Cytochrome c-d regulates developmental apoptosis in the Drosophila retina.
ABSTRACT: The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell-culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c-p and cyt c-d. Loss of cyt c-p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c-d mutants are viable but male sterile. Here, we show that cyt c-d regulates developmental apoptosis in the pupal eye. cyt c-d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinal tissues for the Drosophila homologues of apoptotic protease-activating factor 1 (Ark) and caspase 9 (Dronc). In addition, we found that cyt c-d--as with ark and dronc-regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, our results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells.
Project description:Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, cyt-c-d and cyt-c-p. We show that only cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis.
Project description:Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. Here we demonstrate that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.
Project description:Apoptosis operates to eliminate damaged or potentially dangerous cells. This loss is often compensated by extra proliferation of neighboring cells. Studies in Drosophila imaginal discs suggest that the signal for the additional growth emanates from the dying cells. In particular, it was suggested that the initiator caspase Dronc mediates compensatory proliferation (CP) through Dp53 in wing discs. However, the exact mechanism that governs this CP remained poorly understood. We have previously shown that elimination of misspecified cells due to reduced Dpp signaling is achieved by the interaction of the co-repressor NAB with the transcriptional repressor Brk, which in turn induces Jun N-terminal kinase-dependent apoptosis. Here, we performed a systematic in vivo loss- and gain-of-function analysis to study NAB-induced death and CP. Our findings indicate that the NAB primary signal activates JNK, which in turn transmits two independent signals. One triggers apoptosis through the pro-apoptotic proteins Reaper and Hid, which in turn promote activation of caspases by the apoptosome components Ark and Dronc. The other signal induces CP in a manner that is independent of the death signal, Dronc, or Dp53. Once induced, the apoptotic pathway further activates a CP response. Our data suggest that JNK is the candidate factor that differentiates between apoptosis that involves CP and apoptosis that does not.
Project description:Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome-wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc-mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII-mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death-inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non-phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.
Project description:Caspases play an essential role in the execution of programmed cell death in metazoans. Although 14 caspases are known in mammals, only a few have been described in other organisms. Here we describe the identification and characterization of a Drosophila caspase, DRONC, that contains an amino terminal caspase recruitment domain. Ectopic expression of DRONC in cultured cells resulted in apoptosis, which was inhibited by the caspase inhibitors p35 and MIHA. DRONC exhibited a substrate specificity similar to mammalian caspase-2. DRONC is ubiquitously expressed in Drosophila embryos during early stages of development. In late third instar larvae, dronc mRNA is dramatically up-regulated in salivary glands and midgut before histolysis of these tissues. Exposure of salivary glands and midgut isolated from second instar larvae to ecdysone resulted in a massive increase in dronc mRNA levels. These results suggest that DRONC is an effector of steroid-mediated apoptosis during insect metamorphosis.
Project description:Accidental cell death often leads to compensatory proliferation. In Drosophila imaginal discs, for example, gamma-irradiation induces extensive cell death, which is rapidly compensated by elevated proliferation. Excessive compensatory proliferation can be artificially induced by "undead cells" that are kept alive by inhibition of effector caspases in the presence of apoptotic stimuli. This suggests that compensatory proliferation is induced by dying cells as part of the apoptosis program. Here, we provide genetic evidence that the Drosophila initiator caspase DRONC governs both apoptosis execution and subsequent compensatory proliferation. We examined mutants of five Drosophila caspases and identified the initiator caspase DRONC and the effector caspase DRICE as crucial executioners of apoptosis. Artificial compensatory proliferation induced by coexpression of Reaper and p35 was completely suppressed in dronc mutants. Moreover, compensatory proliferation after gamma-irradiation was enhanced in drice mutants, in which DRONC is activated but the cells remain alive. These results show that the apoptotic pathway bifurcates at DRONC and that DRONC coordinates the execution of cell death and compensatory proliferation.
Project description:Ommatidial rotation is one of the most important events for correct patterning of the Drosophila eye. Although several signaling pathways are involved in this process, few genes have been shown to specifically affect it. One of them is nemo (nmo), which encodes a MAP-like protein kinase that regulates the rate of rotation throughout the entire process, and serves as a link between core planar cell polarity (PCP) factors and the E-cadherin-?-catenin complex. To determine more precisely the role of nmo in ommatidial rotation, live-imaging analyses in nmo mutant and wild-type early pupal eye discs were performed. We demonstrate that ommatidial rotation is not a continuous process, and that rotating and non-rotating interommatidial cells are very dynamic. Our in vivo analyses also show that nmo regulates the speed of rotation and is required in cone cells for correct ommatidial rotation, and that these cells as well as interommatidial cells are less dynamic in nmo mutants. Furthermore, microarray analyses of nmo and wild-type larval eye discs led us to identify new genes and signaling pathways related to nmo function during this process. One of them, miple, encodes the Drosophila ortholog of the midkine/pleiotrophin secreted cytokines that are involved in cell migration processes. miple is highly up-regulated in nmo mutant discs. Indeed, phenotypic analyses reveal that miple overexpression leads to ommatidial rotation defects. Genetic interaction assays suggest that miple is signaling through Ptp99A, the Drosophila ortholog of the vertebrate midkine/pleiotrophin PTP? receptor. Accordingly, we propose that one of the roles of Nmo during ommatial rotation is to repress miple expression, which may in turn affect the dynamics in E-cadherin-?-catenin complexes.
Project description:Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. Here we report the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome.
Project description:Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition ("undead" cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in "undead" cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs.
Project description:Apoptosis is an evolutionary conserved cell death mechanism, which requires activation of initiator and effector caspases. The Drosophila initiator caspase Dronc, the ortholog of mammalian Caspase-2 and Caspase-9, has an N-terminal CARD domain that recruits Dronc into the apoptosome for activation. In addition to its role in apoptosis, Dronc also has non-apoptotic functions such as compensatory proliferation. One mechanism to control the activation of Dronc is ubiquitylation. However, the mechanistic details of ubiquitylation of Dronc are less clear. For example, monomeric inactive Dronc is subject to non-degradative ubiquitylation in living cells, while ubiquitylation of active apoptosome-bound Dronc triggers its proteolytic degradation in apoptotic cells. Here, we examined the role of non-degradative ubiquitylation of Dronc in living cells in vivo, i.e. in the context of a multi-cellular organism. Our in vivo data suggest that in living cells Dronc is mono-ubiquitylated on Lys78 (K78) in its CARD domain. This ubiquitylation prevents activation of Dronc in the apoptosome and protects cells from apoptosis. Furthermore, K78 ubiquitylation plays an inhibitory role for non-apoptotic functions of Dronc. We provide evidence that not all of the non-apoptotic functions of Dronc require its catalytic activity. In conclusion, we demonstrate a mechanism whereby Dronc's apoptotic and non-apoptotic activities can be kept silenced in a non-degradative manner through a single ubiquitylation event in living cells.