To examine the role of Ark isoforms in the induction of cell death, the effect of Ark overexpression in the Drosophila S2 cell line was analyzed. Transient expression of Dapaf-1S (the short isoform of Ark), like C. elegans CED-4, markedly reduces cell viability in S2 cells. However, neither Dapaf-1L (the long isoform of Ark) nor human Apaf-1 could induce cell death. dapaf-1S was transfected with the antiapoptotic genes C. elegans ced-9, baculovirus caspase inhibitor p35, bcl-xL, or diap2. The overexpression of CED-9 and p35 most effectively prevents Dapaf-1S-induced cell death, and Bcl-xL inhibits apoptosis moderately, suggesting that Dapaf-1S activates endogenous caspase(s) in Drosophila. Transfection of diap2 can block reaper-induced cell death in S2 cells, but not Dapaf-1S-induced cell death, indicating that the Dapaf-1S-induced cell death pathway is downstream or independent of DIAP2. Next, either dapaf-1L or dapaf-1S was transfected together with drICE, which encodes a typical DEVDase. The drICE-induced cell death is greatly enhanced by the overexpression of Dapaf-1L. A synergistic effect of Dapaf-1S and drICE coexpression was not observed. In summary, these results show that the each isoform of Ark has distinct cell death-inducing activity in Drosophila cells (Kanuka, 1999b).
Caspase activities were measured in S2 cells expressing Dapaf-1L or Dapaf-1S using two different substrates, which distinguish caspase-1-like proteases (Ac-YVAD-MCA) from caspase-3-like proteases (Ac-DEVD-MCA). High DEVD- but not YVAD-cleaving activities were observed in the cytoplasmic lysates from dapaf-1S-transfected S2 cells, which is consistent with the cell-killing activity of Dapaf-1S. Based on the observation that Dapaf-1L and Dapaf-1S activate distinct types of caspases, the caspase activities present in these lysates were measured using various caspase-specific substrates. This experiment also revealed that Dapaf-1L and Dapaf-1S activate different members of the caspase family (Kanuka, 1999b).
To determine the function of these novel YVADase-type caspases activated by Dapaf-1L, it was first hypothesized that these YVADase activities are required for Dapaf-1L-induced drICE activation. When dapaf-1L and drICE were cotransfected into S2 cells, Dapaf-1L dramatically enhanced the DEVDase activities, including that of drICE. In this case, the addition of a YVADase inhibitor (Ac-YVAD-CMK) into the culture medium effectively blocked the Dapaf-1L-induced DEVDase activation. Since Ac-YVAD-CMK could not inhibit Dapaf-1S function against drICE activation, it was concluded that the novel YVADase activities were required for the sequential activation of drICE and other DEVDases in S2 cells. The Drosophila YVADase activities already had been reported in Drosophila S2 cell lysates overexpressing the cytoplasmic region of Fas protein (Kondo, 1997), and several ESTs (expressed sequence tags) were found that encode Drosophila YVADase-like proteins; thus, candidates for Dapaf-1L-activated YVADases may exist and participate in cell death cascades in Drosophila (Kanuka, 1999b).
Do Dapaf-1L and Dapaf-1S interact with drICE, a Drosophila DEVDase-type caspase, by forming a physical complex, in a manner similar to that reported for CED-4 and proCED-3, or Apaf-1 and procaspase-9? drICE is essential for the apoptosis in Drosophila S2 cells induced by etoposide, cycloheximide, and the overexpression of rpr or C. elegans ced-4. CED-4 has been shown to activate drICE and bind to it directly (Kanuka, 1999a). Immunoprecipitation analysis reveals that overexpressed Dapaf-1S, but not Dapaf-1L, specifically binds to drICE. Dapaf-1L also interacts with Dronc, a long CARD-containing Drosophila caspase in 293 T cells. Interestingly, in this case, the processed form of DRONC was detected in Dapaf-1L-expressing lysates. These data suggest that the distinct caspase-binding affinities of Dapaf-1L and Dapaf-1S are responsible for the activation of different members of the caspase family. The presence of Apaf-1 isoform (Apaf-1S), which lacks WDRs produced by alternative splicing, may suggest the distinct activation mechanisms of caspases by Apaf-1, and Apaf-1S may be also present in mammals (Kanuka, 1999b).
