The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila

The initiator caspase Dronc is the only Drosophila caspase that contains a caspase activation and recruitment domain (CARD). Although Dronc has been implicated as an important effector of apoptosis, the genetic function of dronc in normal development is unclear because dronc mutants have not been available. In an EMS mutagenesis screen, we isolated four point mutations in dronc that recessively suppress the eye ablation phenotype caused by eye-specific overexpression of hid. Homozygous mutant dronc animals die during pupal stages; however, at a low frequency we obtained homozygous adult escapers. These escapers have additional cells in the eye and wings that are less transparent and slightly curved down. We determined that this is due to lack of apoptosis. Our analyses of dronc mutant embryos suggest that dronc is essential for most apoptotic cell death during Drosophila development, but they also imply the existence of a dronc-independent cell death pathway. We also constructed double mutant flies for dronc and the apoptosis inhibitor diap1. dronc mutants can rescue the ovarian degeneration phenotype caused by diap1 mutations, confirming that dronc acts genetically downstream of diap1.     

AP-1, but not NF-kappa B, is required for efficient steroid-triggered cell death in Drosophila

Extensive studies in vertebrate cells have assigned a central role to Rel/NF-kappa B and AP-1 family members in the control of apoptosis. We ask here whether parallel pathways might function in Drosophila by determining if Rel/NF-kappa B or AP-1 family members contribute to the steroid-triggered death of larval salivary glands during Drosophila metamorphosis. We show that two of the three Drosophila Rel/NF-kappa B genes are expressed in doomed salivary glands and that one family member, Dif, is induced in a stage-specific manner immediately before the onset of programmed cell death. Similarly, Djun is expressed for many hours before salivary gland cell death while Dfos is induced in a stage-specific manner, immediately before this tissue is destroyed. We show that null mutations in the three Drosophila Rel/NF-kappa B family members, either alone or in combination, have no apparent effect on this death response. In contrast, Dfos is required for the proper timing of larval salivary gland cell death as well as the proper induction of key death genes. This study demonstrates a role for AP-1 in the stage-specific steroid-triggered programmed cell death of larval tissues during Drosophila metamorphosis.     

Identification of X-linked genes required for migration and programmed cell death of Drosophila melanogaster germ cells

Drosophila germ cells form at the posterior pole of the embryo and migrate to the somatic gonad. Approximately 50% of the germ cells that form reach their target. The errant cells within the embryo undergo developmentally regulated cell death. Prior studies have identified some autosomal genes that regulate germ cell migration, but the genes that control germ cell death are not known. To identify X-linked genes required for germ cell migration and/or death, we performed a screen for mutations that disrupt these processes. Here we report the identification of scattershot and outsiders, two genes that regulate the programmed death of germ cells. The scattershot gene is defined by a mutation that disrupts both germ cell migration and the death of germ cells ectopic to the gonad. Maternal and zygotic expression of scattershot is required, but the migration and cell death functions can be genetically uncoupled. Zygotic expression of wild-type scattershot rescues germ cell pathfinding, but does not restore the programmed death of errant cells. The outsiders gene is required zygotically. In outsiders mutant embryos, the appropriate number of germ cells is incorporated into the gonad, but germ cells ectopic to the gonad persist.     

Genetic control of programmed cell death in Drosophila melanogaster

Apoptosis is a genetically controlled form of cell death that is an important feature of animal development and homeostasis. The genes involved in the control and execution of apoptosis are conserved throughout evolution. However, the actual molecular mechanisms used by these genes vary from species to species. In this review, we focus on the genetic components of apoptosis in the fruit fly Drosophila melanogaster, and compare their mode of action to the one employed by the homologous genes in mammals. We also cover recent advances that show that apoptotic genes have a requirement in processes other than apoptosis.     

