RESUMEN

Homologous recombination (HR) is one of the key pathways to repair double-strand breaks (DSBs). Rad51 serves an important function of catalysing strand exchange between two homologous sequences in the HR pathway. In higher organisms, rad51 function is indispensable with its absence leading to early embryonic lethality, thus precluding any mechanistic probing of the system. In contrast, the absence of Drosophila rad51 (spn-A/rad51) has been associated with defects in the germline, without any reported detrimental consequences to Drosophila somatic tissues. In this study, we have performed a systematic analysis of developmental defects in somatic tissues of spn-A mutant flies by using genetic complementation between multiple spn-A alleles. Our current study, for the first time, uncovers a requirement for spn-A in somatic tissue maintenance during both larval and pupal stages. Also, we show that spn-A mutant exhibits patterning defects in abdominal cuticle in the stripes and bristles, while there appear to be only subtle defects in the adult wing and eye. Interestingly, spn-A mutant shows a discernible phenotype of low temperature sensitivity, suggesting a role of spn-A in temperature sensitive cellular processes. In summary, our study describes the important role played by spn-A/rad51 in Drosophila somatic tissues.

Asunto(s)

Muerte Celular , Daño del ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Mutación , Recombinasa Rad51/metabolismo , Temperatura , Animales , Tipificación del Cuerpo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Células Germinativas , Recombinación Homóloga , Masculino , Meiosis , Fenotipo , Recombinasa Rad51/genética

RESUMEN

Mitochondrial mechanisms and pathways have recently emerged as critical determinants of organismal aging. While nuclear sirtuins have been shown to regulate aging, whether mitochondrial sirtuins do so is still unclear. Here, we report that mitochondrial dSirt4 mediates organismal aging. We establish that absence of dSirt4 leads to reduced lifespan independent of dietary inputs. Further by assaying locomotion, a key correlate of aging, we demonstrate that dSirt4 null flies are severely physically impaired with a significant reduction in locomotion. In summary, we report that mitochondrial dSirt4 is a key determinant of longevity and its loss leads to early aging.

Asunto(s)

Envejecimiento/genética , Drosophila/genética , Longevidad/genética , Sirtuinas/genética , Animales , Núcleo Celular , Drosophila/fisiología , Longevidad/fisiología , Mitocondrias/genética , Condicionamiento Físico Animal/fisiología

RESUMEN

The phosphorylation of the variant histone H2Ax (denoted γH2Ax; γH2Av in flies) constitutes an important signalling event in DNA damage sensing, ensuring effective repair by recruiting DNA repair machinery. In contrast, the γH2Av response has also been reported in dying cells, where it requires activation of caspase-activated DNases (CADs). Moreover, caspases are known to be required downstream of DNA damage for cell death execution. We show here, for the first time, that the Drosophila initiator caspase Dronc acts as an upstream regulator of the DNA damage response (DDR) independently of executioner caspases by facilitating γH2Av signalling, possibly through a function that is not related to apoptosis. Such a γH2Av response is mediated by ATM rather than ATR, suggesting that Dronc function is required upstream of ATM. In contrast, the role of γH2Av in cell death requires effector caspases and is associated with fragmented nuclei. Our study uncovers a novel function of Dronc in response to DNA damage aimed at promoting DDR via γH2Av signalling in intact nuclei. We propose that Dronc plays a dual role that can either initiate DDR or apoptosis depending upon its level and the required threshold of its activation in damaged cells.This article has an associated First Person interview with the first author of the paper.

Asunto(s)

Apoptosis , Caspasas/metabolismo , Daño del ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cisplatino/farmacología , Fragmentación del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Transducción de Señal/efectos de los fármacos

RESUMEN

Endocrine signaling is central in coupling organismal nutrient status with maintenance of systemic metabolic homeostasis. While local nutrient sensing within the insulinogenic tissue is well studied, distant mechanisms that relay organismal nutrient status in controlling metabolic-endocrine signaling are less well understood. Here, we report a novel mechanism underlying the distant regulation of the metabolic endocrine response in Drosophila melanogaster We show that the communication between the fat body and insulin-producing cells (IPCs), important for the secretion of Drosophila insulin-like peptides (dILPs), is regulated by the master metabolic sensor Sir2/Sirt1. This communication involves a fat body-specific direct regulation of the JAK/STAT cytokine upd2 by Sir2/Sirt1. We have also uncovered the importance of this regulation in coupling nutrient inputs with dILP secretion, and distantly controlling insulin/IGF signaling (IIS) in the intestine. Our results provide fundamental mechanistic insights into the top-down control involving tissues that play key roles in metabolic sensing, endocrine signaling and nutrient uptake.

