BACKGROUND
apoptosis, or programmed cell death, is an evolutionarily conserved, genetically regulated process, whereby cells that are no longer needed undergo self-destruction through the activation of a cell suicide program  <cit> .
this cell death program is associated with characteristic morphological alterations, such as condensation of the nucleus and cytoplasm, fragmentation of nuclear dna, reorganization of the cytoskeleton, and reduction of the cell into apoptotic bodies that can be phagocytosed by neighbouring epithelial cells or phagocytes  <cit> .
autophagy is also an evolutionarily conserved mechanism that degrades unnecessary long-lived proteins and organelles.
during autophagy, cellular components are sequestered by double-membrane structures called autophagosomes.
these autophagosomes then fuse with lysosomes to form autolysosomes, where degradation occurs  <cit> .
the autophagy acts as a cellular response against extracellular stresses, such as nutrient starvation, hypoxia, and overcrowding and against intracellar stresses, such as formation of damaged or redundant organelles and cytoplasmic components  <cit> .
even though autophagy can induce a cell-survival response to some conditions, autophagic structures, especially the autophagic vacuoles, are associated with cell death.
this cell death phenomenon is classified as type ii cell death and called autophagic cell death.
in drosophila, loss of function of the atg genes leads to lethality in the transition from the larval to pupal stages, because autophagic cell death is essential for puparium formation  <cit> .
the caspases are a family of ubiquitously expressed cysteine proteases whose prototypic member is the caenorhabditis elegans death effector, ced- <dig>  <cit> .
activation of caspases typically leads to the selective cleavage of a restricted set of target proteins, generally resulting in inactivation of the target proteins.
normally present in cells as inactive precursors, caspases are proteolytically activated following upstream pro-apoptotic signals.
activated caspases cleave their substrates at an aspartic acid residue, and substrate specificity is determined by a four-residue motif n-terminal to the cleavage site  <cit> .
the "initiator" caspases primarily activate the downstream "effector" caspases whose proteolytic activity is directed toward the deconstruction of the cellular machinery during apoptosis.
in drosophila, a normally functioning apoptotic pathway depends critically on caspases, seven of which have been identified in the drosophila melanogaster genome  <cit> .
these drosophila caspase genes include three initiators and four effectors.
the initiators are dronc , dredd , and strica/dream.
the effectors are dcp- <dig> , drice , damm, and decay   <cit> .
these caspases are expected to have functions that lead to apoptosis.
many recent reports have described non-apoptotic functions of caspases, such as the cell proliferation function of dronc  <cit> , spermatid individualization by drice  <cit> , and activation of the drosophila immune system, toll receptor signaling, by dredd  <cit> .
dcp- <dig> proteins cleave cysteine protease substrates and are important for development and oogenesis  <cit> .
dcp- <dig> deletion mutants display a lack of germline cell death phenotype during mid-stage oogenesis in response to nutrient deprivation  <cit> , whereas in normal flies, cell death occurs during mid-stage oogenesis under nutrient-deprived conditions   <cit> .
in contrast, over-expression of a single copy of the truncated n-terminal region of dcp- <dig> , specifically in the eye using the glass multimer reporter  promoter, results in a slightly rough and reduced pigment eye phenotype  <cit> .
in addition to the critical roles of dcp- <dig> and caspase  <dig> in apoptosis, recent studies in mammals and flies suggest that these caspases have many important non-apoptotic roles  <cit> , although how these caspases act in these non-apoptotic responses are incompletely understood.
we hypothesized the existence of unknown effectors for dcp- <dig>  identification and characterization of such proteins would allow us to better understand apoptotic pathways and dcp-1-related non-apoptotic pathways.
since a large-scale genetic screen to identify components of the dcp- <dig> pathway had not been preformed, either in vivo or in vitro, we screened ~ <dig>  genexel ep fly lines.
interestingly, we noticed that autophagy-related genes specifically suppressed the rough eye phenotype caused by dcp- <dig> expression.
in addition to eight autophagy genes and two genes reported to be related to autophagy, we identified five insulin/igf and tor signaling genes, six map kinase and jun n-terminal kinase  signaling, four ecdysone genes, and others .
there were several interesting novel genes among the  <dig> dcp- <dig> genetic interactors.
the identification of many new dcp-1-interacting genes will help clarify the molecular mechanisms by which dcp- <dig> regulates apoptosis and other non-apoptotic cellular processes.
in addition, our findings that autophagy genes influence the roles of the dcp- <dig> caspase in autophagy suggest a relationship between autophagy and apoptosis.
finally, our findings that signaling pathways such as map kinase, jnk, and ecdysone signaling regulate dcp- <dig> indicate that there are other regulatory pathways for caspase functions.
RESULTS
phenotypes caused by ectopic expression of drosophila caspases
to identify genes that interact with fly dcp- <dig>  we first generated transgenic flies that over-express full-length dcp- <dig> using the upstream activation sequence /gal <dig> system.
we obtained  <dig> stable lines by microinjecting the uas-dcp- <dig> construct in w <dig> embryos.
forty out of  <dig> fly lines carrying this construct showed a small, slightly rough and reduced eye pigment phenotype when expressed with the eye-specific gmr-gal <dig> drivers .
among the uas-dcp- <dig> lines we generated, we chose to use uas-dcp-119- <dig> flies in this study for several reasons: first, we found that these flies did not exhibit a lethal phenotype.
second, the uas-dcp- <dig> was inserted between gsts <dig> and cg <dig> without disrupting any flanking genes .
we found that in some other lines the uas-dcp- <dig> construct was inserted into exon regions of genes .
in addition, some transgenic flies showed a male pupal lethal phenotype when induced by gmr-gal <dig> .
lastly, since the gmr-gal4/uas-dcp-119- <dig> flies displayed a modest rough eye phenotype, we reasoned that this fly line was suitable for identifying both suppressors and enhancers of dcp- <dig> from our modifier screen.
the eye phenotype that we observed for the gmr-gal <dig>  uas-dcp- <dig> flies was similar to a previous study in which flies that express one copy each of gmr-Δn-dcp- <dig> and gmr-fl-dcp- <dig>  showed a faintly-colored and ablated eye phenotype  <cit> .
the intermediate phenotype caused by one copy of gmr-gal <dig> and one copy of uas-dcp- <dig> makes these flies very useful for screening for modifiers .
flies carrying two copies of gmr-gal <dig> and uas-dcp- <dig> displayed a much more severe eye phenotype, in general agreement with the phenotype from a previous study using two copies of gmr-Δn-dcp- <dig> .
these flies were semi-lethal and had a short life span.