A loss-of-function mutant in the Ark gene was obtained. Ark is located to the cytological position 53F on the right arm of chromosome II. One preexisting lethal P element insertion, l(2)k11502 (P1041) was found in the first noncoding exon of Ark. After removal of the background lethal mutation, this P element mutant may behave as a putative null allele of Ark. It is referred it as the dapaf-1K1 (dpfK1) allele. The expression of Ark mRNA could not be detected in dpfK1 homozygous embryos and larvae by in situ hybridization and RT-PCR. The homozygotes for the dpfK1 mutation were approximately 25% semilethal in pupal stage, and the outer morphology of larvae and adult flies appeared to be normal, except for adult dorsal bristles (Kanuka, 1999b).
A significant decrease in apoptotic cells stained by TUNEL was observed in developing embryos lacking maternal and zygotic Ark function. During early- and mid-germ band shortening (stages 11-12), cell death is normally seen in the dorsal region of the head and beneath the developing epithelium of the gnathal segments. Remarkably, the TUNEL-positive signals under the epithelium had disappeared in dpfK1/dpfK1 embryos. If Ark is required for caspase activation that leads to completion of the cell death pathway in the embryo, the embryonic caspase activity should be decreased in the dpfK1 mutant. To test this possibility, caspase activities were measured in the lysates of mixed embryos at 6-18 hr after egg laying (AEL) using various caspase-specific substrates. Large amounts of DEVDase and DQTDase activities were observed in developing embryos that contained many dead cells. In the dpfK1/dpfK1 embryos, these caspase activities were markedly decreased to half compared with their original levels. Whereas the increases of DEVDase activity in wild-type and dpfK1/dpfK1 embryos were observed in the initial stage of embryogenesis, at 3 hr AEL, DEVDase activity was continuously increased in wild type, but not in dpfK1/dpfK1 embryos. These results indicate that Ark is required for caspase activation in embryonic cell death (Kanuka, 1999b).
Next it was determined whether cyt c activates caspases in the Drosophila embryo in a Dapaf-1-dependent manner. Although the addition of cyt c and dATP into cytosols of S2 cells could not evoke any caspase activities, a prominent caspase activation (DEVDase) was observed in lysates from wild-type embryos in a cyt c and dATP-dependent manner. The cyt c/dATP-induced caspase activation is not entirely observed in the lysates prepared from dpfK1/dpfK1 homozygous embryos and is effectively blocked by an ATPase inhibitor (FSBA; 5'-p-fluorosulfonylbenzoyl adenosine), which is known to inhibit the function of Apaf-1/CED-4-like molecules. These data strongly suggest that Dapaf-1L/cyt c complex actually contributes to the caspase activation in the embryo (Kanuka, 1999b).
Genetic studies of cell death in Drosophila have led to the identification of three apoptotic activators: rpr, head involution defective (hid), and grim. The deletion of all three genes blocks apoptosis in the Drosophila embryo, and overexpression of any one of them is sufficient to kill cells that would normally live. The products of these genes appear to activate one or more caspases, because cell killing by rpr, hid, and grim is blocked by the caspase inhibitor p35. If Ark actually acts as a caspase activator, like Apaf-1/CED-4 in adult flies, the downstream pathways of one or more of these three gene products should depend on Ark function to activate caspases. dpfK1/dpfK1 flies were crossed to the GMR-rpr and GMR-hid strains to examine whether or not there are any genetic interactions. Compared with GMR-rpr adult flies in a wild-type background, GMR-rpr flies homozygous for dpfK1 show significantly improved eye morphology, but no obvious influence on hid-activated cell killing was observed in this case. drICE and Dredd appear to be activated downstream of Rpr in Drosophila S2 cells, and drICE is an essential caspase in rpr-induced cell death, consistent with observations that Dapaf-1L and Dapaf-1S activate drICE in S2 cells. These results suggest that Ark is involved in the rpr-induced cell death pathway, and the contribution of Ark against hid-induced cell death may not be as high as that of rpr (Kanuka, 1999b).
The genetic evidence that Ark interacts with rpr and the observation that Dapaf-1L contains WDRs strongly imply that cyt c might act as an initiator for Dapaf-1-mediated caspase activation. Overexpression of rpr and treatment with staurosporine or cycloheximide causes rapid caspase activation and increase of cyt c in digitonin-extracted lysates of Drosophila S2 cells. In S2 cells, immunoprecipitation experiments reveal that released cyt c by rpr directly interacts with Dapaf-1L, but Dapaf-1S, which lacks WDRs, binds to cyt c weakly. These observations suggest that one candidate for the internal signaling molecule between Rpr and Ark could be cyt c, and the target of cyt c would be Dapaf-1L, a structural homolog of mammalian Apaf-1 (Kanuka, 1999b).