Asunto(s)

Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Histona Desacetilasas/metabolismo , Insulina/metabolismo , Transducción de Señal , Sirtuina 1/metabolismo , Sirtuinas/metabolismo , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Cuerpo Adiposo/metabolismo , Femenino , Células Secretoras de Insulina/metabolismo , Insulinas/metabolismo , Mucosa Intestinal/metabolismo

RESUMEN

Signaling from a niche consisting of somatic cells is essential for maintenance of germline stem cells (GSCs) in the ovary of Drosophila. Decapentaplegic (Dpp), a type of bone morphogenetic protein (BMP) signal, emanating from the niche, is the most important signal for this process. Cullin proteins constitute the core of a multiprotein E3-ligase important for their functions viz. degradation or modification of proteins necessary for different cellular processes. We have found that a Cullin protein called Cullin-2 (Cul-2) expresses in both somatic and germline cells of the Drosophila ovary. Reduction of Cul-2 in somatic cells causes upregulation of Dpp signal and produces accumulation of extra GSC-like cells inside germarium, the anteriormost structure of the ovary. Our results suggest that Cullin-2 protein present in the somatic cells is involved in a non cell-autonomous regulation of the extent of Dpp signaling and thus controls the differentiation of GSCs to cystoblasts (CBs).

Asunto(s)

Proteínas Cullin/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Ovario/fisiología , Células Madre/citología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Cruzamientos Genéticos , Regulación hacia Abajo , Receptores ErbB/metabolismo , Femenino , Genotipo , Microscopía Fluorescente , Fenotipo , Interferencia de ARN , Transducción de Señal

RESUMEN

Sir2 is an evolutionarily conserved NAD(+)-dependent deacetylase which has been shown to play a critical role in glucose and fat metabolism. In this study, we have perturbed Drosophila Sir2 (dSir2) expression, bidirectionally, in muscles and the fat body. We report that dSir2 plays a critical role in insulin signaling, glucose homeostasis, and mitochondrial functions. Importantly, we establish the nonautonomous functions of fat body dSir2 in regulating mitochondrial physiology and insulin signaling in muscles. We have identified a novel interplay between dSir2 and dFOXO at an organismal level, which involves Drosophila insulin-like peptide (dILP)-dependent insulin signaling. By genetic perturbations and metabolic rescue, we provide evidence to illustrate that fat body dSir2 mediates its effects on the muscles via free fatty acids (FFA) and dILPs (from the insulin-producing cells [IPCs]). In summary, we show that fat body dSir2 is a master regulator of organismal energy homeostasis and is required for maintaining the metabolic regulatory network across tissues.

Asunto(s)

Proteínas de Drosophila/metabolismo , Drosophila/genética , Cuerpo Adiposo/fisiología , Histona Desacetilasas/metabolismo , Mitocondrias/fisiología , Músculos/fisiología , Sirtuinas/metabolismo , Animales , Carnitina/administración & dosificación , Drosophila/fisiología , Proteínas de Drosophila/genética , Compuestos Epoxi/administración & dosificación , Ácidos Grasos no Esterificados/metabolismo , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Prueba de Tolerancia a la Glucosa , Histona Desacetilasas/genética , Homeostasis , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Insulina/metabolismo , Metabolismo de los Lípidos , Potencial de la Membrana Mitocondrial , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Transducción de Señal , Sirtuinas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Sir2, an evolutionarily conserved NAD(+)-dependent deacetylase, has been implicated as a key factor in mediating organismal life span. However, recent contradictory findings have brought into question the role of Sir2 and its orthologs in regulating organismal longevity. In this study, we report that Drosophila Sir2 (dSir2) in the adult fat body regulates longevity in a diet-dependent manner. We used inducible Gal4 drivers to knock down and overexpress dSir2 in a tissue-specific manner. A diet-dependent life span phenotype of dSir2 perturbations (both knockdown and overexpression) in the fat body, but not muscles, negates the effects of background genetic mutations. In addition to providing clarity to the field, our study contrasts the ability of dSir2 in two metabolic tissues to affect longevity. We also show that dSir2 knockdown abrogates fat-body dFOXO-dependent life span extension. This report highlights the importance of the interplay between genetic factors and dietary inputs in determining organismal life spans.

