InteractiveFly: GeneBrief

Death-associated APAF1-related killer : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Death-associated APAF1-related killer

Synonyms - Hac1, Apaf-1-related-killer

Cytological map position - 53E11--F2

Function - signaling, caspase activation

Keywords - apoptosis, programmed cell death

Symbol - Dark

FlyBase ID: FBgn0263864

Genetic map position -

Classification - CED-4 domain protein

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Kang, Y., Neuman, S. D. and Bashirullah, A. (2017). Tango7 regulates cortical activity of caspases during reaper-triggered changes in tissue elasticity. Nat Commun 8(1): 603. PubMed ID: 28928435
Summary:
Caspases perform critical functions in both living and dying cells; however, how caspases perform physiological functions without killing the cell remains unclear. This study identified 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. 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. The 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. In this study the authors show that unique caspase adaptor proteins can regulate caspase activity within mutually-exclusive and independently regulated subcellular domains.
Sinenko, S. A. (2017). Proapoptotic function of deubiquitinase DUSP31 in Drosophila. Oncotarget 8(41): 70452-70462. PubMed ID: 29050293
Summary:
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. A modified genetic screen for deubiquitinases (dsRNA lines) was performed 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.

BIOLOGICAL OVERVIEW

Apaf-1-related-killer (Ark) encodes a Drosophila homolog of mammalian Apaf-1 and Caenorhabditis elegans CED-4, cell-death proteins. Like Apaf-1, but in contrast to CED-4, Ark contains a carboxy-terminal WD-repeat domain necessary for interactions with the mitochondrial protein cytochrome c (see Drosophila Cytochrome c proximal and Cytochrome c distal). Ark selectively associates with another protein involved in apoptosis, the fly apical caspase, Dredd. Ark-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 Ark attenuates programmed cell death during development, causing hyperplasia of the central nervous system, and other abnormalities, including ectopic melanotic tumors and defective wings. Moreover, ectopic cell killing by the Drosophila cell-death activators, Reaper, Grim and Hid, is substantially suppressed in Ark mutants. These findings establish Ark as an important apoptosis effector in Drosophila and raise evolutionary considerations concerning the relationship between mitochondrial components and the apoptosis-promoting machinery (Rodriguez, 1999).

To determine whether Ark expression is sufficient to trigger cell death, conditional expression of the protein was directed in cultured fly cells. Epitope-tagged versions of Ark were transiently transfected into Drosophila Schneider L2 (SL2) cells. Robust killing was induced by expression of the apoptosis activator Grim. In parallel tests, moderate cell killing was associated with expression of full-length Ark, whereas a C-terminal truncation of the WD-repeat region, Ark(1-411), showed markedly enhanced killing activity. In both cases, cell killing was completely suppressed by the caspase inhibitor peptide Z-VAD and moderately attenuated by the Z-DEVD peptide. Thus Ark-mediated cell death requires caspase activity (Rodriguez, 1999).

Coexpression of an active-site C408A mutant of the fly apical caspase, Dredd [producing Dredd(C/A)], substantially attenuates cell killing triggered by Ark. In contrast, a comparable C211A mutation in the putative effector caspase drICE [producing drICE(C/A)] did not have similar effects even though it was prominently expressed. Therefore, Ark-mediated cell killing is generally not suppressed by the coexpression of mutant caspases, and the effect of Dredd(C/A) is specific. These data indicated that the Dredd mutant might exert a dominant-negative effect through a physical interaction with Ark. Whether Ark associates with Dredd was tested. A strong interaction between these proteins was detected when using either Ark(1-411) or the full-length protein. Similar tests with a comparable mutant form of drICE showed no evidence for an interaction between this caspase and Ark. These results do not address the question of whether a cofactor is necessary to regulate the Ark-Dredd interaction, since apoptotic SL2 cells may contain other proteins needed for their association. Nevertheless, Ark specifically interacts with the apical caspase Dredd but not with the effector caspase drICE. These data raise parallels to the binding observed between counterparts in the worm (CED-4 and CED-3) and in mammals (Apaf-1 and caspase-9) (Rodriguez, 1999).

Ark interacts with cytochrome c. Release of cytochrome c from the mitochondrial compartment and its association with Apaf-1 is a common feature of apoptosis in mammalian cells. In Drosophila, changes in cytochrome c also occur but the protein remains tethered to mitochondrial membranes, which are sufficient to trigger cytosolic caspase activation if isolated from apoptotic cells. Tests were performed to see whether Ark, like Apaf-1, might similarly associate with fly cytochrome c. Considerable levels of cytochrome c co-precipitate from Ark-expressing cells. To determine whether Ark's C-terminal WD domain might be important for this association, an identically tagged (3 x Myc) C-terminal truncation version of Ark(1-411) was tested. After transient transfection, substantial expression of Myc3-Ark(1-411) occurred but no co-immunoprecipitation with cytochrome c was observed. Therefore, a specific association of Ark with cytochrome c requires residues mapping between residues 412 and 1,440 in the Ark protein. These results indicate that Ark and Apaf-1 share homologous functions engaged by cytochrome c, and that the apoptosis-inducing activity of cytochrome c, through Apaf-1/Ark-like molecules, may be broadly conserved. Consistent with this idea, purified Drosophila cytochrome c was able to substitute for human cytochrome c in activating caspases via Apaf-1 in vitro. Future studies will determine the subcellular localization of Ark and the functional role of its interaction with cytochrome c (Rodriguez, 1999).

Ark loss-of-function mutations cause pleiotropic defects. To determine the function of Ark, mutants defective at this locus were isolated. Using a nearby P-element (P1041), a genetic screen was initiated to obtain loss-of-function Ark alleles. From ~700 transposition events, three were identified bearing a P-element insertion within the Ark locus. Genomic polymerase chain reaction (PCR) analysis reveals that these alleles [darkCD4 (CD4), darkCD8 (CD8) and darkDD1 (DD1)] contain an insertion in the first intron and retain the original P1041 insertion upstream of the Ark promoter. Consistent with the differential severity of these alleles, CD4 and CD8 map several hundred base pairs downstream of DD1 (Rodriguez, 1999).

To characterize the nature of these mutations further, RNA was studied from CD4, CD8, DD1 and wild-type animals for Ark expression by Northern blot analysis. In the wild type, a single Ark transcript, migrating at ~5 kilobases (kb), was detected at all stages examined. Increased levels of Ark occur at a time coincident with the histolysis of larval tissues (during the third-instar and early pupation stages), whereas lowest expression occurs in embryos and adults. In CD4 animals, Ark messenger RNA is not detected even after long exposures of the blot, so this mutation represents either a null allele or a strong loss-of-function hypomorphic allele. By this criterion, CD8 is a hypomorphic allele because a reduced amount of transcript was found. A slightly larger-sized transcript occurs in DD1 animals, but no significant changes in the levels of Ark expression are seen, consistent with the idea that this insertion represents a weak Ark allele. Most animals homozygous for CD4 and CD8 survive to the adult stage, albeit at lower frequencies relative to heterozygous siblings. Many of these CD4/CD4 and CD8/CD8 animals derived from heterozygous parents exhibit melanotic tumours and abnormalities affecting the wings and/or bristles. Although these flies also have impaired viability and fecundity, progeny derived from homozygous parents suffers from notably increased penetrance and expression of similar defects. Substantial numbers of these F1 homozygotes are either sterile and/or die prematurely within several days. CD4 and CD8 animals show the same classes of phenotypes, albeit at different frequencies. Correlating with this molecular analysis, CD4 and CD8 show the strongest penetrance for all classes of abnormalities whereas DD1 animals (which express Ark RNA at wild-type levels) show milder defects that are limited to extra bristles (no defective wings or melanotic tissue are seen in these mutants). Transheterozygous combinations of the stronger CD4 mutation with the CD8 allele fail to complement, showing that lesions at Ark are the cause of the phenotypes observed (Rodriguez, 1999).

Wing abnormalities in CD4 and CD8 Ark homozygotes fall into different classes. The most affected individuals exhibit severe wing defects similar to a 'gnarled' and or 'wrinkled' phenotype. Other afflicted flies had wing blisters or burnt 'notched' wings. At moderate frequencies, melanotic tumors are also observed protruding from the body of the animal next to the haltere. Melanotic tumors in Drosophila are thought to arise from abnormalities in hematopoietic blood cells during larval growth or autoimmune defects. Among Ark mutants with extra bristles, most have one extra anterior scutellar macrochaetae while a minority have two extra such macrochaete and/or an extra posterior scutellar macrochaetae (Rodriguez, 1999).

Ark mutants are defective in programmed cell death. Neuronal PCD is important in the patterning of a functionally mature nervous system. For instance, Drosophila Df(3L)H99 embryos, which lack cell death, suffer from a greatly enlarged central nervous system (CNS), and mouse strains lacking Apaf-1 exhibit cell-death defects leading to hyperplasia of the nervous system. To determine whether the CD4 insertion also causes defects in the CNS, the brain lobes and ventral ganglion were dissected from wandering third-instar CD4/CD4 larvae. Most Ark mutants had a significantly overgrown CNS as compared with wild type. Occasionally, hyperplasia of the CNS was observed in the brain lobes only, while in other animals the ventral nerve cord (for example, the ganglion) was abnormally extended (Rodriguez, 1999).

Since the hyperplasia of the larval CNS might result from defective cell death earlier in development, the patterns of apoptosis were studied in CD4 embryos using the in situ TUNEL technique. Compared with wild-type embryos, CD4/CD4 homozygotes exhibit markedly reduced levels of apoptosis. Although there is a general decrease in TUNEL labelling throughout these embryos, the reduction in number of apoptotic cells is most noticeable within the CNS and the epidermis (Rodriguez, 1999).

Reduced apoptosis is most easily detected within the ventral nerve cord of Ark mutant embryos, but patterns of cell death are diminished in other tissues as well. For instance, whereas a large number of ventral epidermal cells normally undergo PCD shortly after germ-band shortening, Ark mutant embryos have a greatly reduced number of TUNEL-positive cells during this stage (Rodriguez, 1999).

Mutations in Ark suppress reaper-, grim- and hid-induced apoptosis. Since Ark embryos are defective in apoptosis, attempts were made to determine whether reaper, grim or hid signaling might require Ark function. Directed expression of transgenes expressing reaper (P[GMR-reaper]), grim (P[GMR-grim]) and hid (P[GMR-hid]) in the eye disc triggers ectopic apoptosis and, depending on expression levels, the effect is seen in adults as phenotypes that can range in severity from complete ablation of eye tissue to milder 'rough' eye phenotypes. Alterations in the magnitude of cell-death signaling resulting from such transgenes are an effective means of identifying genetic components that regulate apoptosis. Therefore cell-death phenotypes caused by P[GMR-reaper]-97A, P[GMR-grim]-1 and P[GMR-hid]-1M were examined in backgrounds that were either wild-type, heterozygous or homozygous for darkCD4 (Rodriguez, 1999).

Overt suppression of cell killing was not evident in flies heterozygous for Ark. In contrast, substantial suppression was observed in homozygous individuals. The eyes of tester strains with two copies of P[GMR-grim]-1 were completely ablated, whereas homozygous Ark flies with two copies of P[GMR-grim]-1 retained substantial retinal tissue with many surviving ommatidia that were properly pigmented. Similarly, the P[GMR-hid]-1M phenotype was dramatically suppressed in Ark mutants and, compared with the wild-type tester strain, a large number of retinal cells that would have otherwise died persisted in the Ark background. Parallel tests with P[GMR-reaper]-97A uncovered a similar suppressive effect on reaper signalling. These results indicate that Ark functions as a pro-apoptotic effector of reaper-, grim-, and hid-induced cell killing (Rodriguez, 1999).

These data favor a shared evolutionary lineage for Ark, Apaf-1 and ced-4. Among these, the fly and mammalian genes share considerable homologies not found in ced-4. Therefore, Ark and Apaf-1 probably share the most recent common ancestry. Accordingly, the WD-repeat domain, which may represent the site of engagement by mitochondrial signals, could be a recent acquisition or, alternatively, this domain may have been lost from the worm protein. However, although these are the possibilities suggested at present, 'truer' orthologs of ced-4 in flies and mammals may yet be discovered. Functional studies also support a more recent lineage shared by Ark and Apaf-1. Ark, Apaf-1 and CED-4 each associate with apical caspases, but only the fly and mammalian counterparts are known to associate with cytochrome c, through a WD-repeat domain. Moreover, deletion of this region in Ark and Apaf-1 produces enhanced effects upon cell killing. Therefore, analogous to the scenario that has been proposed for Apaf-1 function, these results indicate that the association between cytochrome c and Ark may function to derepress an inhibitory effect imposed on Ark by the WD-repeat domain. One implication of these results is that an ancient mitochondrial circuit for propagating apoptotic signals (binding of cytochrome c to a WD-repeat domain) is preserved in insects and thus probably existed in a common ancestor of insects and mammals (Rodriguez, 1999).

The identification of Ark also raises important mechanistic questions. For instance, the C. elegans Bcl-2 homolog CED-9 can directly bind and repress the activity of CED-4, and analogous interactions can occur between Bcl2-family members and Apaf-1. However, it is not yet known whether Bcl-2 proteins represent an 'obligate' component of the apoptosome; future studies on Ark and its regulators may shed light on this issue. It will also be interesting to determine whether Ark can engage and/or function to activate other fly caspases (Rodriguez, 1999).

Some aspects of the Ark mutant phenotype suggest striking parallels to those reported for Apaf-1-deficient mice. In both cases, the CNS appears to be preferentially affected and a decrease in apoptosis leads to hyperplasia of this tissue. Although PCD is overtly compromised in darkCD4 embryos, clearly not all deaths are affected. Thus, certain apoptotic deaths may not use an Ark-dependent pathway; similar inferences have been drawn from study of the Apaf-1-deficient mouse embryos (where partial PCD suppression also occurs). Since it is formally possible that darkCD4 is not a null mutation, an absolute requirement for Ark activity in all PCDs cannot be excluded. However, the fact that no lethal complementation groups or stronger phenotypes have been mapped to Ark argues that other CED-4/Apaf-1-like molecules with partially redundant functions may be present in flies (Rodriguez, 1999).

Although the existence of an enlarged CNS can be easily reconciled with reduced apoptosis, the precise origins of other abnormalities are less obvious. Nevertheless, conspicuous parallels to phenotypes associated with mutations in caspases and other cell-death regulators in the fly are apparent. For example, larvae homozygous for loss-of-function mutations in the caspase gene DCP-1 also suffer from melanotic tumours, and dredd-deficient flies have wing and bristle abnormalities. Bristle defects are also associated with mutations at the Drosophila inhibitor of apoptosis-1 gene and wing defects are associated with mutation of a hid allele. Some of these defects (such as melanotic tumors) could arise from the incomplete histolysis of larval tissue during metamorphosis but, for the most part, many defects appear to be confined to tissues derived from the wing disc. It will be interesting to determine whether defects occur in tissues derived from other imaginal tissues (Rodriguez, 1999).

Other questions to be addressed relate to the role of Ark as an effector of signaling by the death activators reaper, grim and hid. Since these proteins can activate both pro-Dredd processing and the accumulation of dredd RNA in cells that are specified to die, it will be interesting to determine the precise role of Ark in these pathways. For example, it should be possible to test directly whether the apoptosis activators function through Ark to trigger Dredd processing. Ark functions as a pro-apoptotic effector of ectopic cell killing by reaper, grim and hid. However, the effect of these cell-death activators is not entirely abolished in Ark homozygotes and, therefore, Ark-independent apoptosis pathways, also triggered by reaper, grim and hid, are implicated. Further investigation into the function of Ark as it relates to signaling by reaper, grim and hid is now possible within the context of normal PCD and cell-type-specific fates (Rodriguez, 1999).


REGULATION

Characterization of Ark activity

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).

Regulation Of DRONC processing by Arc

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).

Response of Ark to irradiation

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).

The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells

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).

Three-dimensional structure of a double apoptosome formed by the Drosophila Apaf-1 related killer

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).

Structure of the Drosophila apoptosome at 6.9 &ARing; resolution

The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. This study shows that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. The 3D structure of a double ring was determined at approximately 6.9 Å resolution, and a model was created of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has 'lost' a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution (Yuan, 2011).

Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila

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. This study reports 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 (Pang, 2015).

This study presents the cryo-EM structures of the Dark apoptosome and the multimeric Dronc-Dark complex at overall resolutions of 4.0 and 4.1 Å, respectively. Notably, the EM density in the central region of the structures exhibits considerably higher resolutions, which allow assignment of specific side chains and atomic interactions. Because the overall domain organization of Dark is identical to that of Apaf-1, the structures reveal for the first time conserved atomic features of an apoptosome from a higher organism. The observed structural features of the Dark apoptosome, most of which are likely preserved in the Apaf-1 apoptosome, reveal the underpinnings of initiator caspase activation. Supporting this analysis, structure of the Dark protomer can be very well aligned with that of the activated Apaf-1 protomer from the Apaf-1 apoptosom (Pang, 2015).

This study presents the cryo-EM structures of the Dark apoptosome and the multimeric Dronc-Dark complex at overall resolutions of 4.0 and 4.1 Å, respectively. Notably, the EM density in the central region of the structures exhibits considerably higher resolutions, which allow assignment of specific side chains and atomic interactions. Because the overall domain organization of Dark is identical to that of Apaf-1, the structures reveal for the first time conserved atomic features of an apoptosome from a higher organism. The observed structural features of the Dark apoptosome, most of which are likely preserved in the Apaf-1 apoptosome, reveal the underpinnings of initiator caspase activation. Supporting this analysis, structure of the Dark protomer can be very well aligned with that of the activated Apaf-1 protomer from the Apaf-1 apoptosome (Pang, 2015).