To reveal the physiological roles of specific caspase-activating cascades initiated by Dapaf-1, morphological defects were sought in the nervous system of the dpfK1 homozygous larvae and adults. At the third-instar larval stage, the brain hemispheres of the dpfK1 mutant are larger than those of the wild-type and contain a markedly decreased number of apoptotic cells. Because the number of cells stained by the antibody against a neural marker (Prospero) actually increases in larval brain in dpfK1/dpfK1, Dapaf-1-dependent cell death might be required for the regulation of the number of neural cells in developing brain (Kanuka, 1999b).
The extra sensory organ on the notum is one of the typical structures of the Drosophila peripheral nervous system (PNS). Four large bristles (macrochaetes) are always observed on the wild-type scutellum. However, extra bristles often appear on the scutellum of dpfK1/dpfK1 flies (48%, n = 54). These ectopic bristles may be induced by defects in caspase activation in the developing scutellum because overexpression of a caspase inhibitor P35 using the GAL4/UAS system also induces a similar phenotype. Expression of P35 in the scutellum with sca-GAL4, dpp-GAL4, and ptc-GAL4 results in the formation of ectopic bristles similar to those observed in the dpfK1 mutants. These observations suggest that Dapaf-1-dependent caspase activation may play roles for control of the sensory organ numbers (Kanuka, 1999b).
The normal ommatidium in the Drosophila eye consists of photoreceptor cells, pigment cells, and cone cells. The exact numbers of these cells are strictly regulated by extracellular and intracellular mechanisms, including apoptotic cell death. In the eyes of the dpfK1 homozygous adults, abnormal ommatidia with one extra photoreceptor cell are frequently observed. The existence of these extra photoreceptor cells is not caused by the mislocation of R7/R8 cells. In addition, morphology of the pigment cell layer is disorganized compared with the regular pattern of the wild type, and the extra pigment cells are often observed in pupal retina of dpfK1 mutant. Since the numbers of pigment cells are regulated by apoptotic cell deaths, blockade of these cell deaths by caspase inhibitor P35 causes survival of extra pigment cells. These data suggest that the control of the number of photoreceptor cells and the pigment cells might depend on Ark function (Kanuka, 1999b).
These findings suggest that the two different caspase activation mechanisms seen in nematodes are both present in Drosophila. The function of Dapaf-1S, fulfilling one of these mechanisms, is to bind to drICE and to activate DEVDase (drICE), resembling the action of CED-4 in C. elegans by which proCED-3 is processed into its mature form (Chinnaiyan, 1997). Dapaf-1L, fulfilling the second of these mechanisms, acts like mammalian Apaf-1, by activating YVADase first, then activating DEVDase (Li, 1997). The mechanism underlying the Dapaf-1L-induced YVADase activation is very similar to that in mammals, which is based on the observation that the inhibition of caspase-1-like protease by the YVAD inhibitor blocks the subsequent activation of caspase-3 in apoptosis induced by Fas antigen, and the observation that Apaf-1 activates procaspase-9, resulting in the subsequent activation of caspase-3 (Li, 1997). These facts lead to an interesting hypothesis that CED-4 acquired WDRs at its C terminus through evolution, which enabled a more advanced regulation of programmed cell death. The WDR of Apaf-1 interacts with cyt c derived from mitochondria in the presence of apoptotic stimuli, and this binding is one of the triggers for Apaf-1-induced caspase activation (Hu, 1998). Cyt c directly interacts with Dapaf-1L and Ark is required for cyt c-dependent caspase activation in lysates from developing embryos. It is possible that cyt c displayed on the surface of mitochondria might activate Dapaf-1. Rpr-induced cyt c release is accelerated by the Rpr-binding protein Scythe in the Xenopus cell-free system. A scythe-like molecule might play a role in Rpr-induced cyt c release in Drosophila. Thus, this finding suggests that the mechanism is evolutionarily conserved by which WDR-containing Apaf-1-like molecules, such as Dapaf-1L, are required for cyt c-dependent caspase activation (Kanuka, 1999b).
