moesin, is involved in mitotic spindle function through stabilizing cell shape and microtubules at the cell cortex. We previously observed that Moesin localizes to the mitotic spindle; hence, we tested for the biological significance of this surprising localization and investigated whether it plays a direct role in spindle function. To separate the cortical and spindle functions of Moesin during mitosis we combined cell biological and genetic methods. We used early Drosophila embryos, in which mitosis occurs in the absence of a cell cortex, and found in vivo evidence for the direct requirement of Moesin in mitotic spindle assembly and function. We also found that the accumulation of Moesin precedes the construction of the microtubule spindle, and the fusiform structure formed by Moesin persists even after the microtubules have disassembled.
Assuntos
Proteínas de Membrana/metabolismo , Fuso Acromático/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Ciclo Celular/fisiologia , Forma Celular/fisiologia , Citoplasma/metabolismo , Drosophila melanogaster , Proteínas de Membrana/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Even though the number of Candida infections due to non-albicans species like C. parapsilosis has been increasing, little is known about their pathomechanisms. Certain aspects of C. parapsilosis and host interactions have already been investigated; however we lack information about the innate cellular responses toward this species. The aim of our project was to dissect and compare the phagocytosis of C. parapsilosis to C. albicans and to another Candida species C. glabrata by murine and human macrophages by live cell video microscopy. We broke down the phagocytic process into three stages: macrophage migration, engulfment of fungal cells and host cell killing after the uptake. Our results showed increased macrophage migration toward C. parapsilosis and we observed differences during the engulfment processes when comparing the three species. The engulfment time of C. parapsilosis was comparable to that of C. albicans regardless of the pseudohypha length and spatial orientation relative to phagocytes, while the rate of host cell killing and the overall uptake regarding C. parapsilosis showed similarities mainly with C. glabrata. Furthermore, we observed difference between human and murine phagocytes in the uptake of C. parapsilosis. UV-treatment of fungal cells had varied effects on phagocytosis dependent upon which Candida strain was used. Besides statistical analysis, live cell imaging videos showed that this species similarly to the other two also has the ability to survive in host cells via the following mechanisms: yeast replication, and pseudohypha growth inside of phagocytes, exocytosis of fungal cells and also abortion of host cell mitosis following the uptake. According to our knowledge this is the first study that provides a thorough examination of C. parapsilosis phagocytosis and reports intracellular survival mechanisms associated with this species.
RESUMO
In recent years, Drosophila melanogaster has become an attractive model organism in which to study the structure and development of the cellular immune components. The emergence of immunological markers greatly accelerated the identification of the immune cells (hemocytes), while the creation of genetic reporter constructs allowed unique insight into the structural organization of hematopoietic tissues. However, investigation of the hemocyte compartments by the means of immunological markers requires dissection and fixation, which regularly disrupt the delicate structure and hamper the microanatomical characterization. Moreover, the investigation of transgenic reporters alone can be misleading as their expression often differs from the native expression pattern of their respective genes. We describe here a method that combines the reporter constructs and the immunological tools in live imaging, thereby allowing use of the array of available immunological markers while retaining the structural integrity of the hematopoietic compartments. The procedure allows the reversible immobilization of Drosophila larvae for high-resolution confocal imaging and the time-lapse video analysis of in vivo reporters. When combined with our antibody injection-based in situ immunostaining assay, the resulting double labeling of the hemocyte compartments can provide new information on the microanatomy and functional properties of the hematopoietic tissues in an intact state. Although this method was developed to study the immune system of Drosophila melanogaster, we anticipate that such a combination of genetic and immunological markers could become a versatile technique for in vivo studies in other biological systems too.
Assuntos
Compartimento Celular , Drosophila melanogaster/citologia , Hemócitos/citologia , Imageamento Tridimensional , Animais , Hematopoese , Imobilização , Imuno-Histoquímica , Larva , Paralisia/patologiaRESUMO
In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general.