DEVELOPMENTAL BIOLOGY

It is thought that the basic cell death program is constitutively expressed, and many cell death genes, including apaf-1, appear to be widely expressed. Ark mRNA is broadly distributed in preblastoderm embryos before the major onset of zygotic transcription, apparently due to maternal contribution. However, by stage 7, the highest levels of Ark mRNA were detected in the ventral neurogenic region, and around several invagination furrows. Interestingly, these are regions in which prominent apoptosis occurs during subsequent development. Later, the highest levels of Ark mRNA were found in the ectoderm and mesoderm of the procephalic region. The expression of Ark in the developing head overlaps significantly with that of the proapoptotic gene hid. Again, abundant apoptosis is subsequently observed in this region. The mRNA distribution of Ark is closely mirrored by the pattern of ß-gal expression from the P element insertion l(2)k11502, which carries a lacZ reporter gene. The l(2)k11502 P element transposon is inserted near the transcriptional start site of Ark, 81 bp upstream of the presumptive TATA box. Although a basal level of lacZ expression could be detected in essentially all cells, high levels of expression are again seen in the procephalic region. At later stages, both lacZ reporter expression and Ark mRNA are detected in a segmentally repeated pattern. Finally, ß-gal immunoreactivity is seen in macrophages starting from late stage 11 to the end of embryonic development. These observations indicate that Ark is abundantly expressed in many but not all dying cells and that its expression is transcriptionally regulated during development (Zhou, 2001).

Apaf-1-related-killer, a putative caspase activator, should be expressed in all cells that have the ability to undergo apoptosis. The distribution of Ark mRNA during Drosophila embryogenesis was determined by in situ hybridization. Ark is maternally expressed in the preblastoderm stage, and its expression continues throughout the embryo as it develops. In the larval stage, Ark is also expressed ubiquitously in eye imaginal discs and wing discs, where cell deaths are known to occur through tissue remodeling. A single 7 kb band was detected in Drosophila RNA by Northern hybridization. Although the expression of dapaf-1S, the alternative-splicing variant of Ark, was not detectable in larval tissues or the S2 cell line, a few dapaf-1S transcripts could be detected in developing embryos. These observations suggest that Dapaf-1L is the predominantly expressed isoform in Drosophila (Kanuka, 1999b).

Drosophila IAP1 (DIAP1/Thread) inhibits cell death to facilitate normal embryonic development. Using RNA interference it has been shown that down-regulation of DIAP1 is sufficient to induce cell death in Drosophila S2 cells. Although this cell death process is accompanied by elevated caspase activity, this activation is not essential for cell death. DIAP1 depletion-induced cell death is strongly suppressed by a reduction in the Drosophila caspase DRONC or Dark. RNA interference studies in Drosophila embryos also have demonstrated that the action of Dark is epistatic to that of DIAP1 in this cell death pathway. The cell death caused by down-regulation of DIAP1 is accelerated by overexpression of DRONC and Dark, and a caspase-inactive mutant form of DRONC can functionally substitute the wild-type DRONC in accelerating cell death. These results suggest the existence of a novel mechanism for cell death signaling in Drosophila that is mediated by DRONC and Dark (Igaki, 2002).

The observation that the pan-caspase inhibitor zD-dcb can not suppress the DIAP1 depletion-induced cell death suggests that DRONC may be able to induce cell death independent of its caspase activity. The observation that the caspase-inactive form of DRONC can functionally substitute the wild-type DRONC in accelerating DIAP1 depletion-induced cell death also supports the idea that the cell death can be mediated through non-caspase mechanisms. DRONC might have a protease-independent cell-killing activity that is activated by Dark. It is possible that DRONC is required simply as a bridging or scaffolding protein to bring other proteins together to transmit the cell death signaling. Although the possibility cannot be excluded that zD-dcb can not completely inhibit the caspase activity of DRONC, it is apparent that the mode of cell death caused by the down-regulation of DIAP1 is distinct from Reaper-induced cell death. The effects were assessed of dsRNAs synthesized from reaper, hid, grim, drob-1, and buffy/dborg-2 cDNAs on the diap1 dsRNA-induced cell death; none of them suppresses the cell death. Further in vivo analysis should help elucidate the role of the caspase-independent cell death pathway regulated by DIAP1 (Igaki, 2002).

Ark and autophagy

Apoptosis and autophagy are morphologically distinct forms of programmed cell death. While autophagy occurs during the development of diverse organisms and has been implicated in tumorigenesis, little is known about the molecular mechanisms that regulate this type of cell death. Steroid-activated programmed cell death of Drosophila salivary glands occurs by autophagy. Expression of p35 prevents DNA fragmentation and partially inhibits changes in the cytosol and plasma membranes of dying salivary glands, suggesting that caspases are involved in autophagy. The steroid-regulated BR-C, E74A and E93 genes are required for salivary gland cell death. BR-C and E74A mutant salivary glands exhibit vacuole and plasma membrane breakdown, but E93 mutant salivary glands fail to exhibit these changes, indicating that E93 regulates early autophagic events. Expression of E93 in embryos is sufficient to induce cell death with many characteristics of apoptosis, but requires the H99 genetic interval that contains the rpr, hid and grim proapoptotic genes to induce nuclear changes diagnostic of apoptosis. In contrast, E93 expression is sufficient to induce the removal of cells by phagocytes in the absence of the H99 genes. These studies indicate that apoptosis and autophagy utilize some common regulatory mechanisms (Lee, 2001).

Morphological studies of developing vertebrate embryos have resulted in the definition of three types of physiological cell death. The first type, widely known as apoptosis, is found in isolated dying cells that exhibit condensation of the nucleus and cytoplasm, followed by fragmentation and phagocytosis by cells that degrade their contents. The second type, known as autophagy, is observed when groups of associated cells or entire tissues are destroyed. These dying cells contain autophagic vacuoles in the cytoplasm that function in the degeneration of cell components. Autophagic cells destroy their own contents, while apoptotic cells depend on phagocytes to accomplish terminal degradation. The third type, known as non-lysosomal cell death, is least common, and is characterized by swelling of cavities with membrane borders followed by degeneration without lysosomal activity. While autophagy fulfills the definition of programmed cell death, occurs during development of diverse organisms, and has been implicated in tumorigenesis, little is known about the molecular genetic mechanisms underlying this type of programmed cell death. The morphological characteristics that distinguish apoptosis and autophagy suggest that these cell deaths are regulated by independent mechanisms. Comparison of biochemical changes during lymphocyte apoptosis and insect intersegmental muscle autophagy also indicate that these physiological cell deaths occur by distinct mechanisms. However, recent studies of steroid-triggered cell death of Drosophila larval salivary glands suggest that these cells utilize genes that are part of the conserved apoptosis pathway, even though these cells exhibit characteristics of autophagy. Specifically, the caspase Dronc and the homolog of ced4/Apaf-1 (Ark), two components of the core apoptotic machinery, increase in transcription immediately prior to salivary gland cell death. Thus, characterization of the mechanisms governing the regulation of autophagy will identify how these cell deaths differ from those that occur by apoptosis (Lee, 2001 and references therein).

Larval salivary glands of Drosophila undergo rapid programmed cell death in response to ecdysone. This cell destruction can be detected using markers that are typically associated with apoptosis including nuclear staining by Acridine Orange, TUNEL to detect DNA fragmentation, and exposure of phosphatidylserine on the outer leaflet of the plasma membrane. The changes in vacuolar structure that immediately precede the synchronous destruction of larval salivary gland cells are clearly more similar to autophagy than heterophagy (apoptosis). Large vacuoles increase in number in prepupal salivary glands, and rearrangement of the cytoskeleton and an increase in acid phosphatase activity are associated with these structures. Dynamic changes in salivary gland structure may reflect important biochemical changes during programmed cell death. Large Eosin-positive vacuoles appear to fragment, a distinct class of Eosin-negative vacuoles are formed that are closely associated with the plasma membrane, and vacuoles containing organelles are observed in the cytoplasm immediately preceding destruction of salivary glands. An increase in transcription of the caspase Dronc occurs at this stage, and inhibition of caspase activity blocks DNA fragmentation and partially prevents changes in vacuoles and plasma membranes, suggesting that these morphological changes may be attributed in part to the activity of enzymes typically associated with apoptosis (Lee, 2001 and references therein).

While morphological analyses of apoptosis and autophagy suggest different mechanisms for these forms of cell death, some genes that function in apoptosis also function during autophagy. Steroid-regulated genes impact distinct cellular changes in dying cells. Ecdysone impacts on the transcription of the cell death genes rpr, hid and diap2. This regulation is mediated by the ecdysone receptor, and a group of ecdysone-activated factors that include the BR-C, E74 and E93 genes. The function of the steroid-regulated BR-C, E74 and E93 genes in salivary gland cell death has been examined. E93 mutant salivary glands exhibit persistence of large vacuoles and plasma membranes, while these structures are destroyed in BR-C and E74A mutants. Two possible explanations exist for the differences in BR-C, E74A and E93 mutant salivary gland cell morphology. E93 mutant salivary glands could be arrested at an earlier stage of cell destruction that is similar to that of 12-hour wild-type cells, while BR-C and E74A mutants are arrested at a stage that is similar to 14.5-hour salivary gland cells. This model is supported by previous studies indicating that E93 function is required for proper regulation of BR-C and E74A transcription. Alternatively, E93 could function to regulate autophagy that results in destruction of vacuoles and plasma membranes, while BR-C and E74A do not function in the regulation of these cellular changes even though these genes are required for salivary gland cell death. The latter interpretation is intriguing when one considers that expression of E93 is sufficient to induce characteristics of apoptosis, and can induce the removal of cells even in the absence of the rpr, hid and grim cell death genes and nuclear apoptotic changes (Lee, 2001).

Several factors indicate that salivary gland autophagy is regulated by genes that also function in apoptosis. (1) Caspases function in salivary gland cell death. Expression of the baculovirus inhibitor of caspases, p35, inhibits destruction of this tissue. Furthermore, p35 expression prevents DNA fragmentation and partially inhibits morphological changes in vacuoles that are associated with autophagy, indicating that caspases are utilized during autophagy. Transcription of the Apaf1 homolog Ark and the caspase, dronc increases immediately preceding salivary gland cell death, and this transcription is blocked in E93 mutants, further supporting that caspases function in salivary gland autophagy. (2) Transcription of the proapoptotic genes, rpr and hid increases immediately prior to salivary gland autophagy, and the transcription of these genes is blocked by mutations in steroid-regulated genes that are involved in this process. Ectopic expression of E93, a critical determinant of salivary gland autophagy, is sufficient to induce cell death with numerous characteristics of apoptosis. In addition, the association of Croquemort (Crq) expression with E93-induced removal of apoptotic cells and autophagy of salivary glands provides yet another link between these morphologically distinct forms of programmed cell death. Combined, these factors indicate that autophagy and apoptosis utilize at least some similar mechanisms (Lee, 2001).

The location and type of cell appears to be an important determinant for the type of programmed cell death that occurs in the context of animal development. Autophagy occurs when groups of cells or entire tissues die, while apoptosis occurs in isolated dying cells. These studies are consistent with these criteria; salivary gland destruction occurs by autophagy and requires E93 function, while ectopic induction of cell death by expression of E93 during embryogenesis has the characteristics of apoptosis. It is hypothesized that this is due to similarities between autophagy and apoptosis. Alternatively, autophagy and apoptosis may be mechanistically distinct, and the ability to induce ectopic cell death by expression of E93 is simply due to activating a death program in different cell types. This explanation is supported by data demonstrating that p35 inhibits salivary gland cell death, but that p35 is not capable of inhibiting E93-induced cell death in embryos. However, several possibilities exist to explain the disparity of these data. (1) Ectopic expression of E93 during embryogenesis may lead to higher than normal levels of this protein. In side-by-side comparisons with the proapoptotic genes rpr and hid, expression of E93 results in greater cell death and lethality. Thus, the strong killing potential of E93 may be sufficient to overcome inhibition of cell death by p35. (2) Other cell death genes are not inhibited by expression of p35, including cell death that is induced by ectopic expression of the caspase Dronc. (3) Inhibition of vacuolar changes by expression of p35 during salivary gland cell death is incomplete, even though DNA fragmentation is inhibited in this tissue. Thus, caspases may play a role in salivary gland cell death, and both p35 experiments and the transcription of dronc during salivary gland autophagy support this conclusion. However, it is possible that other proteolytic mechanisms act in concert with caspases in the bulk degradation of salivary gland cells (Lee, 2001).

It is concluded that Autophagy and apoptosis are morphologically distinct, suggesting that the mechanisms underlying the regulation of these forms of programmed cell death are different. Nearly all of the large polytenized larval cells die during Drosophila metamorphosis. The synchrony and volume of these cell deaths suggests that engulfment of each dying cell may be limited by the number of available phagocytes. One obvious distinction between autophagy and apoptosis is the location of the lysosomal machinery that degrades the dying cell. Autophagic cells destroy their own contents, while apoptotic cells depend on phagocytes to accomplish terminal degradation. This distinction may account for much of the differences in the morphological appearance of these two forms of dying cells, but does not exclude the possibility that a single autophagic cell utilizes the mechanisms that exist in distinct apoptotic and phagocytic cells. The specific expression of Crq during autophagy supports this possibility, but genetic studies of crq function are needed to test this hypothesis. Future studies of autophagy, and its relationship to apoptosis, will illustrate the similarities and differences between these forms of programmed cell death (Lee, 2001).


EFFECTS OF MUTATION

The insertion of P element transposon l(2)k11502 appears to disrupt the expression of Ark since embryos homozygous for the insertion display no head-specific in situ signals above the basal levels derived from maternally contributed mRNA. In order to examine whether zygotic Ark function is required for normal cell death, embryos heterozygous or homozygous for the l(2)k11502 insertion were labeled with TUNEL to visualize apoptotic cells. In wild-type and heterozygous embryos, a large number of TUNEL-positive cells were consistently observed in the head region of stage 12 embryos. In contrast, significantly fewer TUNEL-positive cells were present in the head region of embryos homozygous for l(2)k11502. This phenotype is highly penetrant, but some variation among homozygous mutant embryos is seen, possibly due to differences in the amount or perdurance of maternal Ark. Therefore, although many cells can still die in homozygous mutant embryos, zygotic Ark product is required for the normal pattern of apoptosis (Zhou, 1999).

It is unlikely that elimination of zygotic Ark function in l(2)k11502 homozygous embryos represents the true null phenotype, since Ark mRNA is maternally contributed. To inactivate both zygotic and maternal Ark mRNA, the RNA interference assay (RNAi) was used. Double-stranded Ark RNA corresponding to either the first 723 amino acids, or amino acids 134-332, encompassing the most closely conserved region between APAF-1 and CED-4, was made and injected into syncytial wild-type embryos. The patterns of cell death in these embryos were compared to uninjected and lacZ RNAi control embryos by TUNEL and acridine orange staining. When injected into the posterior of the embryo, both Ark dsRNAs significantly reduce the number of apoptotic cells in the injected part of the embryo. Injecting the anterior of the embryo yields similar results, with the reduction of cell death restricted to the injected half of the embryo. Since RNAi produced a more severe reduction of apoptosis than elimination of zygotic Ark function, it appears that maternal Ark product contribute to the induction of cell death during normal embryonic development (Zhou, 1999).

At least some of the 'undead' cells in the l(2)k11502 mutant embryos appear to develop into extra neurons. When stained with the anti-Elav antibody, which recognizes all differentiated neurons, brains of l(2)k11502 homozygous mutant embryos contain more Elav-positive cells than wild type. This phenotype is very similar to that seen in null mutants of the proapoptotic gene head involution defective (hid). In addition, like loss of hid function, many Ark mutant embryos show defects in head involution. These findings suggest that Ark, like hid, is required to eliminate cells for proper morphogenesis of the Drosophila embryo (Zhou, 1999).

The ectopic expression of reaper, hid, grim, and several Drosophila caspases can lead to ectopic apoptosis. For example, when these genes are expressed in the developing retina under the control of an eye-specific promoter, GMR, cell death is induced in a dosage-dependent manner, and this produces various degrees of eye ablation. This provides a highly sensitized background to investigate genetic interactions with other components of the cell death pathway. For example, eliminating one copy of the antiapoptotic gene diap1 suppresses the eye ablation phenotypes of GMR-reaper and GMR-hid, and mutations in various components of the Ras-signaling pathway dominantly modify the GMR-hid phenotype. Potential interactions of Ark with several other proapoptotic genes were investigated by crossing the l(2)k11502 P insertion strain with transgenic fly strains carrying GMR-reaper, GMR-hid, GMR-grim, and GMR-dcp-1. Heterozygosity for Ark results in only a very mild suppression of the eye phenotypes of GMR-reaper/hid/grim. In contrast, l(2)k11502 robustly suppresses the eye phenotype of GMR-dcp-1. This indicates that endogenous Ark is rate limiting for cell killing by DCP-1, but not for apoptosis induced by reaper, hid, and grim under these circumstances. Similar effects were seen upon expression of a truncated form of dcp-1 lacking the prodomain (GMR-dcp-1-N). This suggests that Ark functions at a step either downstream of, or in parallel to, the removal of the dcp-1 prodomain (Zhou, 1999).

Genetic evidence suggests that reaper and grim induce apoptosis by activating dcp-1. For example, when both reaper and dcp-1 are expressed in the eye, they synergize to induce cell death. Expression of full-length dcp-1 results in a weak phenotype, presumably due to poor activation of the zymogen in these conditions. In contrast, coexpression of reaper and grim with dcp-1 leads to an eye ablation phenotype far more severe than the expression of any of these genes alone. Whether this synergy depends on Ark was investigated. Since the eye phenotype of GMR-dcp-1 is suppressed by heterozygosity for Ark, a significant modification of the GMR-reaper GMR-dcp-1 eye phenotype was expected. However, this was not observed. Upon introduction of l(2)k11502 into a GMR-reaper GMR-dcp-1 background, only a very mild change in eye morphology could be detected. It is concluded that the activation of dcp-1 by reaper is independent of Ark function (Zhou, 1999).