Alternative splicing of Bcl-x, caspase-2, and CED-4 has been reported. In all cases, splicing isoforms show the opposite functions of their parental product. In addition to these findings, it was found that activation of distinct caspases can be regulated by alternative splicing of Dapaf-1. Dapaf-1L seems to be a latent form because it binds to cyt c. However, Dapaf-1S can work as an active form without cyt c and activate distinct caspase from Dapaf-1L/cyt c complex when it is expressed. Thus, at least two caspase activation mechanisms (one is cyt c dependent, another is by alternative splicing) are present in Drosophila. Since Apaf-1S is also found in the mouse, this type of regulatory mechanism may be also conserved through evolution (Kanuka, 1999b).
Programmed cell death via caspase activation is essential for normal development in various species. In C. elegans, CED-4 is thought to be the only molecule responsible for activating CED-3. However, the higher multicellular organisms have acquired more sophisticated caspase-processing procedures in response to various apoptotic demands. In Drosophila, three distinct molecules, Rpr, Hid, and Grim, activate endogenous caspases. The Drosophila caspase activator, Dapaf-1, is shown in this study to be involved in rpr-induced cell death cascades. At the same time, the in vivo function of Ark indicates the existence of complicated caspase activation systems in Drosophila. The Drosophila cell death inducers Rpr and Hid exhibit their functions through caspase activation, but no genetic interaction could be seen between Ark and hid-induced cell death in the compound eye, suggesting that Ark may not contribute so much to caspase activation mechanisms evoked by hid. Although Ark is involved in the execution of the cell death program induced by the overexpression of rpr, the GMR-rpr phenotype could not be completely rescued in a dpfK1 homozygous background. There are two possible interpretions of this result. One is that the allele is not null and therefore, some Rpr-dependent death can still occur. Another interpretation is that the Ark allele is entirely null, and Rpr functions through multiple pathways, one Dapaf-1-dependent and one independent. The latter scenario is preferred. Since no transcripts of Ark in Ark mutant embryo could be detected, and embryonic lysates from dpfK1 homozygous mutant do not respond to cyt c, the Ark allele seems to be null. Since Drosophila cyt c that may be released by rpr binds to Dapaf-1L, Ark could contribute to the cyt c-dependent caspase activation that occurs downstream of Rpr. Multiple caspase activation mechanisms are also suggested by the observation that lysates from the dpfK1 homozygous mutant have half the amounts of activated caspase. These data imply that approximately half of the caspase activity in the embryo is dependent on the Ark functions. However, the rest of the caspases are likely to be activated by another mechanism. Although knockout mice lacking Apaf-1, caspase-3, or caspase-9 exhibit several severe defects in early embryonic development, these phenotypes are observed only in certain tissues and organs, suggesting that Ark and mammalian Apaf-1 participate in caspase activation partially in vivo. The roles of these distinct machineries for caspase activation remain to be elucidated, but because in some cases the expression of both Hid and Rpr is required to kill specific cells or tissues in Drosophila, cumulative caspase activation is probably necessary to induce cell death in some situations (Kanuka, 1999b).
Although loss of the inhibitor of apoptosis (IAP) protein DIAP1 has been shown to result in caspase activation and spontaneous cell death in Drosophila cells and embryos, the point at which DIAP1 normally functions to inhibit caspase activation is unknown. Depletion of the DIAP1 protein in Drosophila S2 cells or the Sf-IAP protein in Spodoptera frugiperda Sf21 cells by RNA interference (RNAi) or cycloheximide treatment results in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc< or dark largely suppresses this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibits Ice processing following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induces DRONC processing, which occurs in two steps. The first step appears to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK, because full-length DRONC accumulates when dark is silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 results in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing is observed only in apoptotic cells. These results indicate that the initial step in DRONC processing occurs continuously via a DARK-dependent mechanism in Drosophila cells and that DIAP1 is required to prevent excess accumulation of this first form of processed DRONC, presumably through its ability to act as a ubiquitin-protein ligase (Muro, 2002).
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. This study demonstrates 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. These 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 (Huh, 2004; full text of article).