as positive controls for the suppressors of dcp- <dig> over-expressing flies, we expressed caspase inhibitor p <dig> and drosophila inhibitor of apoptosis .
the eye phenotype caused by gmr-gal <dig>  uas-dcp-119- <dig> ; gmr-gal <dig> and uas-dcp-119- <dig> are linked on one chromosome) was completely rescued by the expression of the caspase inhibitors .
dcp- <dig> modifier screen
we screened ~ <dig>  ep lines obtained from the szeged stock center, genexel, and cell signaling pathway gene lines from the indiana university bloomington stock center and dr. j. chung's laboratory .
among them,  <dig> transgenic flies showed suppression or enhancement of the rough eye phenotype caused by the dcp- <dig> gf.
false positives were excluded by comparing these lines with the lines induced by gmr-gal4- lines without uas-dcp- <dig>  we confirmed that  <dig> alleles corresponding to  <dig> genes showed specific genetic interaction with dcp- <dig> .
in addition, we verified that the gal <dig> mrna expression was constant even when dcp- <dig> gf was crossed with representative alleles, atg1ep and atg6ep .
we also confirmed that ep insertions induced the genes placed down-stream by the gmr-gal <dig> while not affecting the expression of flanking genes placed at a distance: the expression level of cg <dig>  down-stream gene of atg <dig>  was not affected by ep inserted up-stream of atg <dig> gene  and the expression level of tango <dig>  down-stream gene of atg8a, was not affected by ep inserted up-stream of atg8a gene .
apoptotic cell death pathway
we identified three genes that are known to regulate apoptotic cell death.
expression of diap <dig> suppressed the eye phenotype, whereas ep lines of effete  and faf enhanced the rough eye phenotype .
these results are consistent with the previous reports that diap <dig> is a caspase inhibitor and that eff and faf enhance apoptotic cell death  <cit> .
the fact that we recovered apoptosis-modulating genes with the expected direction shows the efficacy of our genetic screen.
autophagy-related genes
three autophagy-related genes, aut <dig>  snf4/amp-activated protein kinase γ subunit , and blue cheese , were identified as genetic modifiers from unbiased screening: expression of aut <dig> and snf4aγ suppressed the rough eye phenotype caused by dcp- <dig> expression, whereas bchs expression enhanced this phenotype, .
aut <dig> is an important regulator of autophagy that is required for modification of atg <dig>  in a ubiquitin conjugation-like manner  <cit> .
aut <dig> loss of function mutant larvae fail to induce autophagy in the fat body before puparium stage and die during metamorphosis  <cit> .
recently, snf4aγ function in autophagosome formation during larval metamorphosis was revealed through a combination of drosophila mutational and rnai studies  <cit> .
we found that eye-specific expression of snf4aγ suppressed the rough eye phenotype in the dcp- <dig> gf flies .
it has been shown that snf4aγ induces developmental and stress-mediated autophagy  <cit> .
thus, our data suggest that autophagy induced by snf4aγ over-expression negatively modulates the dcp-1-mediated cell death phenotype.
bchs is a drosophila homolog of human alfy , which may serve as a scaffold protein to promote autophagosome-related vesicle trafficking to lysosomes  <cit> .
since we identified three autophagy-related genes from our primary screen and since ep lines of aut <dig>  snf4aγ, and bchs resulted in opposite phenotypes, we examined the ep lines for other autophagy-specific genes, including atg <dig>  atg <dig>  atg <dig>  atg <dig>  atg <dig>  atg8a, and atg <dig> .
we found that  <dig> of  <dig> ep alleles for autophagy-specific genes partially recovered the disordered ommatidia and reduced eye pigment that was caused by dcp- <dig> gf, as shown by introduction of atg2ep, atg6ep, and atg1ep .
atg <dig>  whose activity is regulated by tor , initiates the induction of autophagy.
atg <dig> is important for retrieval of autophagic proteins and vesicles by interacting with membrane protein atg <dig> during autophagosome formation.
atg <dig> plays a role in nucleation of the autophagic vesicles by formation of class iii pi3k complexes  <cit> .
the general process of autophagy is considered to be a survival mechanism against cell death  <cit> .
these data suggest that important components of autophagy generally counteract the cell death caused by dcp-1over-expression.
consistent with this idea, we found that a bchsep line can suppress the rough eye phenotype in an allele-specific manner .
genetic interaction between the insulin-igf pathway or tor pathway and dcp-1
in our screen, we identified a set of genes in the insulin-igf signaling pathway, including inr , pi3k, akt <dig>  s6k , tor , and pten .
insulin-igf signaling is initiated by the activation of inr, which leads to the activation of a downstream kinase cascade including pi3k and akt <dig>  on the other hand, pten phosphatase inhibits this signaling by dephosphorylating 3-phosphoinositides ps), the product of pi3k  <cit> .
tor is an important regulator of nutrient responses, cellular growth, and protein synthesis  <cit> .
tor regulates cellular nutrients by insulin or the amp: atp ratio.
tor is activated by the insulin/igf signaling through the igf, akt <dig>  tsc1/ <dig> and rheb or by the lkb <dig> and ampk signaling pathway through lkb <dig>  ampk, tsc1/ <dig> and rheb.
the signals from these two pathways converge on tsc1/ <dig>  rheb, and tor  <cit> .
one of the important downstream targets of tor is s6k, which is an important regulator of protein translation  <cit> .
we found that the rough eye phenotype caused by dcp- <dig> gf was exacerbated by over-expression of inr, pi3k, akt <dig>  pten, or tor .
the tor hypomorphic mutant tork <dig>  which shows a more moderate phenotype than tor null mutant torΔp  <cit> , recovered the dcp- <dig> gf phenotype .
we also identified several genes in the tor pathway from our screen .
in addition to the main role of s6k in the regulation of cell size  <cit> , recent reports showed that s6k mediates many other physological processes, such as larval feeding behaviour  <cit> , adult lifespan  <cit> , and autophagy  <cit> .
the progeny from the cross of dcp- <dig> gf with the uas-ds6k showed a suppressed rough eye phenotype .
on the other hand, pten is known to antagonize pi3k signaling.
however, our data showed that the eye phenotype of dcp- <dig> gf was exacerbated by co-expression of uas-ptenff <dig> , the line that over-expresses wild type pten.
although this result seems to contradict the enhancement of the rough eye phenotype caused by pi3k expression, recent studies have shown that pten over-expression induces apoptosis in a cell context-dependent manner even though pten functions to alleviate the effect of insulin/igf signaling  <cit> .
thus, it is possible that pten may reinforce the apoptotic effect of dcp- <dig> by acting in some other signaling pathways that are distinct from the insulin pi3k signaling and may worsen the rough eye phenotype caused by dcp- <dig> gf.