Assuntos
Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Interferência de RNA , Transcriptoma , Animais , Divisão Celular/genética , Análise por Conglomerados , Biologia Computacional , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mitose/genética , Fenótipo , Reprodutibilidade dos TestesRESUMO
The first microRNAs were discovered some 20 years ago, but only a small fraction of the microRNA-encoding genes have been described in detail yet. Here we report the molecular analysis of a computationally predicted Drosophila melanogaster microRNA gene, mir-282. We show that the mir-282 gene is the source of a 4.9-kb-long primary transcript with a 5' cap and a 3'-poly(A) sequence and a mature microRNA of â¼25 bp. Our data strongly suggest the existence of an independent mir-282 gene conserved in holometabolic insects. We give evidence that the mir-282 locus encodes a functional transcript that influences viability, longevity, and egg production in Drosophila. We identify the nervous system-specific adenylate cyclase (rutabaga) as a target of miR-282 and assume that one of the main functions of mir-282 is the regulation of adenylate cyclase activity in the nervous system during metamorphosis.
Assuntos
Adenilil Ciclases/genética , Drosophila melanogaster/genética , Longevidade/genética , MicroRNAs/genética , Adenilil Ciclases/metabolismo , Animais , Sobrevivência Celular , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Metamorfose Biológica/genética , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Óvulo/crescimento & desenvolvimentoRESUMO
Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil-DNA glycosylase and dUTPase. Lack of the major uracil-DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200-2,000 uracil/million bases, quantified using a novel real-time PCR-based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil-DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil-DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil-DNA in this evolutionary clade.
Assuntos
DNA/genética , Drosophila melanogaster/genética , Larva/genética , Pirofosfatases , Uracila , Animais , Linhagem Celular , DNA/química , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Inseto , Instabilidade Genômica , Células HeLa , Humanos , Larva/crescimento & desenvolvimento , Pirofosfatases/genética , Interferência de RNA , Uracila/química , Uracila/metabolismo , Uracila/farmacologia , Uracila-DNA Glicosidase/genéticaRESUMO
Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl), was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes.
Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/genética , Genes de Insetos/genética , Genômica , Microscopia , Animais , Padronização Corporal/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/anatomia & histologia , Epitélio/embriologia , Epitélio/metabolismo , Masculino , Interferência de RNA , Transdução de Sinais/genética , Fatores de TempoRESUMO
Much of our knowledge on hematopoiesis, hematopoietic compartments, hematopoietic cell lineages and immunity has been derived from studies on the vertebrate immune system. The sophisticated innate immunity of insects, the phylogenetic conservation and the power of Drosophila genetics allowed the investigation of immune cell (hemocyte) lineage relationships in Drosophila melanogaster. The development of the hemocyte lineages in Drosophila is a result of a precisely regulated succession of intracellular and intercellular events, though the nature and extent of these interactions are not known. We describe here a cell lineage tracing system set up to analyze the development of hemocyte lineages and functionally distinct hemocyte subsets. This system allowed us to distinguish two major embryonic hemocyte lineages, the crq and Dot lineages, in two, physically separated compartments, the embryonic macrophages and the embryonic lymph gland. We followed the fate and development of these lineages in the construction of the larval hematopoietic compartments and during the cell-mediated immune response, the encapsulation reaction. Our results revealed the considerable plasticity and concerted action of the hematopoietic compartments and the hemocyte lineages in the development of the innate immune system and in the course of the cell-mediated immune response in Drosophila.
Assuntos
Linhagem da Célula , Drosophila melanogaster/imunologia , Hematopoese , Hemócitos/fisiologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Hemócitos/citologia , Imunidade Celular , Imunidade Inata , Receptores Depuradores/genéticaRESUMO
Members of the vertebrate ezrin-radixin-moesin (ERM) protein family crosslink the actin cytoskeleton and the cell membrane and are, therefore, considered cytoplasmic regulators of cell adhesion, cell movement and membrane trafficking. Here we demonstrate that besides its cytoplasmic functions Drosophila moesin, the only ERM protein in Drosophila melanogaster, exhibits a dynamic cell cycle-dependent nuclear localization. In a small fraction of cells and at a low level, moesin can be detected in interphase nuclei in regions complementary to the chromatin; its level rapidly increases during prophase and it co-localizes with the actin network surrounding the mitotic spindles throughout mitosis. We also found that the predicted single nuclear localization signal in moesin is not necessary for the nuclear accumulation of the protein. FRAP experiments confirmed this finding and further revealed that the mitotic localization of moesin is highly dynamic. Immuno-histochemical staining for moesin demonstrated the existence of spindle association in wild-type embryos. The biological relevance of this phenomenon is indicated by the mitotic phenotypes detected in S2 cells treated with moesin RNAi, and awaits future exploration.