Strong genetic interactions were observed between Ark and the Drosophila caspase dcp-1, but only weak interactions with reaper, grim, and hid. In particular, the dosage of endogenous Ark is rate limiting for ectopic cell killing in the fly retina induced by dcp-1 but has little effect on killing by reaper, hid, and grim. However, reaper and grim interact genetically with dcp-1. Furthermore, the synergy between reaper and dcp-1 for the induction of cell death in the retina does not depend on the dosage of Ark. The lack of significant genetic interactions between Ark and reaper, hid, and grim indicates that these molecules act at some distance from one another in the cell death pathway, since genetic interactions are typically strongest among proximal components of a pathway. Therefore, it is proposed that Ark promotes caspase activation through a pathway that is distinct from the one used by reaper, hid, and grim. According to this model, the activation of caspases during apoptosis in Drosophila, and presumably also in mammals, is controlled by the simultaneous action of two separate pathways. In one case, Reaper, Hid, and Grim can activate caspases by inhibiting the antiapoptotic activity of diap1, one of the Drosophila IAPs. In the other case, Ark appears to promote caspase activation by a mechanism that is similar to APAF-1. If one activating pathway is strongly induced, for example by overexpression of Reaper, the activity of components in the other pathway may become less important. This would explain why a reduction of Ark dosage has little effect on reaper, grim, and hid-induced eye ablation. In addition, it is considered likely that the activation of dcp-1 by Ark involves one or more upstream caspases. A number of caspase-like sequences have been identified in Drosophila, but the precise order in which they may act within a caspase cascade remains to be determined. Therefore, it is possible that reaper, grim, hid, and Ark initially activate distinct upstream caspases. It is expected that the activity of Ark may also be controlled in a manner similar to that of APAF-1 and CED-4. Specifically, Ark may be regulated by CED-9/BCL-2-like proteins. The recent identification of BCL-2-like sequences in Drosophila should allow a critical test of this hypothesis. Significantly, Ark is also regulated at the transcriptional level. These results demonstrate that zygotic Ark expression is not at all uniform and becomes restricted to specific regions of the embryo. Expression of Ark in the cephalic region is similar to the expression of hid and correlates overall well with regions in which major morphogenetic cell death occurs subsequently. Furthermore, Ark transcription is induced by both X-ray and UV irradiation in a manner similar to what has been previously reported for reaper. Therefore, it appears that at least some proapoptotic stimuli induce cell death by simultaneously turning on two different pathways for caspase activation (Zhou, 1999).

To examine genetic interactions between Nedd2-like caspase (Dronc) and other apoptotic pathway genes, two UAS-dronc transgenic lines (#23 and #80) were chosen that result in relatively low lethality when crossed to GMR-GAL4 and a recombinant second chromosome was generated for each of these transgenes with GMR-GAL4. When GMR-GAL4 UAS-dronc#80 was crossed to wild type w1118 flies at 25°C, adult flies that exhibited slightly rough and mottled eyes were observed. A similar phenotype has been observed in previous studies and has been shown to be due to ablation of the pigment and photoreceptor cells. Similar results were observed for GMR-GAL4, UAS-dronc#23. This phenotype became more severe when expression of dronc via GMR-GAL4 was increased by raising the temperature to 29°C. Because this eye phenotype can be modified by increasing the expression of dronc, it provided a dosage-sensitive system for examining genetic interactions between dronc and other genes of the apoptosis pathway. To test this further, whether co-expression of the baculovirus caspase inhibitor P35 from the GMR enhancer was able to suppress the eye phenotype of GMR-dronc at 29°C was examined. Co-expression of GMR-p35 dramatically improves the eye ablation phenotype of GMR-dronc. Thus, in this system, Dronc is sensitive to P35 in the Drosophila eye (Quinn, 2000).

Whether the GMR-dronc eye phenotype is sensitive to halving the dosage of the various Drosophila apoptosis-regulatory genes was tested. To assess whether the GMR-dronc eye phenotype is sensitive to the dosage of the H99 genes (reaper, hid, and grim), GMR-dronc flies were crossed to a deficiency removing the H99 genes, Df(3L)H99, at 29°C. The H99 deficiency dominantly suppressed the GMR-dronc eye phenotype. Thus, the cell death-inducing activity of dronc is sensitive to the dosage of the H99 genes. Furthermore, halving the dosage of dronc using a deficiency modifies the ablated eye phenotype of GMR-hid and GMR-rpr, suggesting that dronc is downstream of hid and rpr. To determine whether there was a genetic interaction with dronc and dark, whether decreasing the dosage of dark modified the eye phenotype of GMR-dronc at 29°C was examined. Three different P-element alleles of dark (darkCD4, darkCD8, and darkl(2)k11502) show suppression of the GMR-dronc eye phenotype, indicating that Dark plays a role in promoting Dronc-induced cell death in the eye. Halving the dosage of diap1 using deficiencies or the specific allele thread5 dominantly enhances the GMR-dronc eye phenotype at 25°C . In addition, these diap1 mutations dominantly enhance the lethality associated with GMR-dronc, resulting in at least 10-fold lower numbers of GMR-dronc/+; Df(diap1)/+ adult flies than expected. In contrast, a deficiency removing diap2 showed no effect on the GMR-dronc phenotype, and no lethal effects were observed. Thus diap1, but not a deficiency removing diap2, shows a dosage-sensitive interaction with dronc. By contrast, ectopic expression of diap1 or diap2 from the GMR promoter shows suppression of the GMR-dronc ablated eye phenotype, although GMR-diap2 results in much weaker suppression than GMR-diap1. Thus, both Diap1 and Diap2 are capable of directly or indirectly blocking Dronc-mediated cell death (Quinn, 2000).

Mutations that remove DRONC are not available. Therefore, to examine a possible role for DRONC as a cell death effector a form of DRONC, DRONCC318S, was generated in which the active site cysteine was altered to serine. Expression of similar forms of other caspases results in a suppression of caspase activity and caspase-dependent cell death. This may occur as a result of interaction of DRONCC318S with the Drosophila homolog of the caspase-activating protein Apaf-1, thus preventing the Drosophila Apaf-1 from binding to wild type DRONC and promoting its activation in a manner similar to that described for mammalian Apaf-1 and caspase-9. Transgenic Drosophila were generated in which DRONCC318S was expressed under the control of a promoter, known as GMR, that drives transgene expression specifically in the developing fly eye. The eyes of these flies, known as GMR-DRONCC318S flies, appear similar to those of wild type flies. To assay the ability of DRONCC318S to block cell death, GMR-DRONCC318S flies were crossed to flies overexpressing rpr (GMR-rpr), hid (GMR-hid), or grim (GMR-grim) under the control of the same promoter. GMR-driven expression of rpr, hid, or grim results in a small eye phenotype due to activation of caspase-dependent cell death. However, flies coexpressing GMR-DRONCC318S and one of the cell death activators showed a dramatic suppression of the small eye phenotype, indicating that cell death had been suppressed. The possibility cannot be ruled out that this suppression is a result of DRONCC318S forming nonproductive interactions with the Drosophila Apaf-1 that block its ability to activate other long prodomain caspases such as DCP-2/DREDD. However, these possibilities notwithstanding, these results suggest that DRONC activity is important for bringing about rpr-, hid-, and grim-dependent cell death (Hawkins, 2000).

In mammals and Drosophila, apoptotic caspases are under positive control via the CED-4/Apaf-1/Dark adaptors and negative control via IAPs (inhibitor of apoptosis proteins). However, the in vivo genetic relationship between these opposing regulators is not known. In this study, it has been demonstrated that a dark mutation reverses catastrophic defects seen in Diap1 mutants and rescues cells specified for Diap1-regulated cell death in development and in response to genotoxic stress. dark function is required for hyperactivation of caspases that occurs in the absence of Diap1. Since the action of dark is epistatic to that of Diap1, these findings demonstrate that caspase-dependent cell death requires concurrent positive input through Apaf-1-like proteins together with disruption of IAP-caspase complexes (Rodriguez, 2002).

Mutations in dark cause a variety of developmental defects, including an enlarged larval CNS. In the absence of dark function, cells specified to die actually survive and also differentiate. The embryonic CNS midline glia is a well-studied cell lineage in the fly embryo and, though only this particular lineage was studied, it is likely that supernumerary cell types in other lineages also persist in dark mutants. Several important conclusions derive from these observations. (1) The persistence of extra cells excludes trivial explanations for reduced TUNEL labeling in dark mutants (e.g. cell death in the absence of TUNEL labeling or redirected cell fates that prevent the specification of PCD) and reinforces models that favor a fundamental role for dark in embryonic PCD. (2) As shown in C.elegans and in studies of Reaper interval Drosophila mutants, rescue from PCD can uncover a cryptic differentiation program. (3) Most important, the same midline glial cells that fail to die in dark mutants also require the induced action of reaper, grim and hid. There is a requirement for dark function in normal PCD when reaper, grim and/or hid are expressed at physiological levels. It is noteworthy that Diap1 and at least two of the cell death initiators (i.e. reaper and hid) are expressed in pools of progenitor cells, which give rise to the embryonic midline glia and also play an important role in specifying the death of these cells. Thus, although midline glia presumably receive death signals from reaper and hid leading to the disruption of Diap1-caspase interactions, these cells still fail to die and are even capable of differentiating normally in the absence of dark function. This inference supports conclusions from epistasis studies indicating that a disruption of caspase-Diap1 interactions alone is insufficient for apoptosis, and suggests that dark functions as a co-effector of cell death signaling along with reaper, grim and/or hid. It is worth noting that the mammalian ortholog of dark, Apaf-1, functions downstream from the point at which mitochondrial factors including cytochrome c are released from mitochondria. If the same holds true for the fly protein, it follows that embryonic midline glia (and perhaps other cell types) can survive and differentiate beyond this mitochondrial 'point of no return' (Rodriguez, 2002).

Additional evidence that PCD requires coordinated dark-dependent caspase activation in conjuction with the release of IAP-mediated caspase inhibition comes from tests of dark mutants under conditions of stress that elicit apoptotic responses by the embryonic cell death initiators. Like the Reaper interval mutants, dark mutants also exhibit profound failures in cell death in response to ionizing radiation. Interestingly, the dark mutation itself does not interfere with the radiation induction of reaper mRNA. Therefore the possibility that resistance to damage-induced apoptosis evident in irradiated dark mutants is caused by a disruption of upstream elements in the signaling pathway can be excluded. Instead, the results raise the possibility that dark, like reaper, may function as an important effector of Drosophila p53-mediated apoptosis. In addition, these findings reinforce an apoptotic requirement for dark (even when reaper is transcriptionally induced) and provide evidence for models where cell death signals do not converge on Diap1 alone. In future studies, it will be interesting to determine the range of damage signals that can engage dark activity, since this locus might also function in a broader range of damage signals beyond those provoked by radiation (Rodriguez, 2002).

In flies, Diap1 is thought to function as a rate-limiting brake on apoptotic cell death. If, however, Diap1 were the most proximal rate-limiting regulator of apoptosis, then the presence or absence of dark function should have no influence on Diap1-dependent effects. If, however, dark and Diap1 exert interdependent functions, then the opposite outcome is predicted. The results from these studies provide consistent and compelling evidence favoring the latter scenario since, wherever tested, the influence of Diap1 upon apoptotic signaling is heavily dependent upon intact dark function. In the darkCD4 mutant, for instance, the Diap15 mutation fails to enhance grim- and hid-induced cell killing in the eye. These experiments examined Diap1 in the heterozygous condition, but the same outcome is also true when Diap1 is tested in the homozygous state. Early in embryonic development, Diap1 homozygotes exhibit catastrophic phenotypes including morphological arrest soon after gastrulation, extensive TUNEL-positive nuclei and hyperactivation of caspases. Each of these defects is profoundly dependent upon dark function since each is dramatically reversed by the dark mutation. In darkCD4; Diap15 double mutant embryos, no evidence is found for widespread apoptosis and, in fact, the large majority of these double mutants proceed through stages of embryogenesis well beyond the point at which single Diap1 mutants arrest. Thus, loss of dark not only suppresses apoptotic deaths that would otherwise occur, but definitively reverses a profound morphogenetic arrest that ensues in the absence of Diap1. Consequently, homozygosity at dark not only prevents the onset of apoptotic markers (i.e. DNA fragmentation and/or caspase activation), but actually preserves cells that are otherwise fated to die when normal checks upon caspases are removed. Rescue from inappropriate apoptosis in this instance is consistent with what was observed in the midline glia but, rather than preserving cells fated for programmed death, rescue occurs even when signaling from IAP antagonists in the Reaper region is bypassed. A similar result, with similar implications, was obtained in the ovary, where ~90% of heteroallelic Diap16/8 mutants show abnormal degeneration of early staged egg chambers and associated sterility. Again, loss of dark function not only suppresses the pathological effect, but actually reverses these degenerative defects to the extent that half of the darkCD4; Diap16/8 double mutant females produce normal egg chambers and are fertile. Thus, in both the ovary and the embryo, loss of dark rescues functional cells from apoptosis caused by misregulated Diap1 (Rodriguez, 2002).

Reversal of Diap1-dependent defects by dark is also evident when caspase activity is directly assayed in early embryos. In contrast to the 'hyperactivated' caspase levels detected in Diap1 single mutant lysates, Diap15; darkCD4 lysates show levels of caspase activity that are suppressed >90% relative to Diap1 mutants. The unusually high caspase activity detected in Diap1 mutant embryos is thought to reflect the action of these proteolytic enzymes unimpeded by native inhibitors. However, studies here demonstrate that removing a negative regulator alone is not sufficient to achieve caspase hyperactivation, since dark is clearly required for this unrestrained activity. It is worth mentioning that the basal DEVDase activity in double mutants is still somewhat elevated compared with control embryos. This might indicate dark-independent caspase activity due to caspase auto-activation in the absence of Diap1 or, since it is possible that the darkCD4 allele used here is not a complete null, it could reflect hypomorphic dark function. Alternatively, since a role for Diap1 in cytokinesis has not been ruled out, it is possible that this residual caspase activity ensues because of secondary developmental defects leading to cell death. Nevertheless, these enzymatic data extend the analyses to the biochemical level in a manner fully consistent with phenotypic studies (Rodriguez, 2002).

Interestingly, since Apaf-1/Dark adaptor proteins act upon procaspases while IAPs preferentially inhibit processed caspases, these results further suggest that pre-existing levels of processed caspases in most cells are probably not high enough to achieve an apoptotic threshold. Instead, a positive cell death stimulus from dark is required for the unusually high levels of caspase activation seen in Diap1-/- embryos. It is also evident that Dark-dependent 'basal' levels of DEVDase are detected in these assays several hours before the onset of embryonic PCD, and therefore authentic effector caspase (e.g. DrICE, Dcp-1) activity occurs even in the absence of overt apoptotic signals. These data indicate that constitutive levels of active effector caspases, not derived from autoproteolysis but instead promoted by Apaf-1-like adaptor proteins, may exist in many and perhaps all viable cells (Rodriguez, 2002).

Taken together, a strict Diap-1-caspase 'liberation' model does not explain sufficiently the evidence described. The action of dark is epistatic to that of Diap1, demonstrating an order of gene action whereby Dark functions either downstream or parallel to Diap1. Therefore, simple derepression of caspases via an IAP inhibitory bridge does not account adequately for epistasis between Diap1 and dark. Put another way, these results support the notion that apoptotic cell death in vivo results from the simultaneous activation of caspases by dark and the derepression of caspases by reaper, grim and/or hid. Accordingly, the findings are inconsistent with models that presume that Diap1 is the sole effector of reaper, grim and hid and that cells are 'pre-loaded' with sufficient levels of IAP-inhibited processed caspases to achieve cell killing. Instead, a 'gas and brake' model is favored whereby positive input from Apaf-1/Dark adaptors, together with removal of IAP inhibition, drives caspase activation to levels that exceed a threshold necessary for apoptosis (Rodriguez, 2002).

Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).

The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).

Border cell migration in the Drosophila ovary is a relatively simple and genetically tractable model for studying the conversion of epithelial cells to migratory cells. Like many cell migrations, border cell migration is inhibited by a dominant-negative form of the GTPase Rac. To identify new genes that function in Rac-dependent cell motility, a screen was performed for genes that when overexpressed suppressed the migration defect caused by dominant-negative Rac. Overexpression of the Drosophila inhibitor of apoptosis 1 (DIAP1), which is encoded by the thread (th) gene, suppresses the migration defect. Moreover, loss-of-function mutations in th causes migration defects but, surprisingly, did not cause apoptosis. Mutations affecting the Dark protein, an activator of the upstream caspase Dronc, also rescues RacN17 migration defects. These results indicate an apoptosis-independent role for DIAP1-mediated Dronc inhibition in Rac-mediated cell motility (Geisbrecht, 2004).

The observation that two different dark mutant alleles cause mild border cell migration defects suggests that Dronc, which is thought to be constitutively active at a low level in most cells, contributes to normal migration. In fact, caspases have been shown to function in cell proliferation and differentiation in a variety of cell types, in addition to their better known role in promoting apoptosis. In some cases, caspase activity is required for terminal differentiation events that resemble incomplete apoptosis. For example, terminal differentiation of Drosophila sperm requires removal of much of the cytoplasm and requires caspase activity. Similarly, differentiation of mammalian lens cells and erythrocytes requires caspase activity. Other differentiation events, such as those of macrophages and skeletal muscle, do not overtly resemble apoptosis and yet require caspase activity. There must be some mechanism in such cells, and in border cells, to restrict the caspase activity to selected substrates so that apoptosis does not occur (Geisbrecht, 2004).

The two Drosophila cytochrome C proteins can function in both respiration and caspase activation: Analyses of ark and dronc mutants demonstrate that both genes are required for spermatid individualization and that their phenotypes resemble cyt-c-d mutant spermatids and expression of the caspase inhibitor p35 in the testes

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, Cytochrome c proximal and Cytochrome c distal. 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 (Arama, 2006).

In order to identify genes required for caspase activation during spermatid differentiation in Drosophila, attempts were made to identify mutants that lacked activated caspase-3 staining, as detected using CM1 antibody, which detects the active form of the effector casepase drICE. For this purpose, an existing collection was screened of more than 1000 male-sterile mutant lines defective in spermatid individualization that were previously identified among a collection of about 6000 viable mutants. Dissected testes from each line were stained with CM1: 33 lines were identified that were CM1-negative. However, the vast majority of male-sterile lines remained CM1-positive, even though many displayed severe defects in spermatid individualization. Therefore, caspase activation at the onset of spermatid individualization appears to be independent of other aspects of sperm differentiation, such as the assembly of the individualization complex or its movement. One of the mutants, line Z2-1091, failed to complement the sterility of bln1, a P-element insertion in cyt-c-d, and was CM1-negative as a homozygote, in trans to a small deletion removing the cyt-c-d locus [Df(2L)Exel6039], or in trans to the cyt-c-dbln1 allele. In contrast, Z2-1091 complemented the lethality of K13905, a P-element insertion in cyt-c-p, and K13905 complemented the sterility of Z2-1091. Genomic sequence analyses of the transcription units of both cyt-c-d and cyt-c-p in Z2-1091 flies revealed a point mutation of TGG to TGA at codon 62 in cyt-c-d, causing a change of Trp62 into a stop codon that results in a truncation of almost half of the protein. Henceforth this allele will be referred to as cyt-c-dZ2-1091. Given the molecular nature of cyt-c-dZ2-1091, it is very unlikely that this allele affects the function of genes adjacent to cyt-c-d (Arama, 2006).