overall, nine autophagy-specific genes as well as tor, which is a well-known regulator of autophagy, modified dcp- <dig> gf.
the identification of these genes as rescuers of the dcp- <dig> phenotype raised questions about the effect of autophagy genes on dcp- <dig> caspase function.
thus, we performed caspase assays using the autophagy strains that over-expressed dcp- <dig>  we confirmed that over-expression of dcp- <dig> strongly increased caspase activity in dcp- <dig> gf animals .
the increased caspase activity induced by dcp- <dig> over-expression was reduced by the expression of autophagy genes in the co-heterozygotic lines with atg <dig>  tork <dig>  atg <dig>  ds6k, and atg <dig> .
the mapk and jnk pathway
the jnk pathway is a well known signaling cascade that regulates apoptosis.
we found that the rough eye phenotype of dcp- <dig> gf was also severely affected by the expression of many components in the mapk and jnk pathways, such as tak <dig> , mekk <dig> , hep, aop , dominant negative form of bsk , and mkp .
the progeny of the co-heterozygotic lines of the mapk and jnk pathway genes with dcp- <dig> gf showed extensive, although not complete, lethality.
in rare cases, a few progeny reached adulthood although they displayed severe rough eye phenotypes and shortened life span .
our finding that expression of tak <dig>  mekk <dig>  hep, or aop enhanced the rough eye phenotype is consistent with the notion that the jnk pathway positively regulates apoptosis.
paradoxically, we found that expression of the mkp, a negative regulator of jnk, or the dominant negative form of bsk  enhanced apoptosis in the eye, as well.
these data suggest that the interaction between dcp-1-induced apoptosis and the jnk pathway is not simple but complicated .
the ecdysone pathway
we identified the ep lines of four ecdysone-regulated genes,eip74ef , eip78c , broad , or eip55e  from our screen.
ecdysone is a hormone that regulates the metamorphosis of insects and the ecdysone hormonal signaling is known to induce apoptotic and autophagic cell death during metamorphosis  <cit> .
the co-heterozygote of eip74efep in the dcp- <dig> background enhanced the dcp- <dig> gf eye phenotype .
the eip78cep progeny showed a phenotype similar to eip74ef when they were crossed with dcp- <dig> gf .
in contrast, when crossed to one of the eip55e alleles, eip55eep, dcp- <dig> gf phenotype was suppressed .
the co-heterozygote of one of the br alleles, brep, with dcp- <dig> gf rescued the eye phenotype almost completely , while another allele, brep, showed an enhanced phenotype .
we noticed that eip55eep, and brep enhanced the rough eye phenotype of dcp- <dig> gf whereas eip55eep and brep significantly suppressed the rough eye phenotype.
one possibility is that the insertion positions of ep lines may have caused opposite phenotypes.
consistent with this idea, we found that eip55eep and brep have insertions in the exon region of eip55e and the first intron of br, respectively  suggesting that these ep elements may reduce the function of the corresponding genes and suppress the cell death phenotype.
relationship between autophagy and dcp-1-induced apoptosis
our initial characterization of the dcp-1-modifier screen suggested that autophagy specifically interacts with dcp-1-induced apoptosis.
first, we identified many genes directly involved in autophagy.
second, we identified several components in the insulin/igf-1/tor pathway, which has been shown to regulate autophagy.
third, the involvement of ecdysone signaling in autophagic cell death is well documented and we identified several ecdysone-induced proteins in our screen.
these findings led us to contemplate the relationship between autophagy and dcp- <dig>
to verify our finding that autophagy suppresses dcp- <dig>  we over-expressed egfp fused to atg <dig>  and the dcp- <dig> in the eye imaginal disc using the uas/gal <dig> system.
indeed, we found that the eye phenotype was suppressed by egfp-atg <dig> over-expression .
intriguingly, we noticed that the egfp-atg <dig> level was increased in the dcp- <dig> gf animals  compared with control animals , suggesting that dcp- <dig> over-expression may induce autophagic genes and autophagy.
we tested this possibility using lysotracker red staining, which is an autophagy marker.
the level of lysotracker red was higher in the uas-egfp-atg5/dcp- <dig> gf animals  than in the uas-egfp-atg <dig> control animals .
together, these data suggest that there exists an interaction between autophagy and dcp-1-induced apoptosis: autophagy suppresses dcp-1-induced cell death, whereas dcp- <dig> induces autophagy.
to further demonstrate the dcp-1-regulated autophagic gene induction, we used uas-atg8b-gfp as an autophagy marker in the dcp- <dig> gf animals.
similar to atg <dig>  atg8b-gfp was induced in these animals , even though atg8b-gfp over-expression did not suppress the rough eye phenotype .
these data suggest that the dcp- <dig> may be able to increase autophagic gene expression even when autophagy does not suppress dcp-1-induced apoptosis.
next, we confirmed these results using the drosophila s <dig> cell culture system.
we transfected s <dig> cells with the atg8b-gfp construct with or without full-length dcp- <dig> and monitored the pattern of gfp fluorescence to observe the induction of autophagy.
as a positive control, we starved s <dig> cells and found that starvation, which is a known cause of autophagy, induced atg8b-gfp punctate expression .
in cells co-transfected with atg8b-gfp and full-length dcp- <dig>  punctate gfp expression appeared even under replete conditions , suggesting that dcp- <dig> increased autophagy in s <dig> cells.
taken together, our data imply that increased autophagy suppresses dcp-1-induced apoptosis and that dcp- <dig> in turn positively regulates autophagy through a feedback regulation.
discussion
in this study, we identified potentially important genes that interact with fly caspase dcp- <dig> through a large-scale in vivo screen.
we used a gal4-induced dcp-1-over-expressing strain to screen for modifiers of the caspase pathway in drosophila.
recently, various caspase substrates or effectors were identified through a genome-wide microarray analysis using diap <dig> knockdown, a caspase inhibitor, and anti-caspase rnai in drosophila embryo-derived kc cells  <cit> .
to the best of our knowledge, the present study represents the first large-scale in vivo screen with a drosophila caspase.
moreover, we used a full-length form of dcp- <dig>  which has the advantage of mimicking the pre-apoptotic state.
as expected, we identified both enhancer and suppressor genes that exacerbated and ameliorated apoptosis, respectively.
moreover, our genetic screen revealed that the phenotype caused by caspase dcp- <dig> is suppressed by autophagy gene expression and influenced by genes from other pathways.
we showed that dcp- <dig> gf was rescued by the expression of autophagy genes, s6k, ecdysone-inducible genes, such as eip55e and broad, and hypomorphic allele of tor.
our data indicate that dcp- <dig> activity is negatively controlled by autophagy.
