RESUMEN
The last step of cell death is cell clearance, a process critical for tissue homeostasis. For efficient cell clearance to occur, phagocytes and dead cells need to reciprocally signal to each other. One important phenomenon that is under-investigated, however, is that phagocytes not only engulf corpses but contribute to cell death progression. The aims of this study were to determine how the phagocytic receptor Draper non-autonomously induces cell death, using the Drosophila ovary as a model system. We found that Draper, expressed in epithelial follicle cells, requires its intracellular signaling domain to kill the adjacent nurse cell population. Kinases Src42A, Shark and JNK (Bsk) were required for Draper-induced nurse cell death. Signs of nurse cell death occurred prior to apparent engulfment and required the caspase Dcp-1, indicating that it uses a similar apoptotic pathway to starvation-induced cell death. These findings indicate that active signaling by Draper is required to kill nurse cells via the caspase Dcp-1, providing novel insights into mechanisms of phagoptosis driven by non-professional phagocytes.
Asunto(s)
Proteínas de Drosophila , Animales , Femenino , Proteínas de Drosophila/metabolismo , Fagocitosis/fisiología , Receptores Inmunológicos , Drosophila/metabolismo , Muerte Celular , Caspasas , Apoptosis/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)RESUMEN
The clearance of dead cells is a fundamental process in the maintenance of tissue homeostasis. Genetic studies in Drosophila melanogaster, Caenorhabditis elegans, and mammals have identified two evolutionarily conserved signaling pathways that act redundantly to regulate this engulfment process: the ced-1/-6/-7 and ced-2/-5/-12 pathways. Of these engulfment genes, only the ced-7/ABCA1 ortholog remains to be identified in D. melanogaster Homology searches have revealed a family of putative ced-7/ABCA1 homologs encoding ATP-binding cassette (ABC) transporters in D. melanogaster To determine which of these genes functions similarly to ced-7/ABCA1, we analyzed mutants for engulfment phenotypes in oogenesis, during which nurse cells (NCs) in each egg chamber undergo programmed cell death (PCD) and are removed by neighboring phagocytic follicle cells (FCs). Our genetic analyses indicate that one of the ABC transporter genes, which we have named Eato (Engulfment ABC Transporter in the ovary), is required for NC clearance in the ovary and acts in the same pathways as drpr, the ced-1 ortholog, and in parallel to Ced-12 in the FCs. Additionally, we show that Eato acts in the FCs to promote accumulation of the transmembrane receptor Drpr, and promote membrane extensions around the NCs for their clearance. Since ABCA class transporters, such as CED-7 and ABCA1, are known to be involved in lipid trafficking, we propose that Eato acts to transport membrane material to the growing phagocytic cup for cell corpse clearance. Our work presented here identifies Eato as the ced-7/ABCA1 ortholog in D. melanogaster, and demonstrates a role for Eato in Drpr accumulation and phagocytic membrane extensions during NC clearance in the ovary.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Apoptosis/genética , Drosophila melanogaster/genética , Ovario/metabolismo , Transportador 1 de Casete de Unión a ATP/química , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Femenino , Genotipo , Mutación , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Ovario/citología , FenotipoRESUMEN
Billions of cells die in our bodies on a daily basis and are engulfed by phagocytes. Engulfment, or phagocytosis, can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification. In this study, we focus on the last two steps, which can collectively be considered corpse processing, in which the engulfed material is degraded. We use the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. We show that engulfed material is processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, we investigated the role of Draper, integrins, and downstream signaling components in corpse processing. We found that some proteins were required for internalization only, while others had defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. We also performed double mutant analysis and found that combined loss of draper and αPS3 still resulted in a small number of engulfed vesicles. Therefore, we investigated another known engulfment receptor, Crq. We found that loss of all three receptors did not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells.