Effector caspases, such as drICE, can display DEVD cleaving activity. Therefore, it was asked whether wild-type adult testes also contain DEVDase activity, and whether this activity is affected in cyt-c-d mutant testes. Lysates of wild-type testes indeed display detectable levels of DEVDase activity, which were significantly reduced upon treatment with the potent DEVDase inhibitor Z-VAD.fmk. Importantly, this activity was highly reduced in cyt-c-dZ2-1091 mutant testes. These results provide independent evidence for effector caspase activity in wild-type sperm, and they support a role of cytochrome C-d in caspase activation in this system (Arama, 2006).

In mammals, mitochondria are important for the regulation of apoptosis, and it has been shown that they can release several proapoptotic proteins into the cytosol in response to apoptotic stimuli. The best-studied case is the release of cytochrome C, an essential component of the respiratory chain. Cytosolic cytochrome C can bind to and activate Apaf-1, which in turn leads to the activation of caspase-9. However, no comparable role of mitochondrial factors for caspase activation has yet been established in invertebrates. The elimination of cytoplasm during terminal differentiation of spermatids in Drosophila involves an apoptosis-like process that requires caspase activity; a P-element insertion (bln1) in one of the two Drosophila cytochrome c genes, cyt-c-d, has been shown to be associated with male-sterility and loss of effector caspase activation during spermatid individualization. This study demonstrates that the defects in caspase activation and spermatid individualization of bln1 mutant males can be rescued by transgenic expression of the ORF of cyt-c-d. Furthermore, from screening more than a thousand male-sterile lines with defects in sperm individualization for defects in active-caspase (CM1) staining, a nonsense point mutation was identified in cyt-c-d, that recapitulates all the phenotypes observed for bln1. Taken together, these results unequivocally demonstrate that cyt-c-d is necessary for effector caspase activation and sperm terminal differentiation in Drosophila (Arama, 2006).

Two decades ago, the mouse cytochrome c gene was used as a probe for screening a Drosophila genomic library and a fragment was isolated that carried two distinct cytochrome c genes. Northern blot analyses indicated high levels of cyt-c-p expression, while cyt-c-d was reported to be expressed at much lower levels in all stages of development. However, neither the exon/intron organization nor the boundaries of the 5' and 3' UTRs of these genes were determined at the time. As a result, the original Northern analyses were performed with a probe corresponding to the untranscribed genomic region between the two cytochrome c genes that was not suitable to properly assess the size and distribution of cytochrome c transcripts. Unfortunately, this has caused considerable confusion in the field from the start, as even the original report noted that the size of the observed cyt-c-d transcript differed more than two-fold from the predicted size. More recently, relying on the incorrect assumption that cyt-c-d is ubiquitously expressed in the fly, it has been suggested that a loss-of-function mutation in cyt-c-d should lead to severe developmental defects and lethality rather than merely male sterility. However, using a specific cyt-c-d 3' UTR probe reveals a transcript of the predicted size that is absent in cyt-c-dbln1 mutants. Furthermore, the RT-PCR and immunofluorescence analyses presented in this study indicate that cyt-c-d is mainly expressed in the male germ line and is completely absent during embryonic and larval development, while cyt-c-p is expressed in the soma during all stages of development. In light of these findings, it is not surprising that loss-of-function mutations in cyt-c-d cause male sterility, whereas cyt-c-p mutations lead to embryonic lethality. RT-PCR results suggest that cyt-c-p is also expressed in the testis, although to a much lower extent than cyt-c-d. This expression is attributed primarily to the somatic cells of the testis, since no cytochrome C protein is detected in cyt-c-dbln1 elongating spermatids, while cyt-c-p RNA is expressed in cyt-c-dbln1 mutant flies. However, the very low cyt-c-d expression detected in the soma of adult females leaves room for the possibility that cyt-c-d might function in caspase activation in some somatic cells as well (Arama, 2006).

In mammalian cells, release of cytochrome C into the cytosol in response to proapoptotic stimuli can be readily demonstrated. However, previous attempts to detect a similar phenomenon in Drosophila have been unsuccessful. In contrast, apoptotic stimuli can lead to increased cytochrome C immuno-reactivity. A possible limitation is that all these studies were conducted using mammalian antibodies with questionable specificity and sensitivity, and only in a small number of cell types and paradigms. Using an antibody that was raised against Drosophila cytochrome C-d, an increase in a 'grainy signal' was detected upon the onset of individualization, with the highest staining observed in the vicinity of the individualization comple (IC). Since it is highly unlikely that additional cytochrome C-d is being transcribed and imported to the mitochondria at this late stage, the explanation is favored that a conformational change or an exposure of a hidden epitope causes the increase in the intensity of the signal. The activation of Dronc, the Drosophila caspase-9 orthologue, also occurs in association with the IC and depends on the presence of the Drosophila Apaf-1 orthologue, Ark. Moreover, the proapoptotic Hid protein is localized in a similar fashion. What are these structures then, which accumulate apoptotic factors in the vicinity of the IC? One plausible suggestion from the literature is that these structures correspond to 'mitochondrial whorls', which result from the extrusion of material from the minor mitochondrial derivative and constitute the leading component of the IC. These 'whorls' can be labeled using a testes-specific mitochondrial-expressed GFP line. Using this GFP marker, it was found that cytochrome C-d is indeed closely associated with mitochondrial whorls. Therefore, it is possible that an active apoptosome forms in the vicinity of the IC in response to dramatic changes in the mitochondrial architecture that occur at this stage of spermatid differentiation. Similarly, studying the response of Drosophila flight muscle cells to oxygen stress, have recently reported that the cristae within individual mitochondria become locally rearranged in a pattern that they termed a 'swirl'. This process was associated with widespread apoptotic cell death in the flight muscle, which was correlated with a conformational change of cytochrome C manifested by the display of an otherwise hidden epitope. Collectively, these observations suggest that apoptosome-like complexes composed of cytochrome C-d, Ark, and Dronc might be associated with unique mitochondrial swirl-like structures. Consistent with this idea, it was found that the long isoform of Ark that contains the WD40 repeats, the target for cytochrome C binding to mammalian Apaf-1, is the major form detectably expressed in testes (Arama, 2006).

The fact that cytochrome C-d immunoreactivity increases in the vicinity of the IC suggests that the extensive mitochondrial organizations preceding individualization may be partially required for caspase activation. Consistent with this idea, several mutants, such as plnZ2-0516, which display defects in Nebenkern differentiation and caspase activation. However, not all mitochondrial differentiation events are required for caspase activation. For example, CM1 staining is seen in fuzzy onions, a mutant defective in the mitochondrial fusion event that generates the Nebenkern. In contrast, analysis of the pln mutant indicates that proper elongation of the Nebenkern is essential for caspase activation. Therefore, characterization of other mitochondrial mutants may shed light on the connection between mitochondrial organization and caspase activation during sperm differentiation (Arama, 2006).

What are the mechanisms by which cytochrome C-d activates caspases during late spermatogenesis? In vertebrate cells, following its release into the cytosol, cytochrome C binds to the WD40 domain of the adaptor molecule Apaf-1, which in turn multimerizes and recruits the initiator caspase, caspase-9 via interaction of their CARD domains. This complex, known as the apoptosome, further cleaves and activates effector caspases like caspase-3. Although this model has become the prevailing dogma in the field, the phenotype of mice mutant for a Cyt c with drastically reduced apoptogenic function ('KA allele') suggests that the mechanisms for caspase activation may be more complex than what was previously thought. In particular, this study suggests that cytochrome C-independent mechanisms for the activation of Apaf-1 and caspase-9 exist, as well as cytochrome C-dependent but Apaf-1-independent mechanisms for apoptosis. These analyses of ark (Apaf-1) and dronc (caspase-9) loss-of-function mutants demonstrate that both genes are required for spermatid individualization, and that their phenotypes, in particular their failure to properly remove the spermatid cytoplasm into the WB, resemble cyt-c-d mutant spermatids and expression of the caspase inhibitor p35 in the testes. However, some caspase-3-like activity could still be detected in these mutant testes. This may suggest that either the ark and dronc alleles are not null, or that cytochrome C-d also functions in an apoptosome-independent pathway to promote caspase-3 activation. Therefore, the regulation of caspase activation and apoptosis may be more similar between insects and mammals than has been previously appreciated. Further genetic analysis of this pathway in Drosophila may provide general insights into diverse mechanisms of apoptosis activation (Arama, 2006).

Previous observations raised the possibility that the two distinct cytochrome c genes may have evolved to serve distinct functions in respiration and caspase regulation. In order to address this hypothesis, it was asked whether expression of one protein might rescue mutations in the other cytochrome c gene. Surprisingly, it was found that transgenic expression of the cyt-c-p ORF in germ cells rescues caspase activation, spermatid individualization, and sterility of cyt-c-d-/- flies. Therefore, the ability to activate caspases is not restricted to the cytochrome C-d protein, and it is possible that cytochrome C-p functions in apoptosis in at least some somatic cells (Arama, 2006).

Although cyt-c-d is almost exclusively expressed in the male germ cells, ectopic expression of this protein in the soma can rescue the respiration defect and lethality of cyt-c-p-/- mutant flies, demonstrating that cytochrome C-d can function in energy metabolism. This raises the question whether the lack of caspase activation could be due to reduced ATP-levels. Although this is a formal possibility, this explanation is considered very unlikely since mutant spermatids complete many other energy-intensive cellular processes. These include the extensive transformation from round spermatids to 1.8 mm long elongated spermatids, a process that involves extensive remodeling and movement of actin filaments, generation of the axonemal tail, mitochondrial reorganization, plasma/axonemal membranes reorganization, and nuclear condensation and elongation. Since all of these processes can occur in the absence of cytochrome C-d, there is no overt shortage of ATP in cyt-c-d mutants. It is therefore considered very unlikely that ATP has become limiting in these mutant cells. Since earlier stage spermatids express cytochrome C-p, sufficient ATP seems to persist to late developmental stages. In mammalian cells, cellular ATP concentration is sufficiently high (around 2 mM) to keep cultured cell alive for several days upon ATP synthase inhibition. Furthermore, cells in which cytochrome c expression is decreased by RNAi still undergo apoptosis in response to various stimuli. Likewise, it appears that cytochrome C is not essential for the function of mature murine sperm, since mice deficient for the testis specific form of cytochrome C, Cyt cT, are fertile. Taken together, all these observations argue strongly against the possibility that ATP levels in cyt-c-d-/- mutant spermatids would be insufficient for caspase activation (Arama, 2006).

In conclusion, the results presented in this study definitively demonstrate that cytochrome C-d is essential for caspase activation and spermatid individualization. Both cytochrome C proteins of Drosophila are, at least to some extent, functionally interchangeable. The results also indicate that cytochrome C can promote caspase activation in the absence of a functional apoptosome. Given the powerful genetic techniques available, late spermatogenesis of Drosophila promises to be a powerful system to identify novel pathways for mitochondrial regulation of caspase activation (Arama, 2006).

The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death

The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, it has been demonstrated that ARK is essential for most programmed cell death (PCD) during Drosophila development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal is severely delayed in ark mutants. However, PCD occurs normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in Drosophila (Mills, 2006).

ark alleles were obtained in a screen conducted using mitotic recombination for mutations that appear in an increased relative representation of mutant over wild-type (WT) tissue. In these mutants, the mutant clones were larger than the corresponding WT twin spots. The screen of the right arm of chromosome 2 identified mutations in the hippo locus. Four alleles of ark were also obtained from the same screen; these alleles were all lethal at the pupal stage of development as homozygotes or in trans to each other. Sequencing revealed point mutations or deletions in the coding sequence of the ark gene in each of the mutant chromosomes. ark1 had a G to A mutation, resulting in the truncation of the protein after residue 206; ark2 had a C to T mutation, causing protein truncation after residue 660, and ark3 had a deletion after residue 592, generating a frameshift mutation, whereas ark4 possessed a T to G mutation, causing protein truncation after residue 1,357. The mutation in ark1 is predicted to affect both of the reported alternately spliced transcripts of the ark gene. Because all ark mutants were lethal at a similar stage, only ark1 and ark2 were analyzed in these studies (Mills, 2006).

Similar to Apaf-1, ARK consists of a CARD, a nucleotide-binding NB-ARC domain, and multiple WD40 repeats. ark1 mutation truncates the protein in the NB-ARC (CED-4 domain), whereas ark2 leads to a protein lacking most of the WD40 repeats. Both mutants are lethal and have very similar phenotypes, suggesting that they are strong loss-of-function alleles. The phenotypes also indicate that both the NB-ARC and the WD40 domains are essential for ARK function. Unlike the published hypomorphs, all homozygous ark1 and ark2 animals die as pupae. Despite the similar overall phenotypes for ark1 and ark2 alleles, development of ark1 mutants to pupation was significantly delayed when compared with WT or ark2 alleles, suggesting that ark1 may be a stronger allele than ark2. Consequently, the survival of ark1-null animals to early pupae stage was lower than that of the heterozygotes. Although larvae and pupae from both ark mutants appear grossly normal externally, some larval tissues derived from late third instar animals show hyperplasia. For example the larval central nervous system (CNS) was enlarged in both ark mutants. This was particularly evident in the ventral ganglion that appeared to be elongated and contained longer nerve fibers. In ~40% of ark1 and most of the ark2 animals, the wing discs were enlarged. In a small number of both mutants, the eye discs were also enlarged (Mills, 2006).

dronc mutant embryos contain extra cells, and the removal of maternal dronc abolishes most cell death during embryogenesis. dronc-deficient embryos also show an enlargement of the CNS, which is presumably caused by reduced PCD. By staining embryos with anti-embryonic lethal abnormal visual protein (ELAV) antibody to visualize neurons in the CNS and peripheral nervous system, extra neurons were found in chordotonal cell clusters in ark mutant embryos. There were up to three extra cells per cluster in most ark mutant embryos analyzed. Staining of embryos with BP102 antibody, which recognizes CNS axons, showed gross abnormalities in many mutant animals, with ark2 animals often showing more dramatic features. Stronger staining of CNS axons was consistently observed in ark mutant embryos compared with WT animals, which could result from more densely packed axons. In many mutant animals, the ventral nerve cord appeared to be improperly compacted and the spacing between longitudinal axonal tracts was enlarged. This could be attributable to additional cells in the mutants caused by reduced PCD (Mills, 2006).

Since ark mutants essentially phenocopy the loss-of-dronc function, the data argue that these proteins act in a common pathway. Previous experiments using RNA interference have shown that ARK is required for DRONC activation. These results suggest that the primary function of ARK is to facilitate DRONC activation. The observation that metamorphic midgut cell death occurs normally, whereas salivary gland PCD is significantly delayed, suggests that the midgut may provide a model system for studying novel caspase activation and cell death pathways that are independent of the evolutionarily conserved canonical pathway (Mills, 2006).

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, since its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. This study further dissects the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. The induction of apoptosis is dependent in each case on expression of ARK and DRONC and surviving cells continue to proliferate. A difference was noted in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these have dramatic effects to sustain cell survival following depletion of DIAP1, but have only minor effects following cellular stress. These results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted (Kiessling, 2006).

This study further dissected the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. The induction of apoptosis is dependent on expression of the APAF-1 homolog ARK, and the initiator caspase, DRONC. Knock-down of either ARK or DRONC led not only to short term cell survival, as is also observed in mammalian cells lacking APAF-1 or caspase-9, but also to long term survival, seen as cellular accumulation as the cells continued to proliferate. This is in striking contrast to observations in mammalian cells lacking APAF-1 or caspase-9, where cells ultimately succumb to 'caspase-independent cell death' and do not proliferate. This difference is most easily explained by the difference in mitochondrial involvement: in mammals, MOMP is associated with the release of potentially toxic factors, such as AIF, endoG, Omi, and others, and with an eventual loss of mitochondrial function, any of which can contribute to death, even when downstream caspase activation is blocked or defective. The apparent absence of MOMP in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted (Kiessling, 2006).

Studies in ARK mutants clearly demonstrate that ARK is required for cell death in vivo, since these mutants display developmental defects, including an enlarged nervous system, and resist death induced by transgenic expression of Grim. Furthermore, genetic studies revealed an epistatic relationship between ARK and DIAP1 by demonstrating that loss of ARK reverses catastrophic defects seen in DIAP1 mutants and rescues developing tissues that would otherwise die from DAIP1 inactivation. The function of ARK is required for hyperactivation of caspases which occurs in the absence of DIAP-1. One might argue that the current findings are therefore merely confirmatory. However, it should be noted that profound developmental defects are observed in mice lacking APAF-1, caspase-9, or caspase-3, which are nevertheless dispensable for stress or oncogene-induced cell death in MEFs and lymphocytes from these mice in vitro and cells of the interdigital web in vitro or in vivo. In fact, there is currently no evidence that a cell capable of proliferation can do so following MOMP, and alternative explanations of developmental defects in these knockout mice (other than survival and proliferation following MOMP) have been offered (Kiessling, 2006).

A rapid loss of the DIAP1 is observed in the S2 cells, when treated with various stressors. The full length DIAP1 protein disappears rapidly and a smaller, 27 kDa fragment accumulates over time. Interestingly, the broad spectrum caspase inhibitor zVAD-fmk does not suppress the degradation of DIAP1, but the 27 kDa cleavage product could not be detected when caspase activation was inhibited. The differences between DIAP1 degradation with or without caspase activity could be explained by the notion that the degradation of DIAP1 after treatment with apoptosis-inducing stimuli is mediated by a combination of cleavage by caspases and proteasomal degradation. Thus, the continued degradation of DIAP1 in the presence of activated caspases produces the 27 kDa fragment. It has been recently reported that caspase-dependent cleavage of DIAP1 is required for DIAP1 loss in an early stage of apoptosis and that cleavage of DIAP1 is required for degradation. Similarly, it was observed that if caspases are inhibited following apoptosis induction, DIAP1 levels remain unaltered for a number of hours, however, the inhibition of caspases does not block DIAP1 degradation at longer times (Kiessling, 2006).