Asunto(s)

Dieta , Proteínas de Drosophila/metabolismo , Cuerpo Adiposo/metabolismo , Histona Desacetilasas/metabolismo , Longevidad/fisiología , Sirtuinas/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Técnicas de Silenciamiento del Gen , Histona Desacetilasas/genética , Músculos/metabolismo , Sirtuinas/genética

RESUMEN

Sir2 is an evolutionarily conserved NAD+ dependent protein. Although, SIRT1 has been implicated to be a key regulator of fat and glucose metabolism in mammals, the role of Sir2 in regulating organismal physiology, in invertebrates, is unclear. Drosophila has been used to study evolutionarily conserved nutrient sensing mechanisms, however, the molecular and metabolic pathways downstream to Sir2 (dSir2) are poorly understood. Here, we have knocked down endogenous dSir2 in a tissue specific manner using gene-switch gal4 drivers. Knockdown of dSir2 in the adult fatbody leads to deregulated fat metabolism involving altered expression of key metabolic genes. Our results highlight the role of dSir2 in mobilizing fat reserves and demonstrate that its functions in the adult fatbody are crucial for starvation survival. Further, dSir2 knockdown in the fatbody affects dilp5 (insulin-like-peptide) expression, and mediates systemic effects of insulin signaling. This report delineates the functions of dSir2 in the fatbody and muscles with systemic consequences on fat metabolism and insulin signaling. In conclusion, these findings highlight the central role that fatbody dSir2 plays in linking metabolism to organismal physiology and its importance for survival.

Asunto(s)

Proteínas de Drosophila/deficiencia , Drosophila melanogaster/enzimología , Metabolismo Energético , Cuerpo Adiposo/enzimología , Histona Desacetilasas/deficiencia , Insulina/metabolismo , Metabolismo de los Lípidos , Músculos/enzimología , Transducción de Señal , Sirtuinas/deficiencia , Inanición , Adaptación Fisiológica , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Metabolismo Energético/genética , Técnicas de Silenciamiento del Gen , Histona Desacetilasas/genética , Insulinas/metabolismo , Metabolismo de los Lípidos/genética , Interferencia de ARN , Transducción de Señal/genética , Sirtuinas/genética , Inanición/genética , Factores de Tiempo

RESUMEN

Cullins confer substrate specificity to E3-ligases which are multi-protein complexes involved in ubiquitin-mediated protein degradation or modification. There are six cullin genes in Drosophila melanogaster. We have raised an antibody against Cul-5 and demonstrated that it expresses in neuronal and non-neuronal cells throughout development. In the embryonic tracheal system, Cul-5 is enriched at fusion sites together with E-Cadherin and Fasciclin III. Mutations of cul-5 do not affect tracheal development but do show defects in the organization of synaptic boutons at the larval neuromuscular junction where the protein is expressed in a subset of motoneuron terminals. Loss of function of another cullin gene 'cul-2' results in similar defects at the larval neuromuscular junction although cul-2;cul-5 double mutants do not show an enhanced phenotype. Both cul-2 and cul-5 mutants show similar aberrations in the development of female germ line. Our results suggest that both of these cullin proteins participate in similar developmental processes.

Asunto(s)

Proteínas Cullin/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Unión Neuromuscular/crecimiento & desarrollo , Ovario/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Proteínas Cullin/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Componentes del Gen , Técnicas de Inactivación de Genes , Larva/genética , Larva/metabolismo , Masculino , Datos de Secuencia Molecular , Mutagénesis Insercional , Unión Neuromuscular/metabolismo , Oogénesis , Especificidad de Órganos , Ovario/metabolismo , Fenotipo , Alineación de Secuencia , Tráquea/crecimiento & desarrollo , Tráquea/metabolismo

RESUMEN

Naive Drosophila larvae show vigorous chemotaxis toward many odorants including ethyl acetate (EA). Chemotaxis toward EA is substantially reduced after a 5-min pre-exposure to the odorant and recovers with a half-time of ∼20 min. An analogous behavioral decrement can be induced without odorant-receptor activation through channelrhodopsin-based, direct photoexcitation of odorant sensory neurons (OSNs). The neural mechanism of short-term habituation (STH) requires the (1) rutabaga adenylate cyclase; (2) transmitter release from predominantly GABAergic local interneurons (LNs); (3) GABA-A receptor function in projection neurons (PNs) that receive excitatory inputs from OSNs; and (4) NMDA-receptor function in PNs. These features of STH cannot be explained by simple sensory adaptation and, instead, point to plasticity of olfactory synapses in the antennal lobe as the underlying mechanism. Our observations suggest a model in which NMDAR-dependent depression of the OSN-PN synapse and/or NMDAR-dependent facilitation of inhibitory transmission from LNs to PNs contributes substantially to short-term habituation.