relationship between dcp- <dig> and autophagy
while we found that over-expression of autophagy genes suppressed dcp-1-induced apoptosis, we also observed that expression of full-length dcp- <dig> induced autophagy in eye imaginal disc and s <dig> cell.
this autophagy induction caused by full-length dcp- <dig> expression was very similar to the one induced by starvation in s <dig> cell.
it has been shown that rnai knock down of dcp- <dig> reduces autophagy in tumorous larval hemocytes-derived lmbn cells during starvation, indicating that dcp- <dig> is required for autophagy regulation  <cit> .
in addition, the dcp-1prev reduction-of-function mutation has been shown to reduce autophagy in region two germaria and stage  <dig> degenerating egg chambers during drosophila oogenesis  <cit> .
autophagic cell death has also been shown to be associated with the transcriptional up-regulation of pro-apoptotic genes, including dcp- <dig>  in salivary glands and the mid-gut  <cit> .
these results are consistent with our observations that autophagy signals were found in the dcp-1-expressing eye discs and in the full-length dcp- <dig> transfected s <dig> cells.
our findings showed that the dcp- <dig> gf phenotype was rescued by autophagy and that autophagy was in turn induced by dcp- <dig> expression.
this seemingly paradoxical relationship between apoptosis and autophagy is not unprecedented.
it has been shown that the autophagy gene, atg <dig>  also plays important roles in the interplay between autophagy and apoptosis.
the full length atg <dig> protein is an essential component for autophagy induction.
however, when atg <dig> is cleaved by calpain, it loses the ability to induce autophagy.
instead, the truncated atg <dig> interacts with bcl-xl, is translocated to mitochondria, and causes apoptosis  <cit> .
interestingly, we found that full-length dcp- <dig> could also induce autophagy, whereas the truncated active dcp- <dig> resulted in apoptosis.
thus, our results suggest that dcp- <dig> may act as a key protein in the regulation of not only apoptosis but also autophagy, similar to atg <dig>
it remains to be determined how full-length dcp- <dig> induces autophagy.
it is tempting to speculate that full-length dcp- <dig>  which is a less active form, causes autophagy as a defensive mechanism at a very early stage in response to cellular damages.
when the cellular damages reach the threshold level for apoptosis, dcp- <dig> is cleaved to its active form and facilitates full-fledged apoptosis.
consistent with this idea, previous studies have shown that autophagy delays apoptosis, perhaps by eliminating unwanted or damaged molecules, to establish cellular homeostasis at the initial stages of cisplatin injury  <cit> .
further studies are needed to test the interaction between dcp- <dig> and autophagy proteins and to elucidate the molecular mechanisms linking dcp- <dig> and autophagy.
other signaling pathways that interact with dcp-1
our genetic screening of ep lines and their interactions with other candidate lines allowed us to identify genetic pathways other than the classical apoptotic pathway.
the major signal that triggers entry into metamorphosis and the activation of autophagic cell death in drosophila is a high-titer pulse of ecdysone  that occurs during puparium formation  <cit> .
the ecdysone receptor complex and the nuclear receptor competence factor ftz-f <dig> lead to salivary gland death through the transcriptional activation of a set of "early" gene transcription factors, br, eip74ef, and eip93f, which in turn regulate the expression of "late" effector genes that appear to function more directly in apoptosis  <cit> .
mutations in each of ftz-f <dig>  br, eip74ef, and eip93f impair salivary gland degeneration during metamorphosis at different stages in the cell death process.
eip93f seems important in autophagic induction because mutations in this gene produce an early block in the formation of autophagic vacuoles in the salivary gland and midgut cells  <cit> .
in conclusion, apoptotic and autophagic cell deaths during drosophila metamorphosis seem to be either combined or sequential, depending on various factors.
serial analysis of gene expression  and microarray analyses have revealed that eip93f is required for the transcriptional up-regulation of genes that are activated in dying salivary glands, including genes involved in autophagy, apoptosis, non-caspase proteolysis, and cytoskeletal remodelling  <cit> .
a gmr-hid modifier screen and hs-hid suppression assay showed that hid is inactivated by the erk/mapk pathway  <cit> .
even though our data differ from previous findings that dcp- <dig> is down-regulated by reaper and grim but not by hid  <cit> , our screen data uncover a relationship between dcp- <dig> and hid, as well as between dcp- <dig> and mapk.
co-expression of gmr-hid and gmr-full-length dcp- <dig> line leads to a slight increase in eye roughness and pigment loss  <cit> .
a similar phenomenon was observed in the present study with our dcp- <dig> gf/cyo line .
these results suggest that hid affects dcp- <dig>  albeit not as strongly as grim and reaper.
the pro-apoptotic protein hid may also have a role in autophagic induction  <cit> .
dcp- <dig> activity was shown to be regulated by diap <dig> and hid  <cit> .
these reports suggest that autophagic cell death in organs, which undergo metamorphosis, is regulated by ecdysone, eip93f, and pro-apoptotic proteins.
thus, we propose that dcp- <dig> and hid regulate autophagy and that dcp- <dig> may regulate autophagy by receiving a signal from hid through a non-apoptotic pathway.
opposite phenotypes and allelic differences
some of the transgenic lines that we tested in this study showed somewhat unexpected phenotypes.
for example, while the enhancement of the rough eye phenotype by co-expression of tak <dig>  mekk <dig>  hep, or aop is consistent with the notion that the jnk pathway positively regulates apoptosis, the expression of mkp, a negative regulator of jnk, or the dominant negative form of bsk  also enhanced the eye phenotype.
likewise, we found that various reduction-of-function mutations of tor either enhanced or suppressed the rough eye phenotype .
both the jnk pathway and the tor pathway have been shown to affect apoptosis and autophagy  <cit> .
therefore, we speculate that the different expression levels of these signaling pathway genes may lead to survival or death of the cells by differentially affecting autophagy and apoptosis.
for example, a mild increase in autophagy may lead to cell survival by suppressing the apoptosis caused by dcp- <dig> over-expression, whereas strong induction of autophagy may worsen the cell death phenotype through the combination of apoptotic and autophagic cell deaths  <cit> .
consistent with this idea, whereas atg <dig> is essential for autophagy, strong induction of atg <dig> has been shown to result in cell death  <cit> .
thus, it is possible that different levels of autophagic induction in the flies mentioned above may explain the seemingly unexpected phenotypes.
also, we noticed that the bchs alleles  <dig>  g <dig>  and g13044) enhanced the dcp- <dig> gf severely.
however, the bchsep line can suppress the rough eye phenotype .
therefore, we further examined the insertion positions of these ep lines.
bchsep, bchsep, bchsep were inserted at  <dig>   <dig>  and  <dig> base pairs upstream from the start codon, respectively .
we speculate that perhaps the insertion positions determined the expression level of bchs and affected the rough eye phenotype differently.