While a requirement for ARK and DRONC was observed under all of the pro-apoptotic conditions in S2 cells, a difference was noted in the effects of knock-down of the executioner caspases DCP-1 and DRICE. Knock-down of either or both of these has dramatic effects to sustain cell survival following knock-down of DIAP1, but has only minor effects following cellular stress (Kiessling, 2006).

Several possible explanations were envisioned for this difference. One possibility is that knock-down of DIAP1 leads to caspase activation uniquely through permitting DRONC function to activate DCP-1 and DRICE, while stressors somehow engage other caspase activation pathways (and other caspases). This, however, is inconsistent with the observation that stress-induced apoptosis is clearly dependent on ARK and DRONC. Alternatively, it may be that stress-induced death also involves inhibition of other IAPs, such as DIAP-2 and dBRUCE, which may have a wider spectrum of effects to engage additional caspases not affected by DIAP1 alone. Previously, however, it was noted that knock-down of DIAP-2 does not trigger apoptosis, but greatly enhances susceptibility to death induced by stressors such as were used in this study. This argues that DIAP-2 function, at least, continues following such stress (such that its knock-down has an effect) and thus it is less likely to be an important explanation for the current effects (Kiessling, 2006).

The final possibility is perhaps the most interesting. The knock-down of DIAP1 leads to death, presumably through permitting low levels of ongoing (and otherwise repressed) caspase activation to function and any subsequent effect may depend on amplification, as active executioner caspases cleave and activate others. Therefore, knock-down of even one caspase in the cell may dampen this amplification so that cells survive. In contrast, the induction of apoptosis by stress may involve not only blockade of DIAP1 function (through the N-termini of Reaper, Hid, Grim and Sickle) but also another signal that amplifies caspase activation upstream of ARK. Such an upstream effect has been suggested by studies of the so-called 'GH3' region in these proteins, that appears to be required for death in Drosophila cells and can function to promote the mitochondrial pathway in vertebrate systems. Reaper, Hid, Grim, and probably Sickle are necessary for stress-induced apoptosis in Drosophila, and therefore their effects are likely to depend on ARK and ARK-DRONC interactions. Nevertheless, this line of reasoning suggests that they function not only to de-repress caspases (though blocking DIAP1), but also to do something else to bypass full dependence on DCP-1 and DRICE, perhaps by amplifying caspase activation at the level of the ARK-DRONC interaction. While speculative, this possibility is intriguing, and suggests that the induction of apoptosis in Drosophila may prove to be more complex than the simple models indicate (Kiessling, 2006).

A collective form of cell death requires homeodomain interacting protein kinase

Post-eclosion elimination of the Drosophila wing epithelium was examined in vivo where collective 'suicide waves' promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. This phenotype wase leveraged in mosaic animals to discover relevant genes. homeodomain interacting protein kinase (HIPK) was shown to be required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers (Link, 2007).

Elimination of cells by programmed cell death (PCD) is a universal feature of development and aging. In both vertebrates and invertebrates, dying cells often progress through a stereotyped set of transformations referred to as apoptosis. In this form of PCD the nucleus condenses, and the collapsing cell corpse fragments into 'apoptotic bodies' that are engulfed by specialized phagocytes or neighboring cells. Apoptosis requires autonomous genetic functions within the dying cell, and extrinsic cues that elicit apoptosis have been investigated in numerous experimental models. Other forms of death are also thought to contribute during development and differ from apoptosis with respect to cellular morphology, mechanism, or mode of activation. These may include necrosis, characterized by swelling of the plasma membrane, or autophagic cell death, which is linked to extensive vacuolization in the cytoplasm. These forms of cell death can be caspase dependent or independent and may or may not be under deliberate genetic control (Link, 2007).

Two conserved protein families comprise central elements of the apoptotic machinery. Orthologous proteins represented by Ced4 in the nematode, Apaf1 in mammals, and Drosophila Ark (Dark) function as activating adaptors for CARD-containing apical caspases. During apoptosis, Ced4/Apaf1/Dark adaptors associate with pro-caspase partners (Ced3, Caspase 9, and Dronc) in a multimeric complex referred to as the 'apoptosome'. This complex is regulated by Bcl2 proteins, but apparently through a diverse group of mechanisms (Link, 2007).

Components of the Drosophila apoptosome have been genetically examined. dark and dronc are recessive, lethal genes. Both exert global functions during PCD and in stress-induced apoptosis. However, their roles in apoptosis are not absolute because rare cell deaths occurred in embryos lacking maternal and zygotic product of either gene. Elimination of dronc in the wing causes a unique, age-dependent phenotype associated with late-onset blemishing throughout the wing blade (Chew, 2004). This study shows that this progressive phenotype is characteristic for wing epithelia that lack apoptogenic functions and is caused by defects in a communal form of PCD where epithelial cells are collectively and rapidly eliminated. These findings were leveraged to discover additional genes required for PCD, and a limited set of loci, many of which were previously unknown to function in cell death, were recovered. This study establish that homeodomain interacting protein kinase (HIPK) is essential for coordinated death in the wing epithelium and, consistent with PCD functions in earlier developmental stages, regulates proper cell number in diverse tissue types (Link, 2007).

Wings mosaic for dronc- tissue exhibit normal morphology at eclosion but develop progressive, melanized blemishes with age (Chew, 2004). Similar methods were applied to determine whether lesions in other apoptogenic genes present a similar phenotype. After eclosion, wings mosaic for Df(H99), a deletion removing the apoptotic activators reaper (rpr), grim, and hid, were morphologically normal at eclosion, but over 3-7 d, melanized blemishes appeared at random throughout the wing (Link, 2007).

Likewise, homozygous driceDelta1 adult 'escapers' deficient for the effector caspase Drice also presented normal wings at eclosion but developed blemishes with age. Wings mosaic for dark82, a null allele of dark, were indistinguishable from wild-type (WT) at eclosion, but within 4 d developed wing blemishes. These late-onset blemishes became markedly more severe as animals aged. Similar yet less severe wing blemishes occurred in adults homozygous for darkCD4, a hypomorphic allele of dark. Together, these observations establish that late-onset progressive blemishing in mosaic wings is a characteristic phenotype shared among mutants in canonical PCD pathways (Link, 2007).

In the wing of newly eclosed adults, PCD removes the epithelium that forms the dorsal and ventral cuticles. To determine whether the cause of the blemish phenotype might trace to defective death in the wing epithelium, this tissue was examined in dark mutants. For these studies, wings of darkCD4 adults were prepared for light and electron microscopy. Histological analyses at the light level showed that on the first day of eclosion, the dorsal and ventral cuticles of WT animals became tightly merged with no intervening tissue evident between these layers. However, even 14 d after eclosion, cells and cell remnants remained situated between the dorsal and ventral cuticles in dark mutants. This 'undead' tissue was most easily visualized in lateral sections through melanized blemishes. Further examination of the persisting epithelium at the EM level showed evidence of intact cells soon after eclosion and ectopic cellular material 24 h after eclosion (Link, 2007).

To directly examine the death of wing epithelial cells in vivo, a transgenic nuclear DsRed reporter was used that driven by vestigial-Gal4 (vg:DsRed), allowing visualization of the fate of these cells soon after eclosion. Observations with this pan-epithelial marker in the wing confirmed earlier studies. Within 1 h of eclosion, intact epithelial cells are clearly present and regularly patterned throughout the wing. 1-2 h later (2-3 h after eclosion), the entire intervein epithelium disappears, manifested here by the abrupt loss of DsRed throughout the wing blade. Live, real-time imaging of the wing in newly eclosed adults revealed unexpected features associated with elimination of the intervein epithelium. Epithelial cells, labeled by nuclear fluorescence, were arranged in a regular, predictable pattern throughout the wing. Then, consistent with nuclear breakdown, fluorescence became redistributed throughout the cell followed by indications of blebbing and the appearance of fragmenting cells. Occasionally, weak fluorescence enclosed in cell corpses condensed to bright punctate bodies. This series of apoptogenic changes spread extremely rapidly throughout the epithelium, appearing here as a collective wave initiating from the peripheral edge and moving across the wing blade (Link, 2007).

Within just 4 min, virtually all nuclei (~450 cells) within a space of ~114 mm2 converted from viable to apoptotic morphology. The process involved tight coordination at the group level because the likelihood of a single cell apoptosing was clearly linked to similar behaviors by nearest neighboring cells over short time frames. Also, the direction and size of the cell death wave may not be fixed in every region of the wing, but centrally located cell groups were generally eliminated earlier (Link, 2007).

Unlike conventional examples of PCD in development, no indication was found that overt engulfment of apoptotic corpses occurred at the site of death. Instead, DsRed-labeled cell remnants were passively swept en masse toward the nearest wing vein where, apparently under hydrostatic pressure, cell debris streamed proximally toward the body through the wing or along the wing vein. Together, these observations describe a communal form of PCD that rapidly eliminates the wing epithelium through coordinated group behavior (Link, 2007).

The vg:DsRed reporter was used to track the fate of mosaic wing epithelia where mutant clones were induced. In sharp contrast to WT wings, abnormally persisting cells could be readily detected as patches of DsRed in the nuclei of epithelial cells in mosaic tissues. For example, wings mosaic for dronc- clones retained extensive patches of persisting DsRed-labeled cells. Here, cells and nuclei were readily detected 4 d after eclosion, and even at 11 d post-eclosion, extensive evidence of cell debris was seen (not depicted). Wings mosaic for the H99 deletion gave identical results. Likewise, adults mutated for dark exhibited persisting cells throughout the wing blade. Consistent with this, rare driceDelta1 escapers also showed evidence of persisting cells after eclosion. These observations link failures in PCD to progressive melanized wing blemishes, raising the possibility that other apoptogenic mutants might also produce this phenotype (Link, 2007).

Unlike previously described wing defects, which are congenital and evident at eclosion, the age-dependent phenotype described in in this study is characteristic of mutations in genes that function in canonical PCD pathways. Moreover, when the dosage of dronc was reduced by half in darkCD4 adults or if WT Dmp53 was removed from these same animals, melanized blemishes became far more severe. These genetic interactions are highly specific because wing defects were never observed in Dmp53- homozygotes or in dronc51 heterozygotes. Numerous other mutants showed no such effects in combination with a dark hypomorph. It was reasoned that, if genetically eliminated, additional regulators and effectors in PCD pathways should phenocopy wings mosaic for dark- or dronc- tissue. A collection of preexisting transposon mutants was screened to capture insertions that exhibit normal wings at eclosion but develop melanized blemishes with age. This strategy exploits the FLP/FRT system together with wing-specific drivers to interrogate animals bearing wing genotypes mosaic for clones of P element-derived lethal mutations. Progeny with mosaic wings were examined for late-onset wing blemishes at 1, 7, and 14 d post-eclosion. Over 1,000 lethal insertions were screened, representing 356 2nd chromosome mutations and 707 3rd chromosome mutations (Link, 2007).

The majority of insertions (87%) produced no visible defects as wing mosaics. 13% of insertions tested produced abnormalities, and these were scored for the phenotypic categories. Congenital defects including notched, blistered, or wrinkled wings occurred alone or occasionally as compound phenotypes. The candidate strains that developed wing blemishing were further subdivided based on phenotypic severity. Insertions in class A developed pronounced blemishes within a week of eclosion, whereas those in class B developed relatively light-colored patches between 1 and 2 wk after eclosion. Mutant lines exhibiting class A phenotypes were rare (~2%). All members of this class lacked blemishes at eclosion and displayed progressive blemishing occasionally associated with fragile and sometimes broken wings. A new allele of dark (l(2)SH0173) was recovered in this class, providing reassuring validation for the screening strategy. Some members among these classes exhibited congenital notches or blisters, but congenital blemishes present at eclosion were not found (Link, 2007).

Inverse PCR was applied to map or confirm insertion sites of many class A and B strains. In addition to darkl(3)SH0173, several mutations associated with genes previously implicated in PCD were isolated. For example, l(2)SH2275 contains an insertion 2 kb upstream of mir-14, a microRNA capable of modulating Rpr-induced cell death. Likewise, l(3)S048915 maps to the first intron of DIAP1 and may represent a hypermorphic allele at this locus. l(3)S055409 maps near misshapen, a gene implicated in cell killing triggered by Rpr or Eiger, the fly counterpart of TNF. Several insertions map in or near transcriptional or translational regulators that might alter the expression of cell death genes. For example, grunge (l(3)S146907), an Atrophin-like protein, functions as a transcriptional repressor, while belle (l(3)S097074) belongs to the DEAD-box family of proteins often implicated in translational regulation and RNA processing. A portion of the class A and B hits were also directly examined for defective PCD by applying the vg:DsRed reporter in mosaic wings. Of the 29 strains tested, 14 showed obvious evidence for persisting cells in the wing epithelium (Link, 2007).

Mutants identified that exhibit both blemishing and persisting cells are likely candidates for PCD genes. One strain, l(3)S134313, produced severe late-onset blemishing and a persisting cell phenotype. After mapping this insertion to the first intron of the HIPK, null alleles at this locus were produced (Link, 2007).

Two FRT-containing P element insertions flanking the coding region of HIPK were used to generate a novel deletion. PCR verified recombination between P elements, and 8 deletion strains were recovered. These validated alleles eliminate exons 4-12, removing over 92% of coding sequence in the predicted HIPK open reading frame. Deletions at the HIPK locus were uniformly lethal before the 3rd instar stage. However, zygotic HIPK is not essential to complete embryogenesis because ~70% of HIPK homozygotes hatch to 1st instar larvae. HIPKD1 was recombined on the FRT79 chromosome to generate adult wings mosaic for this allele, and like the original insertion, these animals also developed robust progressive blemishes and a persisting cell phenotype. Both phenotypes were more severe than the original P insertion, suggesting that the l(3)S134313 allele is hypomorphic for HIPK. These findings link loss of HIPK function to the query phenotypes, establishing that the action of HIPK is essential for post-eclosion PCD in the wing epithelium (Link, 2007).

Using general stains (acridine orange) or TUNEL methods, embryonic PCD was not overtly disturbed in HIPK mutants. To investigate the possibility of more subtle or specific phenotypes, the nervous system was examined using antibodies that label specific populations of neurons affected by the H99 deletion. Using anti-Kruppel antibody, it was confirmed that stage 14-15 WT embryos contained 9-12 Kruppel-positive cells in the Bolwig's Organ. However, a portion of animals lacking maternal HIPK contained as many as 15 cells per organ at a penetrance comparable to H99 animals, which are completely cell death defective. Neurons expressing dHb9, a homeodomain protein marking a subset of cells that persist in cell death-defective H99 embryos, was examined (Rogulja-Ortmann, 2007). In germline clones, distinct classes of dHb9 staining patterns emerged. A subset of animals exhibited extreme patterning defects. Other animals displayed a striking increase in dHb9-positive cell numbers when compared with WT embryos of the parental strain. These data establish that HIPK fundamentally regulates cell numbers in the nervous system, and because the same subpopulation of cells are affected by the H99 mutation, they implicate HIPK as a more general regulator of PCD (Link, 2007).

The pupal eye undergoes reorganization involving cell death of interommatidial cells after pupation. To determine if HIPK regulates cell death in the retina, whole eye clones were generated and the anti-Dlg (discs large) antibody was used to outline cell borders in dissected pupal eyes after pupation. Extra interommatidial cells were frequently retained in whole eye HIPK- clones. This phenotype is overtly similar to animals lacking the apical caspase Dronc and consistent with an essential role in retinal PCD (Link, 2007).

Elimination of the wing epithelium in newly eclosed adults is predictable, easily visualized, and experimentally tractable. The major histomorphologic events involve cell death, delamination, and clearance of corpses and cell remnants. Recent studies established that post-eclosion PCD is under hormonal control and involves the cAMP/PKA pathway (Kimura, 2004). While dying cells in the adult wing present apoptotic features (e.g., sensitivity to p35 and TUNEL positive), elimination of the epithelium is distinct from classical apoptosis in several important respects. (1) Unlike most in vivo models, overt engulfment of cell corpses does not occur at the site of death. Instead, dead or dying cells and their remnants are washed into the thoracic cavity via streaming of material along and through wing veins. (2) Extensive vacuolization is seen in ultrastructural analyses, which could indicate elevated autophagic activity. (3) Widespread and near synchronous death that occurs in this context defines an abrupt group behavior. The process affects dramatic change at the tissue level, causing wholesale loss of intervein cells and coordinated elimination of the entire layer of epithelium. Rather than die independently, these cells die communally, as if responding to coordinated signals propagated throughout the entire epithelium, perhaps involving intercellular gap junctions. This group behavior contrasts with canonical in vivo models where a single cell, surrounded by viable neighbors, sporadically initiates apoptosis (Link, 2007).

It has been proposed that an epithelial-to-mesenchymal transition (EMT) accounts for the removal of epithelial cells after eclosion (Kiger, 2007). Although the results do not exclude EMT associated changes in the newly eclosed wing epithelium, compelling lines of evidence establish that post-eclosion loss of the wing epithelium occurs by PCD in situ -- before cells are removed from the wing. First, before elimination, wing epithelial cells label prominently with TUNEL. Second, every mutation in canonical PCD genes so far tested failed to effectively eliminate the wing epithelium, and at least two of these were recovered in the screen described in this paper. Third, elimination of the wing epithelium was reversed by induction of p35, a broad-spectrum caspase inhibitor. Fourth, using time-lapse microscopy, condensing or pycnotic nuclei, followed by the rapid removal of all cell debris in time frames was detected that was not consistent with active migration. Instead, removal of cell remnants occurred by a passive streaming process, involving perhaps hydrostatic flow of the hemolymph (Link, 2007).