Asunto(s)
Fagocitos/fisiología , Fagocitosis , Animales , Apoptosis , Caenorhabditis elegans , Drosophila , Endocitosis , Células Epiteliales/metabolismo , Femenino , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Fagosomas/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Programmed cell death occurs in the germline of many organisms, both as an essential part of development and throughout adult life. Germline cell death can be apoptotic or nonapoptotic, depending on the stimulus or stage of development. Here, we focus on the Drosophila ovary, which is a powerful model for studying diverse types of cell death. In Drosophila, the death of primordial germ cells occurs normally during embryonic development, and germline nurse cells are programmed to die during oocyte development in adult flies. Cell death of previtellogenic egg chambers in adults can also be induced by starvation or other environmental cues. Mid-oogenesis seems to be particularly sensitive to such cues and has been proposed to serve as a checkpoint to avoid the energetically expensive cost of egg production. After the germline dies in mid-oogenesis, the remnants are engulfed by an epithelial layer of follicle cells; thus, the fly ovary also serves as a highly tractable model for engulfment by epithelial cells. These examples of cell death in the fly ovary share many similarities to the types of cell death seen in the mammalian germline. Recent progress in elucidating the molecular mechanisms of cell death in the germline is discussed.
Asunto(s)
Drosophila/citología , Ovario/citología , Testículo/citología , Animales , Apoptosis , Muerte Celular , Femenino , Masculino , Mamíferos , Oogénesis , Ovario/fisiologíaRESUMEN
During the final stages of Drosophila melanogaster oogenesis fifteen nurse cells, sister cells to the oocyte, degenerate as part of normal development. This process involves at least two cell death mechanisms, caspase-dependent cell death and autophagy, as indicated by apoptosis and autophagy markers. In addition, mutations affecting either caspases or autophagy partially reduce nurse cell removal, leaving behind end-stage egg chambers with persisting nurse cell nuclei. To determine whether apoptosis and autophagy work in parallel to degrade and remove these cells as is the case with salivary glands during pupariation, we generated mutants doubly affecting caspases and autophagy. We found no significant increase in either the number of late stage egg chambers containing persisting nuclei or in the number of persisting nuclei per egg chamber in the double mutants compared to single mutants. These findings suggest that there is another cell death mechanism functioning in the ovary to remove all nurse cell remnants from late stage egg chambers.
Asunto(s)
Apoptosis/fisiología , Autofagia/fisiología , Caspasas/metabolismo , Drosophila melanogaster/fisiología , Oogénesis/fisiología , Ovario/citología , Animales , Apoptosis/genética , Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia , Caspasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Microscopía Fluorescente , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Drosophila is a powerful model system for the identification of cell death genes and understanding the role of cell death in development. In this chapter, we describe three methods typically used for the detection of cell death in Drosophila. The TUNEL and acridine orange methods are used to detect dead or dying cells in a variety of tissues. We focus on methods for the embryo and the ovary, but these techniques can be used on other tissues as well. The third method is the detection of genetic interactions by expressing cell death genes in the Drosophila eye.
Asunto(s)
Apoptosis , Drosophila/citología , Animales , Muerte Celular , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Ojo/metabolismo , Femenino , Regulación de la Expresión Génica , Genes de Insecto , Ovario/metabolismoRESUMEN
Programmed cell death (PCD) is a highly conserved process that occurs during development and in response to adverse conditions. In Drosophila, most PCDs require the genes within the H99 deficiency, the adaptor molecule Ark, and caspases. Here we investigate 10 cell death genes for their potential roles in two distinct types of PCD that occur in oogenesis: developmental nurse cell PCD and starvation-induced PCD. Most of the genes investigated were found to have little effect on late stage developmental PCD in oogenesis, although ark mutants showed a partial inhibition. Mid-stage starvation-induced germline PCD was found to be independent of the upstream activators and ark although it requires caspases, suggesting an apoptosome-independent mechanism of caspase activation in mid-oogenesis. These results indicate that novel pathways must control PCD in the ovary.