Asunto(s)

Drosophila/fisiología , Plasticidad Neuronal/fisiología , Bulbo Olfatorio/fisiología , Percepción Olfatoria/fisiología , Neuronas Receptoras Olfatorias/fisiología , Sinapsis/fisiología , Adenilil Ciclasas , Animales , Proteínas de Drosophila , Habituación Psicofisiológica , Inmunohistoquímica , Larva , Bulbo Olfatorio/citología

RESUMEN

Most insertional mutagenesis screens of Drosophila performed to date have not used target chromosomes that have been checked for their suitability for phenotypic screens for viable phenotypes. To address this, we have generated a selection of stocks carrying either isogenized second chromosomes or isogenized third chromosomes, in a genetic background derived from a Canton-S wild-type strain. We have tested these stocks for a range of behavioral and other viable phenotypes. As expected, most lines are statistically indistinguishable from Canton-S in most phenotypes tested. The lines generated are now being used as target chromosomes in mutagenesis screens, and the characterization reported here will facilitate their use in screens of these lines for behavioral and other viable phenotypes.

Asunto(s)

Drosophila melanogaster/genética , Isocromosomas/genética , Anestésicos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Benzaldehídos/farmacología , Ritmo Circadiano/genética , Copulación , Elementos Transponibles de ADN/genética , Drosophila melanogaster/efectos de los fármacos , Femenino , Pruebas Genéticas/métodos , Aprendizaje/efectos de los fármacos , Locomoción/efectos de los fármacos , Masculino , Mutación , Parálisis/genética , Fenotipo , Conducta Sexual Animal , Alas de Animales/anatomía & histología

RESUMEN

We describe a developmental analysis of Drosophila Cullin-5 (Cul-5) identified from the genome sequence on the basis of its high degree of homology to vertebrate and worm sequences. The gene is expressed in a restricted manner in ectodermal cells throughout development suggesting pleiotropic functions. We decided to examine the phenotypes of Cul-5 aberrations in two well-studied developmental systems: the neuromuscular junction (NMJ) and the developing sensory organ. Alteration of Cul-5 levels in motoneurons results in an increase in bouton number at the NMJ. The cells of a sensory organ on the adult notum arise from a single progenitor cell by regulated cell division. Aberrations in Cul-5 affect different steps in the lineage consistent with a role in cell fate determination, proliferation, and death. Such phenotypes highlight the multiple cellular processes in which Cul-5 can participate.

Asunto(s)

Proteínas Cullin/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Unión Neuromuscular/embriología , Neuronas Aferentes/citología , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , División Celular , Proteínas Cullin/química , Proteínas Cullin/genética , Proteínas Cullin/ultraestructura , Drosophila/genética , Drosophila/crecimiento & desarrollo , Drosophila/ultraestructura , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/ultraestructura , Embrión no Mamífero , Expresión Génica , Regulación de la Expresión Génica , Genes de Insecto , Larva , Metamorfosis Biológica , Datos de Secuencia Molecular , Mutación , Unión Neuromuscular/citología , Unión Neuromuscular/ultraestructura , Neuronas Aferentes/ultraestructura , Organogénesis , Pupa , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Sinapsis/ultraestructura

RESUMEN

In a previous paper, we showed that weak hypomorphic alleles at the myospheroid (mys) locus, which encodes the beta-subunit of integrin, possess defects in olfactory behavior in both adult and larva. In this paper, we show that another olfactory gene, olfE, exhibits haploinsufficient interactions with recessive alleles at the mys locus. olfE has recently been shown to be an allele of swisscheese and is now designated as sws(olfE). Our findings suggest an interaction between the sws protein and beta-integrin in the development and/or functioning of the olfactory system. Similar interactions were also observed between sws and inflated, a gene encoding the alpha2-subunit of integrin, as well as mys and multiple edematous wing (mew), a gene coding for alpha1 subunit of integrin. This study provides evidence for the roles of different integrin subunits and the sws product in regulating normal olfactory behavior in Drosophila.

Asunto(s)

Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto , Integrinas/fisiología , Olfato/genética , Animales , Expresión Génica , Integrinas/genética , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas/genética