This study sampled over one fifth of all lethal genes and nearly 10% of all genes in the fly genome for the progressive blemish phenotype, a reliable indicator of PCD failure in the wing epithelium. Nearly half of the mutants that produced melanized wing blemishing also displayed a cell death-defective phenotype when examined with the vg:DsRed reporter. The precise link between these defects is unclear, but a likely explanation suggests that as the surrounding cuticle fuses, persisting cells, now deprived for nutrients and oxygen, become necrotic and may initiate melanization. Mutants could arrest at upstream steps, involving the specification or execution of PCD, or they might affect proper clearance of cell corpses from the epithelium. New alleles were recovered of dark (l(2)SH0173) and a likely hypermorph of thread (l(3)S048915), which provides reassuring validation of this prediction (Link, 2007).

By leveraging this distinct phenotype, novel cell death genes were captured, including the Drosophila orthologue of HIPK. Though first identified as an NK homeodomain binding partner, this gene is an essential regulator of PCD and cell numbers in diverse tissue contexts. Of the four mammalian HIPK genes, HIPK2, the predicted ortholog of Drosophila HIPK, has been placed in the p53 stress-response apoptotic pathway (D'Orazi, 2002; Hofmann, 2002; Di Stefano, 2004; Di Stefano, 2005), but whether the Drosophila counterpart similarly impacts this network is not yet known (Link, 2007).

Proteomic survey reveals altered energetic patterns and metabolic failure prior to retinal degeneration

Inherited mutations that lead to misfolding of the visual pigment rhodopsin (Rho) are a prominent cause of photoreceptor neuron (PN) degeneration and blindness. How Rho proteotoxic stress progressively impairs PN viability remains unknown. To identify the pathways that mediate Rho toxicity in PNs, a comprehensive proteomic profiling of retinas was performed from Drosophila transgenics expressing Rh1P37H, the equivalent of mammalian RhoP23H, the most common Rho mutation linked to blindness in humans. Profiling of young Rh1P37H retinas revealed a coordinated upregulation of energy-producing pathways and attenuation of energy-consuming pathways involving target of rapamycin (TOR) signaling, which was reversed in older retinas at the onset of PN degeneration. The relevance of these metabolic changes to PN survival was probed by using a combination of pharmacological and genetic approaches. Chronic suppression of TOR signaling, using the inhibitor rapamycin, strongly mitigated PN degeneration, indicating that TOR signaling activation by chronic Rh1P37H proteotoxic stress is deleterious for PNs. Genetic inactivation of the endoplasmic reticulum stress-induced JNK/TRAF1 axis as well as the APAF-1/caspase-9 axis, activated by damaged mitochondria, dramatically suppressed Rh1P37H-induced PN degeneration, identifying the mitochondria as novel mediators of Rh1P37H toxicity. It is thus proposed that chronic Rh1P37H proteotoxic stress distorts the energetic profile of PNs leading to metabolic imbalance, mitochondrial failure, and PN degeneration and therapies normalizing metabolic function might be used to alleviate Rh1P37H toxicity in the retina. This study offers a glimpse into the intricate higher order interactions that underlie PN dysfunction and provides a useful resource for identifying other molecular networks that mediate Rho toxicity in PNs (Griciuc, 2014).


EVOLUTIONARY HOMOLOGS

CED-4, the C. elegans Apaf-1/Ark homolog

Examining the effects of overexpressing cell-death-related genes in specific C. elegans neurons that normally live, it was demonstrated that the cell-death genes ced-3 (a caspase), ced-4 (an Apaf-1 homolog), and ced-9 (a BCL-2 homolog) all can act cell autonomously to control programmed cell death. Not only the protective activity of ced-9 but also the killer activities of ced-3 and ced-4 are likely to be present in cells that normally live. Killing by overexpression of ced-3 does not require endogenous ced-4 function, whereas killing by overexpression of ced-4 is at least in part dependent on endogenous ced-3 function. These results suggest either that ced-4 acts upstream of ced-3 and ced-4 function can be bypassed by high levels of ced-3 activity or that ced-3 and ced-4 act in parallel, with ced-3 perhaps having a greater ability to kill. The finding that ced-4 appears to facilitate the inhibition of ced-3 by ced-9 suggests that ced-9 acts to negatively regulate ced-4. It is proposed that both in C. elegans and in other organisms a competition between antagonistic protective and killer activities determines whether specific cells will live or die. These results suggest a genetic pathway for programmed cell death in C. elegans in which ced-4 acts upstream of or in parallel with ced-3, and ced-9 negatively regulates the activity of ced-4 (Shaham, 1996a).

ced-4 encodes two transcripts; whereas the major transcript can cause programmed cell death, the minor transcript can act oppositely and prevent programmed cell death, thus defining a novel class of cell death inhibitors. That ced-4 has both cell-killing and cell-protective functions is consistent with previous genetic studies. The dual protective and killer functions of the C. elegans bcl-2-like gene ced-9 are mediated by inhibition of the killer and protective ced-4 functions, respectively. It is proposed that a balance between opposing ced-4 functions influences the decision of a cell to live or to die by programmed cell death and that both ced-9 and ced-4 protective functions are required to prevent programmed cell death (Shaham, 1996b).

Three principal genes are involved in developmental programmed cell death in C. elegans: ced-3 and ced-4 genes are both required for PCD, whereas ced-9 acts to prevent the death-promoting actions of these genes. ced-9 is homologous to the Bcl-2 family, whose role in protecting PCD is illusive; no vertebrate homolog of ced-4 is known. This paper describes the effect of expression of C. elegans ced-4 in yeast. Induction of wild type ced-4 results in rapid focal chromatin condensation and lethality. Mutation of a putative nucleotide binding P-loop motif of CED-4 eliminates the lethal phenotype. Immunolocalization of CED-4 to the condensed chromatin suggests that the phenotype may result from an intrinsic ability of CED-4 to interact with chromatin. Co-expression of ced-9 prevents CED-4-induced chromatin condensation and lethality, and causes the relocalization of CED-4 to endoplasmic reticulum and outer mitochondrial membranes. A direct interaction between CED-4 and CED-9 was confirmed by yeast two-hybrid analysis. It is concluded that CED-4 has a direct role in chromatin condensation. Chromatin condensation is a ubiquitous feature of metazoan apoptosis that has yet to be linked to an effector. Further studies are required to establish whether the CED-9/CED-4 interaction is required for the activation of CED-3, the Caspase cysteine protease (James, 1997).

Genetic studies of the nematode C. elegans have identified three important components of the cell death machinery. CED-3 and CED-4 function to kill cells, whereas CED-9 protects cells from death. In this study, CED-9 and its mammalian homolog Bcl-xL (a member of the Bcl-2 family of cell death regulators) were both found to interact with and inhibit the function of CED-4. In addition, analysis has revealed that CED-4 can simultaneously interact with CED-3 and its mammalian counterparts interleukin-1beta-converting enzyme (ICE) and FLICE. Thus, CED-4 plays a central role in the cell death pathway, biochemically linking CED-9 and the Bcl-2 family to CED-3 and the ICE family of pro-apoptotic cysteine proteases (Chinnaiyan, 1997).

Genetic studies suggest that ced-9 controls programmed cell death by regulating ced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates that in vivo CED-9 forms a multimeric protein complex with CED-4 and CED-3. Expression of wild-type CED-4 promotes the ability of CED-3 in mammalian cells to induce apoptosis otherwise inhibited by CED-9. The pro-apoptotic activity of CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic activation in vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a molecular basis for the control of programmed cell death in C. elegans (Wu, 1997).

CED-4 protein plays an important role in the induction of programmed cell death in Caenorhabditis elegans through the activation of caspases. CED-4 acts as a positive regulator of caspases by enhancing the processing of procaspases to their mature forms. In mammalian 293 cells and insect SF21 cells, the processing of the proform of caspase CED-3 to its mature form is facilitated by CED-4, resulting in the acceleration of CED-3-induced cell death. CED-4 can directly activate CED-3 and ATP hydrolysis associated with the ATP binding site (P-loop) of CED-4 is required for CED-3 processing. Additionally, Apaf-1, a mammalian homolog of CED-4 that also has a P-loop motif, activates caspase-9 in the presence of cytochrome c (see Drosophila Cytochrome c proximal and Cytochrome c distal) and dATP, resulting in the sequential activation of caspase-3 in vitro. The CED-3/CED-4 complex has been shown to be activated by the oligomerization of CED-4. To investigate the conservation of CED-4 function in evolution, transgenic Drosophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induces massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in CED-4's ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Drosophila S2 cells. Immunoprecipitation analysis has shown that in S2 cells, both CED-4 and CED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation. Furthermore, CED-4 (K165R) partially prevents cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 function is evolutionarily conserved in Drosophila, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases (Kanuka, 1999a).

Programmed cell death (PCD) is regulated by multiple evolutionarily conserved mechanisms to ensure the survival of the cell. This study describes pvl-5, a gene that likely regulates PCD in Caenorhabditis elegans. In wild-type hermaphrodites at the L2 stage there are 11 Pn.p hypodermal cells in the ventral midline arrayed along the anterior-posterior axis and 6 of these cells become the vulval precursor cells. In pvl-5(ga87) animals, there are fewer Pn.p cells (average of 7.0) present at this time. Lineage analysis reveals that the missing Pn.p cells die around the time of the L1 molt in a manner that often resembles the programmed cell deaths that occur normally in C. elegans development. This Pn.p cell death is suppressed by mutations in the caspase gene ced-3 and in the bcl-2 homolog ced-9, suggesting that the Pn.p cells are dying by PCD in pvl-5 mutants. Surprisingly, the Pn.p cell death is not suppressed by loss of ced-4 function. ced-4 (Apaf-1) is required for all previously known apoptotic cell deaths in C. elegans. This suggests that loss of pvl-5 function leads to the activation of a ced-3-dependent, ced-4-independent form of PCD and that pvl-5 may normally function to protect cells from inappropriate activation of the apoptotic pathway (Joshi, 2004).

Ceramide biogenesis, CED-4, CED-9 and radiation induced apoptosis in C. elegans

Ceramide engagement in apoptotic pathways has been a topic of controversy. To address this controversy, loss-of-function (lf) mutants of conserved genes of sphingolipid metabolism were tested in C. elegans. Although somatic (developmental) apoptosis was unaffected, ionizing radiation-induced apoptosis of germ cells was obliterated upon inactivation of ceramide synthase and restored upon microinjection of long-chain natural ceramide. Radiation-induced increase in the concentration of ceramide localized to mitochondria and was required for BH3-domain protein EGL-1-mediated displacement of CED-4 (an APAF-1-like protein) from the CED-9 (a Bcl-2 family member)/CED-4 complex, an obligate step in activation of the CED-3 caspase. These studies define CEP-1 (the worm homolog of the tumor suppressor p53)-mediated accumulation of EGL-1 and ceramide synthase-mediated generation of ceramide through parallel pathways that integrate at mitochondrial membranes to regulate stress-induced apoptosis (Deng, 2008).

Although studies that use genetic deficiency in ceramide production support it as essential for apoptosis in diverse models, many have questioned whether ceramide functions as a bona fide transducer of apoptotic signals. One reason for skepticism is that, despite delineation of a number of ceramide-activated proteins, no single protein has been identified as mediator of ceramide-induced apoptosis. Recent studies have suggested an alternate mode of ceramide action, based on its capacity to self-associate and locally rearrange membrane bilayers into ceramide-rich macrodomains (1 to 5 µm in diameter), which are sites of protein concentration and oligomerization. Ceramide may thus mediate apoptosis through its ability to reconfigure membranes, coordinating protein complexation at critical junctures of signaling cascades (Deng, 2008).

To establish the role of ceramide definitively, a model of radiation-induced apoptosis was used in C. elegans germ cells. Germline stem cells, located at the distal gonad tip, divide incessantly throughout adult life, with daughter cells arresting in meiotic prophase. Upon exiting prophase, germ cells become sensitive to radiation-induced apoptosis, detected morphologically just proximal to the bend of the gonadal arm. This apoptotic pathway is antagonized by the ABL-1 tyrosine kinase, requiring sequentially the cell cycle checkpoint genes rad-5, hus-1, and mrt-2; the C. elegans p53 homolog cep-1; and the genes making up the conserved apoptotic machinery, the caspase ced-3, the apoptotic protease activating factor 1-like protein ced-4, the Bcl-2 protein ced-9, and the BH3-domain protein egl-1. This pathway differs from apoptotic somatic cell death, which is not subject to upstream checkpoint regulation via the CEP-1 pathway (Deng, 2008).

This study has identified conserved genes that regulate C. elegans sphingolipid intermediary metabolism and has tested deletion alleles. Screening for mutants resistant to radiation-induced germ cell apoptosis revealed apoptosis suppression in only deletion mutants of hyl-1 and lagr-1, two of the three ceramide synthase (CS) genes. CS gene products regulate de novo ceramide biosynthesis, acylating sphinganine to form dihydroceramide that is subsequently converted to ceramide by a desaturase. CSs contain six to seven putative transmembrane domains and a Lag1p motif [which confers enzyme activity, regions conserved in the C. elegans orthologs. The deleted CS sequences in hyl-1(ok976) and lagr-1(gk327) result in frameshifts that disrupt the Lag1p motifs. An ~1.6-kb hyl-1 transcript was detected in wild-type (WT) worms and a smaller ~1.35-kb transcript in hyl-1(ok976), whereas an ~1.4-kb lagr-1 transcript was detected in WT worms and a ~1.25-kb transcript in lagr-1(gk327). In contrast, a deletion mutant of the third C. elegans CS, hyl-2(ok1766), lacking a 1626-base pair fragment of the hyl-2 gene locus that eliminates exons 2 to 5 corresponding to 74% of the coding sequence, displayed no defect in germ cell death (Deng, 2008).

In N2 WTstrain young adults, apoptotic germ cells gradually increased in abundance with age from a baseline of 0.7 ± 0.1 to 1.8 ± 0.2 corpses per distal gonad arm over 48 hours. Exposure to a 120-gray (Gy) ionizing radiation dose increased germ cell apoptosis to 5.2 ± 0.3 cells 36 to 48 hours after treatment. In contrast, in hyl-1 (ok976) and lagr-1(gk327) animals, age-dependent and radiation-induced germ cell apoptosis were nearly abolished. Similar effects were observed in the lagr-1(gk327);hyl-1(ok976) double mutant. The rate of germ cell corpse removal was unaffected in CS mutants, excluding the possibility that defective corpse engulfment elevated corpse numbers. In contrast, loss-of-function (lf) mutations of hyl-1 or lagr-1 did not affect developmental somatic cell death, nor did the lf hyl-2(ok1766) mutation. These studies indicate a requirement for two C. elegans CS genes for radiation-induced germline apoptosis (Deng, 2008).

To confirm ceramide as critical for germline apoptosis, C16-ceramide was injected into gonads of young adult WT worms. C16-ceramide is the predominant ceramide species in apoptosis induction by diverse stresses in multiple organisms. C16-ceramide microinjection resulted in time- and dose-dependent increases in germ cell apoptosis, with a median effective dose of ~0.05 µM gonadal ceramide. Peak effect occurred at ~0.1 µM gonadal ceramide at 36 hours, qualitatively and quantitatively mimicking the 120-Gy effect in WT worms. In contrast, C16-dihydroceramide, which differs from C16-ceramide in a trans double bond at sphingoid base position four to five, was without effect, indicating specificity for ceramide in apoptosis induction. Furthermore, C16-ceramide microinjection into lagr-1(gk327);hyl-1(ok976) animals (~1 µM gonadal ceramide) resulted in a 5.7-fold increase in germ cell apoptosis. Note that the baseline level of apoptosis in lagr-1(gk327);hyl-1(ok976) was less than one-half that in WT worms. Moreover, ~0.005 µM gonadal ceramide, a concentration without impact on germ cell apoptosis, completely restored radiation (120 Gy)-induced apoptosis, an effect inhibitable in a lf ced-3 background. C16-ceramide's ability to bypass the genetic defect and restore the radiation-response phenotype is strong evidence that hyl-1 and lagr-1 represent legitimate C. elegans CS genes. Animals with sphk-1(ok1097), a null allele of sphingosine kinase (SPHK), which prevents conversion of ceramide to its anti-apoptotic derivative sphingosine 1-phosphate (S1P), displayed high baseline germ cell death and were hypersensitive to radiation-induced germ cell apoptosis, inhibitable (by 85 ± 9%) in a lagr-1(gk327);sphk-1(ok1097) double mutant. Collectively, these studies identify ceramide as a critical effector of radiation-induced germ cell apoptosis, although they do not define its mode of engaging the apoptotic pathway (Deng, 2008).

Inactivation of the C. elegans ABL-1 ortholog in the lf mutant abl-1(ok171) (or by RNA interference) increases baseline and post-radiation germ cell apoptosis, modeling radiation hypersensitivity phenotypes. To order CS action relative to ABL-1, hyl-1(ok976);abl-1(ok171) and lagr-1(gk327);abl-1(ok171) and a triple mutant lagr-1(gk327);hyl-1(ok976);abl-1(ok171) were generated. lf hyl-1 or lagr-1 in an abl-1(ok171) genetic background prevented the time-dependent increase in physiologic germ cell apoptosis and completely blocked radiation-induced apoptosis. Similarly, lagr-1(gk327);hyl-1(ok976);abl-1(ok171) displayed inhibition of baseline and radiation-induced germ cell apoptosis. Thus, increased germ cell apoptosis in irradiated abl-1(ok171) depends on the CS genes hyl-1 and lagr-1 (Deng, 2008).

In C. elegans, DNA damage activates the p53 homolog CEP-1, which is required for transcriptional up-regulation of the BH3-only proteins, EGL-1 and CED-13, that in turn activate the core apoptotic machinery (CED-9, CED-4, and CED-3). Exposure of hyl-1(ok976) and lagr-1 (gk327) to 120 Gy increased egl-1 transcripts four- to fivefold at 9 hours after irradiation, whereas ced-13 expression was enhanced five- to sixfold -- levels comparable to those detected in irradiated WTworms. Thus, the loss of CS did not affect CEP-1 activation upon irradiation, suggesting that ceramide and CEP-1 might function in parallel, coordinately conferring radiation-induced germ cell death (Deng, 2008).