CONCLUSIONS
in this study, we aimed to identify genes that modify dcp- <dig> function in a large-scale genetic screen.
we demonstrate that the effector caspase dcp- <dig> regulates and/or is regulated by autophagy, ecdysone signals, ubiquitination signals, jnk, mapk, and various transcription factors and cell death signals.
our genetic screen provides a wealth of information on various genes and pathways that may regulate caspase functions for future research.
furthermore, our findings on the integrated regulation between autophagy and dcp- <dig> could aid the elucidation of molecular mechanisms connecting autophagy and apoptosis.

 apoptosis, or programmed cell death, is an evolutionarily conserved, genetically regulated process, whereby cells that are no longer needed undergo self-destruction through the activation of a cell suicide program. this cell death program is associated with characteristic morphological alterations, such as condensation of the nucleus and cytoplasm, fragmentation of nuclear dna, reorganization of the cytoskeleton, and reduction of the cell into apoptotic bodies that can be phagocytosed by neighbouring epithelial cells or phagocytes. autophagy is also an evolutionarily conserved mechanism that degrades unnecessary long-lived proteins and organelles. during autophagy, cellular components are sequestered by double-membrane structures called autophagosomes. these autophagosomes then fuse with lysosomes to form autolysosomes, where degradation occurs. the autophagy acts as a cellular response against extracellular stresses, such as nutrient starvation, hypoxia, and overcrowding and against intracellar stresses, such as formation of damaged or redundant organelles and cytoplasmic components. even though autophagy can induce a cell-survival response to some conditions, autophagic structures, especially the autophagic vacuoles, are associated with cell death. this cell death phenomenon is classified as type ii cell death and called autophagic cell death. in drosophila, loss of function of the atg genes leads to lethality in the transition from the larval to pupal stages, because autophagic cell death is essential for puparium formation. the caspases are a family of ubiquitously expressed cysteine proteases whose prototypic member is the caenorhabditis elegans death effector, ced-. activation of caspases typically leads to the selective cleavage of a restricted set of target proteins, generally resulting in inactivation of the target proteins. normally present in cells as inactive precursors, caspases are proteolytically activated following upstream pro-apoptotic signals. activated caspases cleave their substrates at an aspartic acid residue, and substrate specificity is determined by a four-residue motif n-terminal to the cleavage site. the "initiator" caspases primarily activate the downstream "effector" caspases whose proteolytic activity is directed toward the deconstruction of the cellular machinery during apoptosis. in drosophila, a normally functioning apoptotic pathway depends critically on caspases, seven of which have been identified in the drosophila melanogaster genome. these drosophila caspase genes include three initiators and four effectors. the initiators are dronc, dredd, and strica/dream. the effectors are dcp-, drice, damm, and decay. these caspases are expected to have functions that lead to apoptosis. many recent reports have described non-apoptotic functions of caspases, such as the cell proliferation function of dronc, spermatid individualization by drice, and activation of the drosophila immune system, toll receptor signaling, by dredd. dcp- proteins cleave cysteine protease substrates and are important for development and oogenesis. dcp- deletion mutants display a lack of germline cell death phenotype during mid-stage oogenesis in response to nutrient deprivation, whereas in normal flies, cell death occurs during mid-stage oogenesis under nutrient-deprived conditions. in contrast, over-expression of a single copy of the truncated n-terminal region of dcp-, specifically in the eye using the glass multimer reporter promoter, results in a slightly rough and reduced pigment eye phenotype. in addition to the critical roles of dcp- and caspase in apoptosis, recent studies in mammals and flies suggest that these caspases have many important non-apoptotic roles, although how these caspases act in these non-apoptotic responses are incompletely understood. we hypothesized the existence of unknown effectors for dcp- identification and characterization of such proteins would allow us to better understand apoptotic pathways and dcp-1-related non-apoptotic pathways. since a large-scale genetic screen to identify components of the dcp- pathway had not been preformed, either in vivo or in vitro, we screened ~ genexel ep fly lines. interestingly, we noticed that autophagy-related genes specifically suppressed the rough eye phenotype caused by dcp- expression. in addition to eight autophagy genes and two genes reported to be related to autophagy, we identified five insulin/igf and tor signaling genes, six map kinase and jun n-terminal kinase signaling, four ecdysone genes, and others. there were several interesting novel genes among the dcp- genetic interactors. the identification of many new dcp-1-interacting genes will help clarify the molecular mechanisms by which dcp- regulates apoptosis and other non-apoptotic cellular processes. in addition, our findings that autophagy genes influence the roles of the dcp- caspase in autophagy suggest a relationship between autophagy and apoptosis. finally, our findings that signaling pathways such as map kinase, jnk, and ecdysone signaling regulate dcp- indicate that there are other regulatory pathways for caspase functions.  phenotypes caused by ectopic expression of drosophila caspases to identify genes that interact with fly dcp- we first generated transgenic flies that over-express full-length dcp- using the upstream activation sequence /gal system. we obtained stable lines by microinjecting the uas-dcp- construct in w embryos. forty out of fly lines carrying this construct showed a small, slightly rough and reduced eye pigment phenotype when expressed with the eye-specific gmr-gal drivers. among the uas-dcp- lines we generated, we chose to use uas-dcp-119- flies in this study for several reasons: first, we found that these flies did not exhibit a lethal phenotype. second, the uas-dcp- was inserted between gsts and cg without disrupting any flanking genes. we found that in some other lines the uas-dcp- construct was inserted into exon regions of genes. in addition, some transgenic flies showed a male pupal lethal phenotype when induced by gmr-gal. lastly, since the gmr-gal4/uas-dcp-119- flies displayed a modest rough eye phenotype, we reasoned that this fly line was suitable for identifying both suppressors and enhancers of dcp- from our modifier screen. the eye phenotype that we observed for the gmr-gal uas-dcp- flies was similar to a previous study in which flies that express one copy each of gmr-Δn-dcp- and gmr-fl-dcp- showed a faintly-colored and ablated eye phenotype. the intermediate phenotype caused by one copy of gmr-gal and one copy of uas-dcp- makes these flies very useful for screening for modifiers. flies carrying two copies of gmr-gal and uas-dcp- displayed a much more severe eye phenotype, in general agreement with the phenotype from a previous study using two copies of gmr-Δn-dcp-. these flies were semi-lethal and had a short life span. as positive controls for the suppressors of dcp- over-expressing flies, we expressed caspase inhibitor p and drosophila inhibitor of apoptosis. the eye phenotype caused by gmr-gal uas-dcp-119-; gmr-gal