Internal ribosome entry site-mediated translation of Apaf-1, but not XIAP, is regulated during UV-induced cell death

Components of the cellular translation machinery are targets of caspase-mediated cleavage during apoptosis that correlates with the inhibition of protein synthesis, which accompanies apoptosis. Paradoxically, protein synthesis is required for apoptosis to occur in many experimental settings. Previous studies showed that two proteins that regulate apoptosis by controlling caspase activity, XIAP and Apaf-1, are translated by a unique, cap-independent mechanism mediated by an internal ribosome entry site (IRES) that is used preferentially under conditions in which normal cap-dependent translation is repressed. We investigated the regulation of XIAP and Apaf-1 following UVC irradiation. We show that UVC irradiation leads to the inhibition of translation and cell death. Furthermore, IRES-mediated translation of Apaf-1, but not XIAP, is enhanced by UVC irradiation, and this increase in Apaf-1 translation correlated with cell death. The enhanced Apaf-1 IRES-mediated translation is caspase-independent but is negatively modulated by the eIF2alpha kinase protein kinase RNA-like endoplasmic reticulum kinase. These data suggest that progression of UV-induced apoptosis requires IRES-mediated translation of Apaf-1 to ensure continuous levels of Apaf-1 despite an overall suppression of protein synthesis.     

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.     

Role of mitochondrial remodeling in programmed cell death in Drosophila melanogaster

The role of mitochondria in Drosophila programmed cell death remains unclear, although certain gene products that regulate cell death seem to be evolutionarily conserved. We find that developmental programmed cell death stimuli in vivo and multiple apoptotic stimuli ex vivo induce dramatic mitochondrial fragmentation upstream of effector caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. Unlike genotoxic stress, a lipid cell death mediator induced an increase in mitochondrial contiguity prior to fragmentation of the mitochondria. Using genetic mutants and RNAi-mediated knockdown of drp-1, we find that Drp-1 not only regulates mitochondrial fission in normal cells, but mediates mitochondrial fragmentation during programmed cell death. Mitochondria in drp-1 mutants fail to fragment, resulting in hyperplasia of tissues in vivo and protection of cells from multiple apoptotic stimuli ex vivo. Thus, mitochondrial remodeling is capable of modifying the propensity of cells to undergo death in Drosophila.     

The role of Apaf-1 in programmed cell death: from worm to tumor

Apoptosis or programmed cell death is an important process to eliminate unnecessary or hazardous cells. Apaf-1, a mammalian homologue of CED-4 of C. elegans, is the essential adaptor molecule in the mitochondrial pathway of apoptosis. Mice lacking Apaf-1 show accumulation of neurons in the developing central nervous system due to reduced apoptosis. Apaf-1-deficient cells are remarkably resistant to various apoptotic stimuli. Apaf-1-mediated apoptosis plays a role in the prevention of tumorigenesis. However, Apaf-1-independent cell death pathways are also indicated. In this review, we will summarize what has been learned about the role of Apaf-1 by biochemical and genetical approaches.     

ARK, the Apaf-1 related killer in Drosophila, requires diverse domains for its apoptotic activity

In mammals and Drosophila, apoptotic caspases are under positive control of the CED-4-like proteins Apaf-1 and ARK, respectively. In an EMS-mutagenesis screen, we isolated 33 ark mutants as recessive suppressors of hid-induced apoptosis. The ark mutants are loss-of-function alleles characterized by reduced developmental apoptosis. Using the phenotypic series of these alleles, we identified helical domain I in the nucleotide oligomerization domain as critical for ARK's apoptotic activity. Interestingly, the WD40 region may also have an unanticipated positive requirement for the apoptotic activity of ARK. Considering structural information, we discuss the roles of these domains for assembly and activity of the ARK apoptosome, and propose that the WD40 region is anti-apoptotic in the absence of apoptotic signals, and pro-apoptotic in the presence of such signals. Furthermore, a defined null allele reveals that ark is required for most, but not all apoptosis suggesting the existence of an ARK-independent apoptotic pathway.     

Apaf1-dependent programmed cell death is required for inner ear morphogenesis and growth

During inner ear development programmed cell death occurs in specific areas of the otic epithelium but the significance of it and the molecules involved have remained unclear. We undertook an analysis of mouse mutants in which genes encoding apoptosis-associated molecules have been inactivated. Disruption of the Apaf1 gene led to a dramatic decrease in apoptosis in the inner ear epithelium, severe morphogenetic defects and a significant size reduction of the membranous labyrinth, demonstrating that an Apaf1-dependent apoptotic pathway is necessary for normal inner ear development. This pathway most probably operates through the apoptosome complex because caspase 9 mutant mice suffered similar defects. Inactivation of the Bcl2-like (Bcl2l) gene led to an overall increase in the number of cells undergoing apoptosis but did not cause any major morphogenetic defects. In contrast, decreased apoptosis was observed in specific locations that suffered from developmental deficits, indicating that proapoptotic isoform(s) produced from Bcl2l might have roles in inner ear development. In Apaf1(-/-)/Bcl2l(-/-) double mutant embryos, no cell death could be detected in the otic epithelium, demonstrating that the cell death regulated by the anti-apoptotic Bcl2l isoform, Bcl-X(L) in the otic epithelium is Apaf1-dependent. Furthermore, the otic vesicle failed to close completely in all double mutant embryos analyzed. These results indicate important roles for both Apaf1 and Bcl2l in inner ear development.     

Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway

Here we identify a new gene, dark, which encodes a Drosophila homologue of mammalian Apaf-1 and Caenorhabditis elegans CED-4, cell-death proteins. Like Apaf-1, but in contrast to CED-4, Dark contains a carboxy-terminal WD-repeat domain necessary for interactions with the mitochondrial protein cytochrome c. Dark selectively associates with another protein involved in apoptosis, the fly apical caspase, Dredd. Dark-induced cell killing is suppressed by caspase-inhibitory peptides and by a dominant-negative mutant Dredd protein, and enhanced by removal of the WD domain. Loss-of-function mutations in dark attenuate programmed cell deaths during development, causing hyperplasia of the central nervous system, and other abnormalities including ectopic melanotic tumours and defective wings. Moreover, ectopic cell killing by the Drosophila cell-death activators, Reaper, Grim and Hid, is substantially suppressed in dark mutants. These findings establish dark as an important apoptosis effector in Drosophila and raise profound evolutionary considerations concerning the relationship between mitochondrial components and the apoptosis-promoting machinery.     

The Drosophila epsin 1 is required for ubiquitin-dependent synaptic growth and function but not for synaptic vesicle recycling

The ubiquitin-proteasome system plays an important role in synaptic development and function. However, many components of this system, and how they act to affect synapses, are still not well understood. In this study, we use the Drosophila neuromuscular junction to study the in vivo function of Liquid facets (Lqf), a homolog of mammalian epsin 1. Our data show that Lqf plays a novel role in synapse development and function. Contrary to prior models, Lqf is not required for clathrin-mediated endocytosis of synaptic vesicles. Lqf is required to maintain bouton size and shape and to sustain synapse growth by acting as a specific substrate of the deubiquitinating enzyme Fat facets. However, Lqf is not a substrate of the Highwire (Hiw) E3 ubiquitin ligase; neither is it required for synapse overgrowth in hiw mutants. Interestingly, Lqf converges on the Hiw pathway by negatively regulating transmitter release in the hiw mutant. These observations demonstrate that Lqf plays distinct roles in two ubiquitin pathways to regulate structural and functional plasticity of the synapse.     

Drosophila non-muscle alpha-actinin is localized in nurse cell actin bundles and ring canals, but is not required for fertility

The single copy Drosophila alpha-actinin gene is alternatively spliced to generate three different isoforms that are expressed in larval muscle, adult muscle and non-muscle cells, respectively. We have generated novel alpha-actinin alleles, which specifically remove the non-muscle isoform. Homozygous mutant flies are viable and fertile with no obvious defects. Using a monoclonal antibody that recognizes all three splice variants, we compared alpha-actinin distribution in wild type and mutant embryos and ovaries. We found that non-muscle alpha-actinin was present in young embryos and in the embryonic central nervous system. In ovaries, non-muscle alpha-actinin was localized in the nurse cell subcortical cytoskeleton, cytoplasmic actin cables and ring canals. In the mutant, alpha-actinin expression remained in muscle tissues, but also in a subpopulation of epithelial cells in both embryos and ovaries. This suggests that various populations of non-muscle cells regulate alpha-actinin expression in different ways. We also show that ectopically expressed adult muscle-specific alpha-actinin localizes to all F-actin containing structures in the nurse cells in the absence of endogenous non-muscle alpha-actinin.     

Ethylene is required for elicitin-induced oxidative burst but not for cell death induction in tobacco cell suspension cultures

The signal compound ethylene and its relationships with oxidative burst and cell death were analyzed in cultured tobacco cells treated with the proteinaceous elicitor quercinin. Quercinin belongs to the protein family of elicitins and was isolated from the soil-born oak pathogen Phytophthora quercina. It was shown to induce a dose-dependent oxidative burst in tobacco cell culture in concentrations from 0.05 to 0.5 nM, and subsequently, cell death. The characteristics of quercinin-induced cell death included both membrane damage and DNA fragmentation in tobacco cell culture.