Asunto(s)
Apoptosis/genética , Apoptosomas/genética , Proteínas de Drosophila/fisiología , Drosophila/crecimiento & desarrollo , Oogénesis , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Femenino , Genes de Insecto/fisiología , Mutación , Transducción de SeñalRESUMEN
Organized boundaries between different cell fates are critical in patterning and organogenesis. In some tissues, long-range signals position a boundary, and local Notch signaling maintains it. How Notch activity is restricted to boundary regions is not well understood. During Drosophila oogenesis, the long-range signals EGF and Dpp regulate expression of bunched (bun), which encodes a homolog of mammalian transcription factors TSC-22 and GILZ. Here, we show that bun establishes a boundary for Notch signaling in the follicle cell epithelium. Notch signaling is active in anterior follicle cells and is required for concurrent follicle cell reorganizations including centripetal migration and operculum formation. bun is required in posterior columnar follicle cells to repress the centripetal migration fate, including gene expression, cell shape changes and accumulation of cytoskeletal components. bun mutant clones adjacent to the centripetally migrating follicle cells showed ectopic Notch responses. bun is necessary, but not sufficient, to down-regulate Serrate protein levels throughout the follicular epithelium. These data indicate that Notch signaling is necessary, but not sufficient, for centripetal migration and that bun regulates the level of Notch stimulation to position the boundary between centripetally migrating and stationary columnar follicle cells.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Folículo Ovárico/fisiología , Receptores Notch/metabolismo , Factores de Transcripción/fisiología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Movimiento Celular , Forma de la Célula , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-1 , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oogénesis , Folículo Ovárico/metabolismo , Receptores Notch/genética , Proteínas Serrate-Jagged , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Drosophila has unique genetic and cell biological advantages as a model system for the study of apoptosis. Many cell death genes are evolutionarily conserved between flies and mammals. Cell death can be induced by environmental stimuli and normally occurs during diverse developmental processes in Drosophila. Here, we review several approaches for detecting cell death in Drosophila. We provide detailed protocols for labeling apoptotic cells in the embryo and ovary using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and acridine orange. Additionally, we describe methods for ectopically expressing cell death genes in the eye and the use of transgenic flies for the detection of genetic interactions among cell death genes.
Asunto(s)
Apoptosis/fisiología , Drosophila melanogaster/fisiología , Naranja de Acridina/metabolismo , Animales , Animales Modificados Genéticamente , ADN/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Ojo/metabolismo , Femenino , Colorantes Fluorescentes/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Ovario/citología , Ovario/metabolismoAsunto(s)
Apoptosis/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Animales , Anexina A5/análisis , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Ojo/citología , Ojo/embriología , Femenino , Histocitoquímica/métodos , Etiquetado Corte-Fin in Situ , Indicadores y Reactivos , Larva , Ovario/citología , Ovario/embriologíaRESUMEN
Germline cell death in Drosophila oogenesis is controlled by distinct signals. The death of nurse cells in late oogenesis is developmentally regulated, whereas the death of egg chambers during mid-oogenesis is induced by environmental stress or developmental abnormalities. P-element insertions in the caspase gene dcp-1 disrupt both dcp-1 and the outlying gene, pita, leading to lethality and defective nurse cell death in late oogenesis. By isolating single mutations in the two genes, we have found that the loss of both genes contributes to this ovary phenotype. Mutants of pita, which encodes a C2H2 zinc-finger protein, are homozygous lethal and show dumpless egg chambers and premature nurse cell death in germline clones. Early nurse cell death is not observed in the dcp-1/pita double mutants, suggesting that dcp-1+ activity is required for the mid-oogenesis cell death seen in pita mutants. dcp-1 mutants are viable and nurse cell death in late oogenesis occurs normally. However, starvation-induced germline cell death during mid-oogenesis is blocked, leading to a reduction and inappropriate nuclear localization of the active caspase Drice. These findings suggest that the combinatorial loss of pita and dcp-1 leads to the increased survival of abnormal egg chambers in mutants bearing the P-element alleles and that dcp-1 is essential for cell death during mid-oogenesis.