and uas-dcp-119- are linked on one chromosome) was completely rescued by the expression of the caspase inhibitors. dcp- modifier screen we screened ~ ep lines obtained from the szeged stock center, genexel, and cell signaling pathway gene lines from the indiana university bloomington stock center and dr. j. chung's laboratory. among them, transgenic flies showed suppression or enhancement of the rough eye phenotype caused by the dcp- gf. false positives were excluded by comparing these lines with the lines induced by gmr-gal4- lines without uas-dcp- we confirmed that alleles corresponding to genes showed specific genetic interaction with dcp-. in addition, we verified that the gal mrna expression was constant even when dcp- gf was crossed with representative alleles, atg1ep and atg6ep. we also confirmed that ep insertions induced the genes placed down-stream by the gmr-gal while not affecting the expression of flanking genes placed at a distance: the expression level of cg down-stream gene of atg was not affected by ep inserted up-stream of atg gene and the expression level of tango down-stream gene of atg8a, was not affected by ep inserted up-stream of atg8a gene. apoptotic cell death pathway we identified three genes that are known to regulate apoptotic cell death. expression of diap suppressed the eye phenotype, whereas ep lines of effete and faf enhanced the rough eye phenotype. these results are consistent with the previous reports that diap is a caspase inhibitor and that eff and faf enhance apoptotic cell death. the fact that we recovered apoptosis-modulating genes with the expected direction shows the efficacy of our genetic screen. autophagy-related genes three autophagy-related genes, aut snf4/amp-activated protein kinase γ subunit, and blue cheese, were identified as genetic modifiers from unbiased screening: expression of aut and snf4aγ suppressed the rough eye phenotype caused by dcp- expression, whereas bchs expression enhanced this phenotype,. aut is an important regulator of autophagy that is required for modification of atg in a ubiquitin conjugation-like manner. aut loss of function mutant larvae fail to induce autophagy in the fat body before puparium stage and die during metamorphosis. recently, snf4aγ function in autophagosome formation during larval metamorphosis was revealed through a combination of drosophila mutational and rnai studies. we found that eye-specific expression of snf4aγ suppressed the rough eye phenotype in the dcp- gf flies. it has been shown that snf4aγ induces developmental and stress-mediated autophagy. thus, our data suggest that autophagy induced by snf4aγ over-expression negatively modulates the dcp-1-mediated cell death phenotype. bchs is a drosophila homolog of human alfy, which may serve as a scaffold protein to promote autophagosome-related vesicle trafficking to lysosomes. since we identified three autophagy-related genes from our primary screen and since ep lines of aut snf4aγ, and bchs resulted in opposite phenotypes, we examined the ep lines for other autophagy-specific genes, including atg atg atg atg atg atg8a, and atg. we found that of ep alleles for autophagy-specific genes partially recovered the disordered ommatidia and reduced eye pigment that was caused by dcp- gf, as shown by introduction of atg2ep, atg6ep, and atg1ep. atg whose activity is regulated by tor, initiates the induction of autophagy. atg is important for retrieval of autophagic proteins and vesicles by interacting with membrane protein atg during autophagosome formation. atg plays a role in nucleation of the autophagic vesicles by formation of class iii pi3k complexes. the general process of autophagy is considered to be a survival mechanism against cell death. these data suggest that important components of autophagy generally counteract the cell death caused by dcp-1over-expression. consistent with this idea, we found that a bchsep line can suppress the rough eye phenotype in an allele-specific manner. genetic interaction between the insulin-igf pathway or tor pathway and dcp-1 in our screen, we identified a set of genes in the insulin-igf signaling pathway, including inr, pi3k, akt s6k, tor, and pten. insulin-igf signaling is initiated by the activation of inr, which leads to the activation of a downstream kinase cascade including pi3k and akt on the other hand, pten phosphatase inhibits this signaling by dephosphorylating 3-phosphoinositides ps), the product of pi3k. tor is an important regulator of nutrient responses, cellular growth, and protein synthesis. tor regulates cellular nutrients by insulin or the amp: atp ratio. tor is activated by the insulin/igf signaling through the igf, akt tsc1/ and rheb or by the lkb and ampk signaling pathway through lkb ampk, tsc1/ and rheb. the signals from these two pathways converge on tsc1/ rheb, and tor. one of the important downstream targets of tor is s6k, which is an important regulator of protein translation. we found that the rough eye phenotype caused by dcp- gf was exacerbated by over-expression of inr, pi3k, akt pten, or tor. the tor hypomorphic mutant tork which shows a more moderate phenotype than tor null mutant torΔp, recovered the dcp- gf phenotype. we also identified several genes in the tor pathway from our screen. in addition to the main role of s6k in the regulation of cell size, recent reports showed that s6k mediates many other physological processes, such as larval feeding behaviour, adult lifespan, and autophagy. the progeny from the cross of dcp- gf with the uas-ds6k showed a suppressed rough eye phenotype. on the other hand, pten is known to antagonize pi3k signaling. however, our data showed that the eye phenotype of dcp- gf was exacerbated by co-expression of uas-ptenff, the line that over-expresses wild type pten. although this result seems to contradict the enhancement of the rough eye phenotype caused by pi3k expression, recent studies have shown that pten over-expression induces apoptosis in a cell context-dependent manner even though pten functions to alleviate the effect of insulin/igf signaling. thus, it is possible that pten may reinforce the apoptotic effect of dcp- by acting in some other signaling pathways that are distinct from the insulin pi3k signaling and may worsen the rough eye phenotype caused by dcp- gf. overall, nine autophagy-specific genes as well as tor, which is a well-known regulator of autophagy, modified dcp- gf. the identification of these genes as rescuers of the dcp- phenotype raised questions about the effect of autophagy genes on dcp- caspase function. thus, we performed caspase assays using the autophagy strains that over-expressed dcp- we confirmed that over-expression of dcp- strongly increased caspase activity in dcp- gf animals. the increased caspase activity induced by dcp- over-expression was reduced by the expression of autophagy genes in the co-heterozygotic lines with atg tork atg ds6k, and atg. the mapk and jnk pathway the jnk pathway is a well known signaling cascade that regulates apoptosis. we found that the rough eye phenotype of dcp- gf was also severely affected by the expression of many components in the mapk and jnk pathways, such as tak, mekk, hep, aop, dominant negative form of bsk, and mkp. the progeny of the co-heterozygotic lines of the mapk and jnk pathway genes with dcp- gf showed extensive, although not complete, lethality. in rare cases, a few progeny reached adulthood although they displayed severe rough eye phenotypes and shortened life span. our finding that expression of tak mekk hep, or aop enhanced the rough eye phenotype is consistent with the notion that the jnk pathway positively regulates apoptosis. paradoxically, we found that expression of the mkp, a negative