At higher quercinin concentrations (2 nM), H₂O₂ formation and ethylene biosynthesis were inhibited. Ethylene at low concentrations proved to be necessary for induction and maintenance of H₂O₂ production in tobacco cells treated with quercinin. It was demonstrated that external addition of inhibitors of ethylene biosynthesis such as -amino-oxy-acetic acid (AOA) and CoCl₂ also decreased or even inhibited the quercinin-induced oxidative burst, but did not influence cell death induction. These results demonstrate evidence for a requirement of the plant hormone ethylene for the onset of the quercinin-induced oxidative burst.     

MAP kinase phosphorylation is dispensable for cell division,but required for cell growth in Drosophila

Proper activation of the Ras/MAPK pathway is broadly required during development, and in many cases, signal transduction downstream of the receptor is linear. Thus, different mechanisms exist to properly regulate the large number of specific developmental outputs that are required by the activation of this pathway. Previously, we have reported a regulated cytoplasmic sequestration of phosphorylated MAPK (pMAPK) in developing Drosophila compound eyes and wings “called MAPK Cytoplasmic Hold”. In the developing wing, we have shown that cytoplasmic hold promotes the differentiation of wing vein tissue, while pMAPK nuclear translocation regulates growth and division. We had also suggested that the Ras pathway signals for inducing cell growth and cell division split upstream of the nuclear translocation of MAPK itself. Here, we further refine the role of MAPK in Drosophila. We report evidence that suggests, for the first time, that the phosphorylation of MAPK is itself another step in the regulation of cell growth and division in both Drosophila wing and eye cells. We show that inhibition of MAPK phosphorylation, or pMAPK nuclear translocation, is sufficient to block cell growth, but not cell division. These data suggest that non-phosphorylated MAPK is sufficient to induce cell division, but not cell growth, once inside the nucleus of the cell.Key words: Drosophila, MAPK, growth, division, proliferation, phosphorylation     

The Drosophila parkin homologue is required for normal mitochondrial dynamics during spermiogenesis

Drosophila parkin, the ortholog of the human parkin gene, responsible for a familiar form of autosomal recessive juvenile parkinsonism, has been shown previously to be involved in Drosophila male fertility. Loss-of-function mutations in the parkin gene cause failure of spermatid individualization by affecting the proper progression of the actin-based investment cones that assemble in the nuclear region, but fail to translocate in synchrony down the cyst. In parkin mutants, the investment cones are scattered along the post-elongated spermatid bundles and fail to act properly in the process of sperm individualization. Using phase-contrast and electron microscopy analysis, we demonstrate that the parkin spermatids assemble a seemingly normal onion-stage nebenkern, but when the axoneme elongates only one mitochondrial derivative unfurls from the nebenkern. This unique mitochondrial derivative undergoes abnormal shaping and condensation during spermatid elongation. Our results indicate that parkin gene function is necessary for mitochondrial morphogenesis during earlier and later phases of spermiogenesis. The failure of cyst individualization may be due to the sensitivity of investment cone movement to the perturbation of mitochondrial morphology during spermatid elongation.     

Hid can induce, but is not required for autophagy in polyploid larval Drosophila tissues

The major cell death pathways are apoptosis and autophagy-type cell death in Drosophila. Overexpression of proapoptotic genes in developing imaginal tissues leads to the activation of caspases and apoptosis, but most of them show no effect on the polytenic cells of the fat body during the last larval stage. Surprisingly, overexpression of Hid induces caspase-independent autophagy in the fat body, as well as in most other larval tissues tested. Hid mutation results in inhibition of salivary gland cell death, but the disintegration of the larval midgut is not affected. Electron microscopy shows that autophagy is normally induced in fat body, midgut and salivary gland cells of homozygous mutant larvae, suggesting that Hid is not required for autophagy itself. Constitutive expression of the caspase inhibitor p35 produces identical phenotypes. Our results show that the large, post-mitotic larval cells do not react or activate autophagy in response to the same strong apoptotic stimuli that trigger apoptosis in small, mitotically active imaginal disc cells.