The mRNA distribution of Ark during embryonic development suggests that the expression of this gene is developmentally regulated. This prompted an examination of whether Ark expression can be induced by other death-inducing stimuli, such as DNA-damaging agents. Ionizing radiation induces apoptosis and expression of reaper in Drosophila embryos. Significantly, X-ray and UV irradiation also induces Ark expression. Embryos from wild-type or l(2)k11502/CyO flies were irradiated with X-ray (4000 rads) or UV (50 mJ/cm2 or 500 mJ/cm2). In control embryos, expression above basal levels is only detected in the head region. In contrast, ectopic Ark expression is detected within 45 min after radiation exposure in the ectoderm, mesoderm in the trunk, and endoderm. In embryos carrying the l(2)k11502 P element insertion, the lacZ reporter gene of the P element is also induced by X-ray irradiation in an essentially identical pattern. Interestingly, Ark expression is normally not detected in mesodermal and endodermal cells. This strongly suggests that the increased levels of Ark mRNA are the result of de novo transcription, and not reduced mRNA turnover. Furthermore, the pattern of ectopic Ark expression corresponds very well with the pattern of TUNEL labeling of embryos subjected to the same radiation treatment, suggesting that the observed induction is functionally relevant. It appears that embryos between stages 8 and 11 are most sensitive to X-ray irradiation, since there is no detectable induction before stage 8, and induction in embryos after stage 12 is much weaker (Zhou, 1999).
The expression of Ark is also induced in response to UV irradiation. Because UV rays do not penetrate far into the embryo, Ark expression is only induced on the exposed side. Again the ectopic Ark expression correlates very well with TUNEL labeling of identically treated embryos. However, there is one significant difference between UV and X-ray induction of Ark expression. While X-ray irradiation fails to induce Ark transcription prior to germ band elongation (stage 8), UV irradiation leads to increased Ark RNA as early as the blastoderm stage. This suggests the possibility that distinct pathways are used for the induction of Ark expression upon UV and X-ray irradiation (Zhou, 1999).
In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Silencing expression of either or both DC3 and DC4 has no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. Loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 fail to activate caspases in Drosophila cell lysates, but, remarkably, induce caspase activation in extracts from human cells. Overall, these results argue that DARK-mediated DRONC activation occurs independently of cytochrome c (Dorstyn, 2004).
The data clearly show that neither of the two cytochrome c species in Drosophila are required for caspase activation or apoptosis. Previous studies reported that a P-element insertion in the dc3 gene (bln1) results in loss of DRICE activity in testis (Arama, 2003). However, a recent report indicates that the bln1 P-element insertion also disrupts a number of other genes (Huh, 2004), thus questioning whether DC3 is responsible for DRICE activity. Additionally, DRICE activation during spermatogenesis appears to be independent of DARK and DRONC (Huh, 2004). If DC3 is required for caspase activation in Drosophila, a loss of function mutation in dc3 should lead to severe developmental defects and lethality. Furthermore, although a tissue-specific function has been suggested for DC3, it is unlikely that DC3 functions only during spermatogenesis, given its ubiquitous expression. Although disruption of the dc4 gene is embryonic lethal, DC4 cannot induce caspase activation and apoptosis in Drosophila cells (Dorstyn, 2004).
The question remains: how does DARK mediates DRONC activation? One possibility is that other factors can substitute for cytochrome c function during apoptosis. Alternatively, removal of DIAP1 from DRONC may be sufficient to allow an interaction with DARK and activation. Given that transcription plays a major role in developmental PCD in Drosophila, changes in the concentration of DIAP1, DRONC, and DARK proteins could facilitate caspase activation in the fly. These studies, combined with published work, demonstrate that Drosophila and mammalian cytochrome c proteins are functionally similar since they can both mediate respiration and Apaf-1 activation in mammalian cell lysates. Therefore, the requirement for cytochrome c in caspase activation in mammals is likely to have evolved late in evolution (Dorstyn, 2004).
The Drosophila Apaf-1 related killer (Dark) forms an apoptosome that activates Dronc, an apical procaspase in the intrinsic cell death pathway. To study this process, a large Dark complex was assembled in the presence of dATP. Remarkably, it was found that cytochrome c was not required for assembly and when added, cytochrome c did not bind to the Dark complex. A 3D structure of the Dark complex was determined at 18.8Å resolution using electron cryo-microscopy and single particle methods. In the structure, eight Dark subunits form a wheel-like particle and two of these rings associate face-to-face. In contrast, Apaf-1 forms a single ring that is comprised of seven subunits and each Apaf-1 binds a molecule of cytochrome c. Relevant crystal structures were used to model the Dark complex. This analysis shows that a single Dark ring and the Apaf-1 apoptosome share many key features. When taken together, the data suggest that a single ring in the Dark complex may represent the Drosophila apoptosome. Thus, this analysis provides a domain model of this complex and gives insights into its function (Yu, 2006).
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