regulator of jnk, or the dominant negative form of bsk enhanced apoptosis in the eye, as well. these data suggest that the interaction between dcp-1-induced apoptosis and the jnk pathway is not simple but complicated. the ecdysone pathway we identified the ep lines of four ecdysone-regulated genes,eip74ef, eip78c, broad, or eip55e from our screen. ecdysone is a hormone that regulates the metamorphosis of insects and the ecdysone hormonal signaling is known to induce apoptotic and autophagic cell death during metamorphosis. the co-heterozygote of eip74efep in the dcp- background enhanced the dcp- gf eye phenotype. the eip78cep progeny showed a phenotype similar to eip74ef when they were crossed with dcp- gf. in contrast, when crossed to one of the eip55e alleles, eip55eep, dcp- gf phenotype was suppressed. the co-heterozygote of one of the br alleles, brep, with dcp- gf rescued the eye phenotype almost completely, while another allele, brep, showed an enhanced phenotype. we noticed that eip55eep, and brep enhanced the rough eye phenotype of dcp- gf whereas eip55eep and brep significantly suppressed the rough eye phenotype. one possibility is that the insertion positions of ep lines may have caused opposite phenotypes. consistent with this idea, we found that eip55eep and brep have insertions in the exon region of eip55e and the first intron of br, respectively suggesting that these ep elements may reduce the function of the corresponding genes and suppress the cell death phenotype. relationship between autophagy and dcp-1-induced apoptosis our initial characterization of the dcp-1-modifier screen suggested that autophagy specifically interacts with dcp-1-induced apoptosis. first, we identified many genes directly involved in autophagy. second, we identified several components in the insulin/igf-1/tor pathway, which has been shown to regulate autophagy. third, the involvement of ecdysone signaling in autophagic cell death is well documented and we identified several ecdysone-induced proteins in our screen. these findings led us to contemplate the relationship between autophagy and dcp- to verify our finding that autophagy suppresses dcp- we over-expressed egfp fused to atg and the dcp- in the eye imaginal disc using the uas/gal system. indeed, we found that the eye phenotype was suppressed by egfp-atg over-expression. intriguingly, we noticed that the egfp-atg level was increased in the dcp- gf animals compared with control animals, suggesting that dcp- over-expression may induce autophagic genes and autophagy. we tested this possibility using lysotracker red staining, which is an autophagy marker. the level of lysotracker red was higher in the uas-egfp-atg5/dcp- gf animals than in the uas-egfp-atg control animals. together, these data suggest that there exists an interaction between autophagy and dcp-1-induced apoptosis: autophagy suppresses dcp-1-induced cell death, whereas dcp- induces autophagy. to further demonstrate the dcp-1-regulated autophagic gene induction, we used uas-atg8b-gfp as an autophagy marker in the dcp- gf animals. similar to atg atg8b-gfp was induced in these animals, even though atg8b-gfp over-expression did not suppress the rough eye phenotype. these data suggest that the dcp- may be able to increase autophagic gene expression even when autophagy does not suppress dcp-1-induced apoptosis. next, we confirmed these results using the drosophila s cell culture system. we transfected s cells with the atg8b-gfp construct with or without full-length dcp- and monitored the pattern of gfp fluorescence to observe the induction of autophagy. as a positive control, we starved s cells and found that starvation, which is a known cause of autophagy, induced atg8b-gfp punctate expression. in cells co-transfected with atg8b-gfp and full-length dcp- punctate gfp expression appeared even under replete conditions, suggesting that dcp- increased autophagy in s cells. taken together, our data imply that increased autophagy suppresses dcp-1-induced apoptosis and that dcp- in turn positively regulates autophagy through a feedback regulation. discussion in this study, we identified potentially important genes that interact with fly caspase dcp- through a large-scale in vivo screen. we used a gal4-induced dcp-1-over-expressing strain to screen for modifiers of the caspase pathway in drosophila. recently, various caspase substrates or effectors were identified through a genome-wide microarray analysis using diap knockdown, a caspase inhibitor, and anti-caspase rnai in drosophila embryo-derived kc cells. to the best of our knowledge, the present study represents the first large-scale in vivo screen with a drosophila caspase. moreover, we used a full-length form of dcp- which has the advantage of mimicking the pre-apoptotic state. as expected, we identified both enhancer and suppressor genes that exacerbated and ameliorated apoptosis, respectively. moreover, our genetic screen revealed that the phenotype caused by caspase dcp- is suppressed by autophagy gene expression and influenced by genes from other pathways. we showed that dcp- gf was rescued by the expression of autophagy genes, s6k, ecdysone-inducible genes, such as eip55e and broad, and hypomorphic allele of tor. our data indicate that dcp- activity is negatively controlled by autophagy. relationship between dcp- and autophagy while we found that over-expression of autophagy genes suppressed dcp-1-induced apoptosis, we also observed that expression of full-length dcp- induced autophagy in eye imaginal disc and s cell. this autophagy induction caused by full-length dcp- expression was very similar to the one induced by starvation in s cell. it has been shown that rnai knock down of dcp- reduces autophagy in tumorous larval hemocytes-derived lmbn cells during starvation, indicating that dcp- is required for autophagy regulation. in addition, the dcp-1prev reduction-of-function mutation has been shown to reduce autophagy in region two germaria and stage degenerating egg chambers during drosophila oogenesis. autophagic cell death has also been shown to be associated with the transcriptional up-regulation of pro-apoptotic genes, including dcp- in salivary glands and the mid-gut. these results are consistent with our observations that autophagy signals were found in the dcp-1-expressing eye discs and in the full-length dcp- transfected s cells. our findings showed that the dcp- gf phenotype was rescued by autophagy and that autophagy was in turn induced by dcp- expression. this seemingly paradoxical relationship between apoptosis and autophagy is not unprecedented. it has been shown that the autophagy gene, atg also plays important roles in the interplay between autophagy and apoptosis. the full length atg protein is an essential component for autophagy induction. however, when atg is cleaved by calpain, it loses the ability to induce autophagy. instead, the truncated atg interacts with bcl-xl, is translocated to mitochondria, and causes apoptosis. interestingly, we found that full-length dcp- could also induce autophagy, whereas the truncated active dcp- resulted in apoptosis. thus, our results suggest that dcp- may act as a key protein in the regulation of not only apoptosis but also autophagy, similar to atg it remains to be determined how full-length dcp- induces autophagy. it is tempting to speculate that full-length dcp- which is a less active form, causes autophagy as a defensive mechanism at a very early stage in response to cellular damages. when the cellular damages reach the threshold level for apoptosis, dcp- is cleaved to its active form and facilitates full-fledged apoptosis. consistent with this idea, previous studies have shown that autophagy delays