It was reasoned that in contrast to radiation-induced germ cell apoptosis, which apparently requires increased abundance of both BH3-only proteins and ceramide, C16-ceramide provided exogenously might act independent of p53-mediated egl-1 expression by maximizing the effect of baseline EGL-1. In fact, microinjected C16-ceramide partially restored germ cell death in cep-1(gk138) from 0.4 ± 0.13 to 2.5 ± 0.32 corpses per distal gonad arm. Since C16-ceramide is inactive in the lf egl-1 mutant egl-1(n1084n3082), it appears that there is a requirement for at least a baseline level of BH3-only proteins for ceramide-induced apoptosis. Consistent with this notion, C16-ceramide administration did not increase egl-1 and ced-13 transcription. Furthermore, inactivating the core apoptotic machinery in lf ced-3(n717) and ced-4(n1162) or in gain-of-function ced-9(n1950) animals, which abolish radiation-induced germline apoptosis, similarly abolished C16-ceramide-induced death. Collectively, these data indicate that ceramide acts in conjunction with BH3-only proteins upstream of the mitochondrial commitment step of apoptosis in the C. elegans germ line (Deng, 2008).

Since these studies point to a mitochondrial site of ceramide action, an immune histochemical approach was devised to evaluate whether ceramide might increase in the mitochondria of C. elegans germ cells. Advantage was taken of the increased frequency of germ cell apoptosis in abl-1(ok171), anticipating a maximized ceramide signal upon irradiation in this strain. Gonads from unirradiated or irradiated worms were dissected, opened by freeze-cracking, and then stained with MID15B4, a specific anti-ceramide antibody. Mitochondria were localized with an antibody to the mitochondrial marker protein OxPhos Complex IV subunit I (COX-IV) or by Rhodamine B staining. COX-IV staining (green) before and after irradiation displayed a prominent perinuclear distribution reminiscent of mitochondrial topography in some mammalian cell systems. Ceramide staining (red) displayed a similar profile and at baseline was faint, increasing 2.4-fold at 24 hours post-irradiation. Merging the two signals (red and green) revealed that ceramide accumulation was distinctively mitochondrial (yellow). Radiation-induced ceramide accumulation was abrogated in lagr1(gk327);hyl-1(ok976);abl-1(ok171) animals. Similarly, ceramide increase was abrogated in irradiated lagr-1(gk327);hyl-1(ok976) as compared with WT animals. These results define ionizing radiation-induced ceramide accumulation in the C. elegans germ line as mitochondrial in origin, mediated via the classic ceramide biosynthetic pathway (Deng, 2008).

Whether mitochondrial ceramide accumulation was required for CED-4 redistribution to nuclear membranes was examined. In nonapoptotic somatic cells, CED-4 is sequestered to mitochondria by binding CED-9. When displaced by EGL-1, CED-4 targets nuclear membranes and activates caspase CED-3, necessary for the effector phase of apoptosis. For these studies, abl-1(ok171) and lagr-1(gk327);hyl-1(ok976);abl-1(ok171) animals were exposed to 120 Gy, and germ cells were released from gonads and stained with antibodies against C. elegans CED-4 and Ce-lamin, a nuclear membrane marker. CED-4 and Ce-lamin colocalization by confocal microscopy (yellow merged signal) served as readout for nuclear CED-4 redistribution. After irradiation nuclear CED-4 staining intensity increased 4.3-fold in abl-1(ok171). Consistent with reduced germ cell apoptosis, nuclear CED-4 staining is significantly reduced in lagr-1(gk327);hyl-1(ok976);abl-1(ok171). Specifically, baseline CED-4 intensity at the nuclear membrane is lower in lagr-1(gk327);hyl-1(ok976);abl-1(ok171) than in abl-1(ok171), increasing post-irradiation only to the control level of unirradiated abl-1(ok171) worms, an effect probably of biologic relevance as the biophysical effects of ceramide on membrane structure are concentration-dependent (Deng, 2008).

This study also used opls219 worms, a strain expressing a CED-4::GFP fusion protein (where GFP is green fluorescent protein), which permits in vivo detection of CED-4 trafficking. opls219 worms were cultured on plates containing Rhodamine B to stain mitochondria (red). Merged images detect mitochondrial CED-4 as a yellow signal (red and green overlay), whereas nonmitochondrial CED-4 appears green. Although a low-intensity green CED-4 signal was detected in nuclear membranes of unirradiated germ cells, the large majority of CED-4 was present in mitochondria before irradiation. At 36 hours postradiation, the CED-4 signal was markedly reduced in mitochondria, relocalizing primarily to nuclear membranes as bright green platformlike structures. In eight worms, overall reduction in CED-4 mitochondrial colocalization upon irradiation was ~50%, abrogated in lagr-1(gk327);opls219. Consistent with the anti-CED-4 antibody staining, the loss of mitochondrial CED-4 signal in opls219 was accompanied by a twofold increase in nuclear CED-4 signal, blocked entirely in lagr-1(gk327);opls219. These results indicate that mitochondrial ceramide contributes substantively to CED-4 displacement from mitochondrial membranes during radiation-induced germ cell apoptosis (Deng, 2008).

These data indicate that the ceramide synthetic pathway is required for radiation-induced apoptosis of C. elegans germ cells. The most parsimonious molecular ordering suggests that CS (as well as its enzymatic product ceramide) functions on a pathway that is parallel to the CEP-1/p53-EGL-1 system. The coordinated function of these two pathways occurs at the mitochondrial commitment step of the apoptotic process. It is hypothesized that ceramide may recompartmentalize the mitochondrial outer membrane, yielding a permissive microenvironment for EGL-1-mediated displacement of CED-4, the trigger for the effector stage of the apoptotic process (Deng, 2008).

Isolation and characterization of mammalian Apaf-1 family proteins

Apaf-1 (apoptotic protease activating factor-1), a novel 130 kd protein from HeLa cell cytosol, participates in the cytochrome c-dependent activation of caspase-3. The release of cytochrome c from mitochondria in response to apoptotic stimuli is blocked in cells overexpresssing Bcl-2. The NH2-terminal 85 amino acids of Apaf-1 show 21% identity and 53% similarity to the NH2-terminal prodomain of the Caenorhabditis elegans caspase, CED-3. However, Apaf-1 does not seem to be a caspase, since the conserved active site pentapeptide that is present in all identified caspases is not present in Apaf-1. The caspase homologous region is followed by 320 amino acids that show 22% identity and 48% similarity to CED-4, a protein that is believed to initiate apoptosis in C. elegans. The COOH-terminal region of Apaf-1 comprises multiple WD repeats, which are proposed to mediate protein-protein interactions. Cytochrome c binds to Apaf-1, an event that may trigger the activation of caspase-3, leading to apoptosis (Zou, 1997).

The deduced amino acid sequences have been reported of two alternately spliced isoforms, designated DEFCAP-L and -S, that differ in 44 amino acids and encode a novel member of the mammalian Ced-4 family of apoptosis proteins. Similar to the other mammalian Ced-4 proteins (Apaf-1 and Nod1), DEFCAP contains a caspase recruitment domain (CARD) and a putative nucleotide binding domain, signified by a consensus Walker's A box (P-loop) and B box [Mg(2+)-binding site]. Like Nod1, but different from Apaf-1, DEFCAP contains a putative regulatory domain containing multiple leucine-rich repeats (LRR). However, a distinguishing feature of the primary sequence of DEFCAP is that DEFCAP contains at its NH(2) terminus a pyrin-like motif and a proline-rich sequence, possibly involved in protein-protein interactions with Src homology domain 3-containing proteins. By using in vitro coimmunoprecipitation experiments, both long and short isoforms were capable of strongly interacting with caspase-2 and exhibited a weaker interaction with caspase-9. Transient overexpression of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma cells MCF7 resulted in significant levels of apoptosis. In vitro death assays with transient overexpression of deletion constructs of both isoforms using beta-galactosidase as a reporter gene in MCF7 cells suggest the following: (1) the nucleotide binding domain may act as a negative regulator of the killing activity of DEFCAP; (2) the LRR/CARD represents a putative constitutively active inducer of apoptosis; (3) the killing activity of LRR/CARD is inhibitable by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone, and (4) the CARD is critical for killing activity of DEFCAP. These results suggest that DEFCAP is a novel member of the mammalian Ced-4 family of proteins capable of inducing apoptosis, and understanding its regulation may elucidate the complex nature of the mammalian apoptosis-promoting machinery (Hlaing, 2001).

Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of procaspase-9, involves association with an Apaf-1-related molecule. A human Apaf-1-related protein, Ipaf, has been identified that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells. These results suggest that Ipaf is a specific and direct activator of procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli (Poyet, 2001).

Is the short form of apaf-1 also present in mammals? The exon/intron boundary was found at a similar position to that of Drosophila. If apaf-1 short (apaf-1S) is conserved through evolution, a similar type of splicing variant could be expected to be present in mammals. Semiquantitative RT-PCR was performed using specific primers that could differentiate apaf-1 (281 bp PCR product) and apaf-1S (242 bp PCR product). As in Drosophila, the expression of apaf-1S was found to be present in embryonic brain but was hard to detect in adult brain. These results suggest that splicing variant apaf-1S is present through evolution (Kanuka, 1999b).

Apaf-1 plays an essential role in apoptosis. In the presence of cytochrome c and dATP, Apaf-1 assembles into an oligomeric apoptosome; this is responsible for the activation of procaspase-9 and the maintenance of the enzymatic activity of the processed caspase-9. Regulation of apoptosome assembly by other cellular factors is poorly understood. This study reports that physiological concentrations of calcium ion negatively affect the assembly of apoptosome by inhibiting nucleotide exchange in the monomeric, autoinhibited Apaf-1 protein. Consequently, calcium blocks the ability of Apaf-1 to activate caspase-9. These observations suggest an important role of calcium homeostasis on the Apaf-1-dependent apoptotic pathway (Bao, 2007).

Mutation of Apaf-1

The cytosolic protein APAF1, a human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. A null allele of the murine Apaf1 was generated by a gene trap. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. These findings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals (Cecconi, 1998).

Apoptosis is essential for the precise regulation of cellular homeostasis and development. The role in vivo of Apaf1, a mammalian homolog of C. elegans CED-4, was investigated in gene-targeted Apaf1-/- mice. Apaf1-deficient mice exhibit reduced apoptosis in the brain and striking craniofacial abnormalities with hyperproliferation of neuronal cells. Apaf1-deficient cells are resistant to a variety of apoptotic stimuli, and the processing of Caspases 2, 3, and 8 is impaired. However, both Apaf1-/- thymocytes and activated T lymphocytes are sensitive to Fas-induced killing, showing that Fas-mediated apoptosis in these cells is independent of Apaf1. These data indicate that Apaf1 plays a central role in the common events of mitochondria-dependent apoptosis in most death pathways and that this role is critical for normal development (Yoshida, 1998).

The forebrain overgrowth mutation (fog) was originally described as a spontaneous autosomal recessive mutation mapping to mouse chromosome 10 that produces forebrain defects, facial defects, and spina bifida. Although the fog mutant has been characterized and available to investigators for several years, the underlying mutation causing the pathology has not been known. Because of its phenotypic resemblance to apoptotic protease activating factor-1 (Apaf-1) knockout mice, the possibility that the fog mutation is in the Apaf-1 gene was investigated. Allelic complementation, Western blot analysis, and caspase activation assays indicate that fog mutant mice lack Apaf-1 activity. Northern blot and reverse transcription-PCR analysis show that Apaf-1 mRNA is aberrantly processed, resulting in greatly reduced expression levels of normal Apaf-1 mRNA. These findings are strongly suggestive of the fog mutation being a hypomorphic Apaf-1 defect and implicate neural progenitor cell death in the pathogenesis of spina bifida, a common human congenital malformation. Because a complete deficiency in Apaf-1 usually results in perinatal lethality and fog/fog mice more readily survive into adulthood, these mutants serve as a valuable model with which apoptotic cell death can be studied in vivo (Honarpour, 2001).

Transcriptional regulation of Apaf-1

Loss of function of the retinoblastoma protein, pRB, leads to lack of differentiation, hyperproliferation and apoptosis. Inactivation of pRB results in deregulated E2F activity, which in turn induces entry to S-phase and apoptosis. Induction of apoptosis by either the loss of pRB or the deregulation of E2F activity occurs via both p53-dependent and p53-independent mechanisms. The mechanism by which E2F induces apoptosis is still unclear. E2F1 directly regulates the expression of Apaf-1, the gene for apoptosis protease-activating factor 1. These results provide a direct link between the deregulation of the pRB pathway and apoptosis. Furthermore, because the pRB pathway is functionally inactivated in most cancers, the identification of Apaf-1 as a transcriptional target for E2F might explain the increased sensitivity of tumor cells to chemotherapy. Independently of the pRB pathway, Apaf-1 is a direct transcriptional target of p53, suggesting that p53 might sensitize cells to apoptosis by increasing Apaf-1 levels (Moroni, 2001).

Translational regulation of Apaf

Polypyrimidine tract binding protein 1 (PTB: Drosophila homolog Hephaestus) binds and activates the Apaf-1 internal ribosome entry segment (IRES) when the protein upstream of N-ras (unr; a single-stranded RNA binding protein which contains five cold shock domains, Drosophila homolog CG7015) is prebound. The Apaf-1 IRES is highly active in neuronal-derived cell lines due to the presence of the neuronal-enhanced version of PTB, nPTB. The unr and PTB/nPTB binding sites have been located on the Apaf-1 IRES RNA, and a structural model for the IRES bound to these proteins has been derived. The ribosome landing site has been located to a single-stranded region, and this is generated by the binding of the nPTB and unr to the RNA. These data suggest that unr and nPTB act as RNA chaperones by changing the structure of the IRES into one that permits translation initiation (Mitchell, 2003).

The regulatory mechanisms controlling cell death are complex, and in addition to control of transcription, the expression of proteins that are involved in apoptosis is regulated by control of translation. Indeed, many mRNAs whose protein products are involved in apoptosis are translated by the alternative mechanism of internal ribosome entry. This process is mediated by a complex RNA structural element located in the 5' untranslated region (UTR) of the mRNA termed an internal ribosome entry segment (IRES). During apoptosis cap-dependent translation initiation is very much reduced, yet expression of certain key proteins required for this process is maintained by internal ribosome entry. Thus c-myc, DAP5, and XIAP IRESes function to maintain expression of these proteins following apoptosis. Apaf-1 translation is solely initiated by internal ribosome entry, but to date the only situation where a small increase in Apaf-1 IRES function has been observed is following genotoxic stress. Given the importance of Apaf-1 during brain development, it is possible that the Apaf-1 IRES is required for expression of this protein in the developing brain. In this regard the FGF-2 IRES has been shown to be active in adult brain while in developing embryos both the FGF-2 and c-myc IRESes are active. This suggests that certain IRES trans-acting factors (ITAFs) are not present in the fully differentiated cell types, and these and additional studies have demonstrated that the function of certain cellular IRESes varies considerably with cell type. Most cellular IRESes are inactive in vitro, again suggesting an absolute requirement for ITAFs that are not present in these systems. However, very few ITAFs have been identified for cellular IRESes although the auto-antigen La has been shown to interact with the XIAP IRES and hnRNPC has been shown to interact with the PDGF IRES. The Apaf-1 IRES requires both polypyrimidine tract binding protein (PTB; a protein that has a role in regulating splicing as well as aiding internal ribosome entry of certain viral IRESes and upstream of N-ras). PTB only binds to the Apaf-1 IRES RNA if unr is prebound suggesting that unr is required to attain the correct structural conformation of the Apaf-1 IRES (Mitchell, 2003).

Apaf-1 IRES is most active in cell lines of neural origin and this activity correlates with the neuronal enhanced version of PTB, nPTB. The PTB/nPTB binding sites on the Apaf-1 RNA have been located, and by chemical and enzymatic probing of the RNA a structural model for the IRES has been derived in the presence of these binding factors. To test this model, specific structural alterations were made to the Apaf-1 IRES, and these abolished the binding of trans-acting factors while retaining and even increasing IRES activity. The region in which the ribosome lands has been localized, and it would appear that in the presence of unr and nPTB a region of single-stranded RNA becomes accessible to the ribosome. This would suggest that unr and nPTB act as RNA chaperones, changing the structure of the Apaf-1 IRES into one that is functionally competent for 48S formation (Mitchell, 2003).

Apaf-1 interaction with cytochrome

In the apoptosis pathway in mammals, cytochrome c and dATP are critical cofactors in the activation of caspase 9 by Apaf-1. Until now, the detailed sequence of events in which these cofactors interact has been unclear. This study shows, through fluorescence polarization experiments, that cytochrome c can bind to Apaf-1 in the absence of dATP; when dATP is added to the cytochrome c.Apaf-1 complex, further assembly occurs to produce the apoptosome. These findings, along with the discovery that the exposed heme edge of cytochrome c is involved in the cytochrome c.Apaf-1 interaction, are confirmed through enhanced chemiluminescence visualization of native PAGE gels and through acrylamide fluorescence quenching experiments. The cytochrome c.Apaf-1 interaction depends highly on ionic strength, indicating that there is a strong electrostatic interaction between the two proteins (Purring-Koch, 2000).

As components of the apoptosome, a caspase-activating complex, cytochrome c (Cyt c) and Apaf-1 are thought to play critical roles during apoptosis. Due to the obligate function of Cyt c in electron transport, its requirement for apoptosis in animals has been difficult to establish. 'Knockin' mice were generated expressing a mutant Cyt c (KA allele), which retains normal electron transfer function but fails to activate Apaf-1. Most KA/KA mice displayed embryonic or perinatal lethality caused by defects in the central nervous system, and surviving mice exhibited impaired lymphocyte homeostasis. Although fibroblasts from the KA/KA mice were resistant to apoptosis, their thymocytes were markedly more sensitive to death stimuli than Apaf-1(-/-) thymocytes. Upon treatment with gamma irradiation, procaspases were efficiently activated in apoptotic KA/KA thymocytes, but Apaf-1 oligomerization was not observed. These studies indicate the existence of a Cyt c- and apoptosome-independent but Apaf-1-dependent mechanism(s) for caspase activation (Hao, 2005).

Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-1

Apoptosis in metazoans is executed intracellular caspases. One of the caspase-activating pathways in mammals is initiated by the release of cytochrome c from mitochondria to cytosol, where it binds to Apaf-1 to form a procaspase-9-activating heptameric protein complex named apoptosome. This study reports the reconstitution of this pathway with purified recombinant Apaf-1, procaspase-9, procaspase-3, and cytochrome c from horse heart. Apaf-1 contains a dATP as a cofactor. Cytochrome c binding to Apaf-1 induces hydrolysis of dATP to dADP, which is subsequently replaced by exogenous dATP. The dATP hydrolysis and exchange on Apaf-1 are two required steps for apoptosome formation (Kim, 2005; full text of article).

Apaf-1 generated in insect cells contains dATP as a cofactor. The bound dATP then undergoes one round of hydrolysis to dADP, a process that is stimulated by cytochrome c. This hydrolysis appears to serve two roles: (1) it provides energy for the conformational change need for Apaf-1 to transient from the inactive monomeric state to the oligomeric state, and (2) it allows exogenous dATP (or ATP) to exchange for the dADP that has lower binding affinity for Apaf-1, a critical step for Apaf-1 to form functional apoptosome rather than nonfunctional aggregates. This hydrolysis only happens in one round. Exogenously added dATP will then bind Apaf-1, but remains unhydrolyzed during apoptosome formation (Kim, 2005).

The formation of the nonfunctional Apaf-1 aggregate is also induced by cytochrome c binding to Apaf-1. Cytochrome c was actually reisolated as an inhibitor of Apaf-1 activity when dATP or ATP was not included in the caspase-3-activating reaction. Such aggregates may be identical to the large, inactive Apaf-1 complex first observed in human monocytic tumor cells (Kim, 2005).

Interestingly, the crystal structure of a WD-40-truncated Apaf-1 revealed Apaf-1 in an inactive configuration with an ADP bound to it. Because WD-40 repeats serve as an autoinhibitory role, Apaf-1 without this region should be active, yet may easily become inactive if the exogenous dATP or ATP level is low. This finding is consistent with the observation that WD-40-less Apaf-1 is indeed active in promoting procaspase-9/3 activation but is rather unstable and the only stable configuration might be the dADP or ADP binding form. It is also interesting that this form of Apaf-1 expressed in bacteria contains an ADP but not dADP, whereas full-length Apaf-1 expressed in insect cells contains exclusively dATP. The measured difference between dATP and ATP in binding affinity to Apaf-1 is 10-fold, not enough to explain this exclusive binding of dATP because intracellular ATP level is several orders of magnitude higher than dATP. One possibility is that dATP level in E. coli is very low. Another possibility could be that mammalian and insect cells contain a dATP-specific loading factor for Apaf-1 that is absent in bacteria. Such a factor could potentially function in conjunction with prothymosin- and PHAPI, two proteins that regulate apoptosome activity (Kim, 2005).

The role of cytochrome c in apoptosome formation also becomes clear through the current study. Upon binding to Apaf-1, cytochrome c releases the autoinhibition imposed by the WD-40 repeats and allows Apaf-1 to hydrolyze the bound dATP. This role of cytochrome c also suggests that its simple release from mitochondria and binding to Apaf-1 may not necessarily result in the activation of caspase-9/3. Without exogenous dATP or ATP exchange, cytochrome c binding to Apaf-1 will irreversibly deplete the Apaf-1 protein in cells without activating caspases. Consistently, when intracellular ATP is depleted, cells undergo necrosis in response to stimuli that normally induce apoptosis, and when the cellular ATP levels are restored, the response shifts back to apoptosis. Therefore, it is hypothesized that the nucleotide exchange on Apaf-1 may provide another regulatory step for apoptosis (Kim, 2005).

The question remains whether endogenous Apaf-1 in mammalian cells also exclusively binds dATP. Addressing this question requires improvement on LC-MS method so that the nucleotide bound to endogenous Apaf-1 can be identified by using much less available material (Kim, 2005).

Apaf-1 and the activation of caspases

This paper reports the purification of Apaf-3, a third protein factor that participates in caspase-3 activation in vitro. Apaf-3 is a member of the caspase family: specifically, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. In turn, activated caspase-9 cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminishes caspase-3 activation. Mutation of the active site of caspase-9 attenuates the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade triggered by cytochrome c and dATP. Several caspases, including caspase-9, have long prodomains at their NH2 termini. This domain has been proposed to function as a caspase recruitment domain (CARD), allowing proteins with such domains to interact with each other. Although Apaf-1 is not a caspase, its NH2-terminal region contains a CARD, suggesting that Apaf-1 may recruit caspase-9 through their respective CARDs. The data suggest that, within the context of full-length Apaf-1, the CARD is not accessible for caspase-9 binding. Cytochrome c and dATP may induce a conformational change in Apaf-1 that exposes its CARD (P. Li, 1997).

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. A mammalian homolog of CED-4, Apaf-1, can associate with several death proteases in mammalian cells, including caspase-4, caspase-8, caspase-9, and nematode CED-3. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhances the killing activity of caspase-9, which requires the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promotes the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, has been shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL is mediated through both domains of Apaf-1: the CED-4-like domain and the C-terminal domain, which contains WD-40 repeats. Expression of Bcl-XL inhibits the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibits Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL fails to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans (Hu, 1998).

The exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homolog, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and three others (caspases-1, -4, and -5) fail to be activated under the same conditions. In vitro association assays confirm that caspase-9 selectively binds to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 do not. Depletion of caspase-9 from cell extracts abrogates cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enables an ordering of the sequence of caspase activation events downstream of caspase-9 and reveals the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9 (Slee, 1999).

The reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9 is reported. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1.cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3 (Zou, 1999).

Apaf-1 plays a critical role in apoptosis by binding to and activating procaspase-9. A novel Apaf-1 cDNA encoding a protein of 1248 amino acids has been identified, containing an insertion of 11 residues between the CARD and ATPase domains and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activates procaspase-9 in a cytochrome c and dATP/ATP-dependent manner. This Apaf-1 was used to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to procaspase-9. A P-loop mutant (Apaf-1K160R) is unable to associate with Apaf-1 or bind to procaspase-9. Mutation of Met368 to Leu enables Apaf-1 to self-associate and bind procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibits greater ability to induce apoptosis compared with the wild-type Apaf-1. Procaspase-9 can recruit procaspase-3 to the Apaf-1-procaspase-9 complex. Apaf-1(1-570), a mutant lacking the WD-40 repeats, associates with and activated procaspase-9, but fails to recruit procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in procaspase-9-mediated procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate procaspase-9 and induce apoptosis (Hu, 1999).

Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large (on the order of approximately 1.4 MDa). dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M(r) values of approximately 1.4 MDa and approximately 700 kDa. Kinetic analysis demonstrates that in vitro the approximately 700-kDa complex is produced more rapidly than the approximately 1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the approximately 700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processes effector caspases. Thus, the approximately 700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis (Cain, 2000).

During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome has been examined. A multi-step process is proposed, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex (Bratton, 2001).

Apoptotic protease-activating factor-1 (Apaf-1), a key regulator of the mitochondrial apoptosis pathway, consists of three functional regions: (1) an N-terminal caspase recruitment domain (CARD) that can bind to procaspase-9, (2) a CED-4-like region enabling self-oligomerization, and (3) a regulatory C terminus with WD-40 repeats masking the CARD and CED-4 region. During apoptosis, cytochrome c and dATP can relieve the inhibitory action of the WD-40 repeats and thus enable the oligomerization of Apaf-1 and the subsequent recruitment and activation of procaspase-9. Different apoptotic stimuli induce the caspase-mediated cleavage of Apaf-1 into an 84-kDa fragment. The same Apaf-1 fragment is obtained in vitro by incubation of cell lysates with either cytochrome c/dATP or caspase-3 but not with caspase-6 or caspase-8. Apaf-1 is cleaved at the N terminus, leading to the removal of its CARD H1 helix. An additional cleavage site is located within the WD-40 repeats and enables the oligomerization of p84 into a approximately 440-kDa Apaf-1 multimer even in the absence of cytochrome c. Due to the partial loss of its CARD, the p84 multimer is devoid of caspase-9 or other caspase activity. Thus, these data indicate that Apaf-1 cleavage causes the release of caspases from the apoptosome in the course of apoptosis (Lauber, 2001).

Bcl-2 family members as regulators of the cell death hierarchy: Bcl-2 interacts with Apaf-1

The Bcl-2 family of proteins regulates apoptosis, the cell death program triggered by activation of certain proteases (caspases). An attractive model for how Bcl-2 and its closest relatives prevent caspase activation is that they bind to and inactivate an adaptor protein required for procaspase processing. That model has been supported by reports that mammalian prosurvival Bcl-2 relatives bind the adaptor Apaf-1, which activates procaspase-9. However, the in vivo association studies reported here with both overexpressed and endogenous Apaf-1 challenge this notion. Apaf-1 can be immunoprecipitated together with procaspase-9, and the Apaf-1 caspase-recruitment domain is necessary and sufficient for their interaction. However, Apaf-1 does not bind to any of the six known mammalian prosurvival family members [Bcl-2, Bcl-x(L), Bcl-w, A1, Mcl-1, or Boo], or their viral homologs adenovirus E1B 19K and Epstein-Barr virus BHRF-1. Endogenous Apaf-1 also fails to coimmunoprecipitate with endogenous Bcl-2 or Bcl-x(L), or with two proapoptotic relatives (Bax and Bim). Moreover, apoptotic stimuli do not induce Apaf-1 to bind to these family members. Thus, the prosurvival Bcl-2 homologs do not appear to act by sequestering Apaf-1 and probably instead constrain its activity indirectly (Moriishi, 1999).

The C. elegans Bcl-2-like protein CED-9 prevents programmed cell death by antagonizing the Apaf-1-like cell-death activator CED-4. Endogenous CED-9 and CED-4 proteins localize to mitochondria in wild-type embryos, in which most cells survive. By contrast, in embryos in which cells have been induced to die, CED-4 assumes a perinuclear localization. CED-4 translocation induced by the cell-death activator EGL-1 (EGL-1 protein contains a Bcl-2 homology 3 domain and can physically interact with CED-9) is blocked by a gain-of-function mutation in ced-9 but is not dependent on ced-3 function, suggesting that CED-4 translocation precedes caspase activation and the execution phase of programmed cell death. Thus, a change in the subcellular localization of CED-4 may drive programmed cell death (Chen, 2000).

The death-promoting proteins Bax and BAD, which like EGL-1 contain BH3 domains, translocate to mitochondria and bind anti-apoptotic Bcl-2 family members in response to apoptotic signals. Whether and how this translocation promotes cell death is unknown. The results presented here suggest that Bax and BAD may act to release Apaf-1 or another CED-4-like protein, allowing it to activate caspase processing. Some caspase precursors, specifically procaspases-2, and -3, are present in mitochondria and upon activation translocate to nuclei. It is possible that this movement of caspases involves the translocation of a complex that includes a CED-4-like protein. By analogy, the translocation of a CED-4-CED-3 complex from mitochondria to the nuclear envelope could provide access for the active caspase to both the nucleus and the cytosol, thereby fulfilling the roles of the multiple, differentially localized mammalian caspases (Chen, 2000).

Bcl-x(L), an antiapoptotic Bcl-2 family member, is postulated to function at multiple stages in the cell death pathway. The possibility that Bcl-x(L) inhibits cell death at a late (postmitochondrial) step in the death pathway is supported by this report of a novel apoptosis inhibitor, Aven, which binds to both Bcl-x(L) and the caspase regulator, Apaf-1. Identified in a yeast two-hybrid screen, Aven is broadly expressed and is conserved in other mammalian species. Only those mutants of Bcl-x(L) that retain their antiapoptotic activity are capable of binding Aven. Aven interferes with the ability of Apaf-1 to self-associate, suggesting that Aven impairs Apaf-1-mediated activation of caspases. Consistent with this idea, Aven inhibits the proteolytic activation of caspases in a cell-free extract and suppresses apoptosis induced by Apaf-1 plus caspase-9. Thus, Aven represents a new class of cell death regulator (Chau, 2000).

Apaf-1 interaction with heat-shock proteins

The release of cytochrome c from mitochondria results in the formation of an Apaf-1-caspase-9 apoptosome and induces the apoptotic protease cascade by activation of procaspase-3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf-1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf-1 and thereby inhibits cytochrome c-mediated activation of caspase-9. Addition of purified Apaf-1 to Hsp90-depleted cytosolic extracts restores cytochrome c-mediated activation of procaspase-9. Hsp90 inhibits cytochrome c-mediated oligomerization of Apaf-1 and thereby activation of procaspase-9. Furthermore, treatment of cells with diverse DNA-damaging agents dissociates the Hsp90-Apaf-1 complex and relieves the inhibition of procaspase-9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c-dependent apoptosis and for involvement of a chaperone in the caspase cascade (Pandey, 2000).

Release of cytochrome c from mitochondria by apoptotic signals induces ATP/dATP-dependent formation of the oligomeric Apaf-1-caspase-9 apoptosome. The documented anti-apoptotic effect of the principal heat-shock protein, Hsp70, is mediated through its direct association with the caspase-recruitment domain (CARD) of Apaf-1 and through inhibition of apoptosome formation. The interaction between Hsp70 and Apaf-1 prevents oligomerization of Apaf-1 and association of Apaf-1 with procaspase-9. On the basis of these results, it is proposed that resistance to apoptosis exhibited by stressed cells and some tumors, which constitutively express high levels of Hsp70, may be due in part to modulation of Apaf-1 function by Hsp70 (Saleh, 2000).

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Using a cell-free system, it has been shown that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome (Beere, 2001).

Miscellaneous Apaf-1 interactions

Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotide-binding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation (Chu, 2001).

Apaf-1, apoptosis and development

Caspase 9 (Casp9)/Apaf3, a 45 kDa protein (also known as ICE-LAP-6 or Mch6) forms a multiprotein complex containing Apaf1 and cytochrome c. It has been proposed that cytochrome c initiates apoptosis by inducing the formation of the Casp9/Apaf1 complex. Physical association of Casp9 and Apaf1 is mediated by the interaction of their respective caspase recruitment domains (CARDs). CARDs are also found in other caspases with large prodomains, such as Casp4 and Casp8, that can associate with Apaf1 in mammalian cells. The antiapoptotic protein Bcl-XL has also been shown to interact with Casp9 and Apaf1, resulting in the inhibition of Casp9 activation. The association of Casp9 with antiapoptotic as well as proapoptotic proteins suggests a major role for Casp9 in the control of apoptosis in vivo. Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. The absence of Casp9 leads to a dramatic disturbance of telencephalic development that apparently results from decreased apoptosis in this region. The gross morphological features observed in Casp9-/- mice are remarkably similar to those observed in mice lacking Casp3. Both mutants exhibit a profound disturbance of cortical morphology, an expanded germinal zone, and hydrocephaly, suggesting that these mutations affect a common cellular apoptotic pathway dependent on both Casp9 and Casp3. Casp9-/- embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9-/- thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9-/- ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. The Casp9-dependent, Casp3-independent apoptotic pathway is preferentially triggered in thymocytes in response to dexamethasone. However, the fact that Casp3 is still processed in dexamethasone-treated Casp9-/- thymocytes suggests that dexamethasone also activates a Casp9-independent and Casp3-dependent apoptotic pathway in these cells. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of multiple apoptotic pathways in mammalian cells (Hakem, 1998).

During inner ear development, programmed cell death occurs in specific areas of the otic epithelium but the significance of this death and the molecules involved have remained unclear. An analysis was undertaken of mouse mutants in which genes encoding apoptosis-associated molecules have been inactivated. Disruption of the Apaf1 gene leads 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 suffer similar defects. Inactivation of the Bcl2-like (Bcl2l) gene leads to an overall increase in the number of cells undergoing apoptosis but does not cause any major morphogenetic defects. In contrast, decreased apoptosis is observed in specific locations that suffer 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-XL) in the otic epithelium is Apaf1-dependent. Furthermore, the otic vesicle fails to close completely in all double mutant embryos analyzed. These results indicate important roles for both Apaf1 and Bcl2l in inner ear development (Cecconi, 2004).

Apaf-1 and oncogenesis

The ability of p53 to promote apoptosis in response to mitogenic oncogenes appears to be critical for its tumor suppressor function. Caspase-9 and its cofactor Apaf-1 are essential downstream components of p53 in Myc-induced apoptosis. Like p53 null cells, mouse embryo fibroblast cells deficient in Apaf-1 and caspase-9, and expressing c-Myc, are resistant to apoptotic stimuli that mimic conditions in developing tumors. Inactivation of Apaf-1 or caspase-9 substitutes for p53 loss in promoting the oncogenic transformation of Myc-expressing cells. These results imply a role for Apaf-1 and caspase-9 in controlling tumor development (Soengas, 1999).

Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic program in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. It is concluded that Apaf-1 is inactivated in metastatic melanomas: this leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumor type (Soengas, 2001).

Apoptosis via the mitochondrial pathway requires release of cytochrome c into the cytosol to initiate formation of an oligomeric apoptotic protease-activating factor-1 (APAF-1) apoptosome. The apoptosome recruits and activates caspase-9, which in turn activates caspase-3 and -7, which then kill the cell by proteolysis. Because inactivation of this pathway may promote oncogenesis, 10 ovarian cancer cell lines were examined for resistance to cytochrome c-dependent caspase activation using a cell-free system. Strikingly, it was found that cytosolic extracts from all cell lines had diminished cytochrome c-dependent caspase activation, compared with normal ovarian epithelium extracts. The resistant cell lines expressed APAF-1 and caspase-9, -3, and -7; however, each demonstrated diminished APAF-1 activity relative to the normal ovarian epithelium cell lines. A competitive APAF-1 inhibitor may account for the diminished APAF-1 activity because no dominant APAF-1 inhibitors, altered APAF-1 isoform expression, or APAF-1 deletion, degradation, or mutation was detected. Lack of APAF-1 activity correlates in some but not all cell lines with resistance to apoptosis. These data suggest that regulation of APAF-1 activity may be important for apoptosis regulation in some ovarian cancers (Wolf, 2001).


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Search PubMed for articles about Drosophila Death-associated APAF1-related killer

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