Tango7 regulates cortical activity of caspases during reaper-triggered changes in tissue elasticity.
ABSTRACT: Caspases perform critical functions in both living and dying cells; however, how caspases perform physiological functions without killing the cell remains unclear. Here we identify a novel physiological function of caspases at the cortex of Drosophila salivary glands. In living glands, activation of the initiator caspase dronc triggers cortical F-actin dismantling, enabling the glands to stretch as they accumulate secreted products in the lumen. We demonstrate that tango7, not the canonical Apaf-1-adaptor dark, regulates dronc activity at the cortex; in contrast, dark is required for cytoplasmic activity of dronc during salivary gland death. Therefore, tango7 and dark define distinct subcellular domains of caspase activity. Furthermore, tango7-dependent cortical dronc activity is initiated by a sublethal pulse of the inhibitor of apoptosis protein (IAP) antagonist reaper. Our results support a model in which biological outcomes of caspase activation are regulated by differential amplification of IAP antagonists, unique caspase adaptor proteins, and mutually exclusive subcellular domains of caspase activity.Caspases are known for their role in cell death, but they can also participate in other physiological functions without killing the cells. Here the authors show that unique caspase adaptor proteins can regulate caspase activity within mutually-exclusive and independently regulated subcellular domains.
Project description:Apoptosis is induced by caspases, which are members of the cysteine protease family. Caspases are synthesized as inactive zymogens and initiator caspases first gain activity by associating with an oligomeric complex of their adaptor proteins, such as the apoptosome. Activated initiator caspases subsequently cleave and activate effector caspases. Although such a proteolytic cascade would predict that a small number of active caspases could irreversibly amplify caspase activity and trigger apoptosis, many cells can maintain moderate levels of caspase activity to perform non-apoptotic roles in cellular differentiation, shape change and migration. Here we show that the Drosophila melanogaster apoptosome engages in a feedback inhibitory loop, which moderates its activation level in vivo. Specifically, the adaptor protein Apaf-1 lowers the level of its associated initiator caspase Dronc, without triggering apoptosis. Conversely, Dronc lowers Apaf-1 protein levels. This mutual suppression depends on the catalytic site of Dronc and a caspase cleavage site within Apaf-1. Moreover, the Drosophila inhibitor of apoptosis protein 1 (Diap1) is required for this process. We speculate that this feedback inhibition allows cells to regulate the degree of caspase activation for apoptotic and non-apoptotic purposes.
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: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:Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-? to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.
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:Most developmentally programmed cell death in metazoans is mediated by caspases. During Drosophila metamorphosis, obsolete tissues, including the midgut and salivary glands, are removed by programmed cell death . The initiator caspase Dronc and its activator Ark are required for the death of salivary glands, but not for midgut removal [2, 3]. In addition to caspases, complete removal of salivary glands requires autophagy . However, the contribution of autophagy to midgut cell death has not been explored. Examination of combined mutants of the main initiator and effector caspases revealed that the canonical apoptotic pathway is not required for midgut cell death. Further analyses revealed that the caspase Decay is responsible for most of the caspase activity in dying midguts, yet inhibition of this activity has no effect on midgut removal. By contrast, midgut degradation was severely delayed by inhibition of autophagy, and this occurred without a decrease in caspase activity. Surprisingly, the combined inhibition of caspases and autophagy did not result in an additional delay in midgut removal. Together, our results indicate that autophagy, not caspases, is essential for midgut programmed cell death, providing the first in vivo evidence of caspase-independent programmed cell death that requires autophagy despite the presence of high caspase activity.
Project description:Drosophila have been used to identify new components in apoptosis regulation. The Drosophila protein Dark forms an octameric apoptosome complex that induces the initiator caspase Dronc to trigger the caspase cell death pathway and, therefore, plays an important role in controlling apoptosis. Caspases and Dark are constantly expressed in cells, but their activity is blocked by DIAP1 E3 ligase-mediated ubiquitination and subsequent inactivation or proteasomal degradation. One of the regulatory mechanisms that stabilize proapoptotic factors is the removal of ubiquitin chains by deubiquitinases. In this study performed a modified genetic screen for deubiquitinases (dsRNA lines) to identify those involved in stabilizing proapoptotic components. Loss-of-function alleles of deubiquitinase DUSP31 were identified as suppressors of the Dronc overexpression phenotype. DUSP31 deficiency also suppresses apoptosis induced by the RHG protein, Grim. Genetic analysis revealed for the first time that DUSP31 deficiency sufficiently suppresses the Dark phenotype, indicating its involvement in the control of Dark/Dronc apoptosome function in invertebrate apoptosis.
Project description:While apoptosis regulation has been studied extensively in Drosophila melanogaster, similar studies in other insects, including disease vectors, lag far behind. In D. melanogaster, the inhibitor of apoptosis (IAP) protein DIAP1 is the major negative regulator of caspases, while IAP antagonists induce apoptosis, in part, by binding to DIAP1 and inhibiting its ability to regulate caspases. In this study, we characterized the roles of two IAP antagonists, Michelob_x (Mx) and IMP, in apoptosis in the yellow fever mosquito Aedes aegypti. Overexpression of Mx or IMP caused apoptosis in A. aegypti Aag2 cells, while silencing expression of mx or imp attenuated apoptosis. Addition of recombinant Mx or IMP, but not cytochrome c, to Aag2 cytosolic extract caused caspase activation. Consistent with this finding, AeIAP1 bound and inhibited both initiator and effector caspases from A. aegypti, and Mx and IMP competed with caspases for binding to AeIAP1. However, a difference was observed in the BIR domains responsible for Dronc binding by AeIAP1 versus DIAP1. These findings demonstrate that the mechanisms by which IAP antagonists regulate apoptosis are largely conserved between A. aegypti and D. melanogaster, although subtle differences exist.
Project description:Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro.
Project description:Although essential in mammals, in flies the importance of mitochondrial outer membrane permeabilization for apoptosis remains highly controversial. Herein, we demonstrate that Drosophila Omi (dOmi), a fly homologue of the serine protease Omi/HtrA2, is a developmentally regulated mitochondrial intermembrane space protein that undergoes processive cleavage, in situ, to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the proapoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. In summary, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis but also results in the release of a bona fide proapoptotic protein.