apoptosis, perhaps by eliminating unwanted or damaged molecules, to establish cellular homeostasis at the initial stages of cisplatin injury. further studies are needed to test the interaction between dcp- and autophagy proteins and to elucidate the molecular mechanisms linking dcp- and autophagy. other signaling pathways that interact with dcp-1 our genetic screening of ep lines and their interactions with other candidate lines allowed us to identify genetic pathways other than the classical apoptotic pathway. the major signal that triggers entry into metamorphosis and the activation of autophagic cell death in drosophila is a high-titer pulse of ecdysone that occurs during puparium formation. the ecdysone receptor complex and the nuclear receptor competence factor ftz-f lead to salivary gland death through the transcriptional activation of a set of "early" gene transcription factors, br, eip74ef, and eip93f, which in turn regulate the expression of "late" effector genes that appear to function more directly in apoptosis. mutations in each of ftz-f br, eip74ef, and eip93f impair salivary gland degeneration during metamorphosis at different stages in the cell death process. eip93f seems important in autophagic induction because mutations in this gene produce an early block in the formation of autophagic vacuoles in the salivary gland and midgut cells. in conclusion, apoptotic and autophagic cell deaths during drosophila metamorphosis seem to be either combined or sequential, depending on various factors. serial analysis of gene expression and microarray analyses have revealed that eip93f is required for the transcriptional up-regulation of genes that are activated in dying salivary glands, including genes involved in autophagy, apoptosis, non-caspase proteolysis, and cytoskeletal remodelling. a gmr-hid modifier screen and hs-hid suppression assay showed that hid is inactivated by the erk/mapk pathway. even though our data differ from previous findings that dcp- is down-regulated by reaper and grim but not by hid, our screen data uncover a relationship between dcp- and hid, as well as between dcp- and mapk. co-expression of gmr-hid and gmr-full-length dcp- line leads to a slight increase in eye roughness and pigment loss. a similar phenomenon was observed in the present study with our dcp- gf/cyo line. these results suggest that hid affects dcp- albeit not as strongly as grim and reaper. the pro-apoptotic protein hid may also have a role in autophagic induction. dcp- activity was shown to be regulated by diap and hid. these reports suggest that autophagic cell death in organs, which undergo metamorphosis, is regulated by ecdysone, eip93f, and pro-apoptotic proteins. thus, we propose that dcp- and hid regulate autophagy and that dcp- may regulate autophagy by receiving a signal from hid through a non-apoptotic pathway. opposite phenotypes and allelic differences some of the transgenic lines that we tested in this study showed somewhat unexpected phenotypes. for example, while the enhancement of the rough eye phenotype by co-expression of tak mekk hep, or aop is consistent with the notion that the jnk pathway positively regulates apoptosis, the expression of mkp, a negative regulator of jnk, or the dominant negative form of bsk also enhanced the eye phenotype. likewise, we found that various reduction-of-function mutations of tor either enhanced or suppressed the rough eye phenotype. both the jnk pathway and the tor pathway have been shown to affect apoptosis and autophagy. therefore, we speculate that the different expression levels of these signaling pathway genes may lead to survival or death of the cells by differentially affecting autophagy and apoptosis. for example, a mild increase in autophagy may lead to cell survival by suppressing the apoptosis caused by dcp- over-expression, whereas strong induction of autophagy may worsen the cell death phenotype through the combination of apoptotic and autophagic cell deaths. consistent with this idea, whereas atg is essential for autophagy, strong induction of atg has been shown to result in cell death. thus, it is possible that different levels of autophagic induction in the flies mentioned above may explain the seemingly unexpected phenotypes. also, we noticed that the bchs alleles g and g13044) enhanced the dcp- gf severely. however, the bchsep line can suppress the rough eye phenotype. therefore, we further examined the insertion positions of these ep lines. bchsep, bchsep, bchsep were inserted at and base pairs upstream from the start codon, respectively. we speculate that perhaps the insertion positions determined the expression level of bchs and affected the rough eye phenotype differently.  in this study, we aimed to identify genes that modify dcp- function in a large-scale genetic screen. we demonstrate that the effector caspase dcp- regulates and/or is regulated by autophagy, ecdysone signals, ubiquitination signals, jnk, mapk, and various transcription factors and cell death signals. our genetic screen provides a wealth of information on various genes and pathways that may regulate caspase functions for future research. furthermore, our findings on the integrated regulation between autophagy and dcp- could aid the elucidation of molecular mechanisms connecting autophagy and apoptosis. 
caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts dcp-1-induced apoptosis. our results showing that dcp- and autophagy-related genes influence each other will aid future investigations of the complicated relationships between apoptosis and autophagy. recent reports have described non-apoptotic functions of caspases, including autophagy. taken together, these data suggest that autophagy suppresses dcp-1-mediated apoptotic cell death, whereas dcp- positively regulates autophagy, possibly through feedback regulation. consistent with this idea, we show that expression of egfp-atg rescued the eye phenotype caused by dcp- gf. we screened ~ ep lines and identified dcp-1-interacting genes that were classified into groups based on their functions and pathways: apoptosis signaling genes, autophagy genes, insulin/igf and tor signaling pathway genes, map kinase and jnk signaling pathway genes, ecdysone signaling genes, ubiquitination genes, various developmental signaling genes, transcription factors, translation factors, and other unclassified genes including functionally undefined genes. among them, insulin/igf and tor signaling pathway, map kinase and jnk signaling pathway, and ecdysone signaling are known to be involved in autophagy. we identified a number of dcp- modifiers that genetically interact with dcp-1-induced cell death.
 caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. recent reports have described non-apoptotic functions of caspases, including autophagy. in this report, we searched for novel modifiers of the phenotype of dcp- gain-of-function animals by screening promoter element- inserted drosophila melanogaster lines.  we screened ~ ep lines and identified dcp-1-interacting genes that were classified into groups based on their functions and pathways: apoptosis signaling genes, autophagy genes, insulin/igf and tor signaling pathway genes, map kinase and jnk signaling pathway genes, ecdysone signaling genes, ubiquitination genes, various developmental signaling genes, transcription factors, translation factors, and other unclassified genes including functionally undefined genes. among them, insulin/igf and tor signaling pathway, map kinase and jnk signaling pathway, and ecdysone signaling are known to be involved in autophagy. together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts dcp-1-induced apoptosis. consistent with this idea, we show that expression of egfp-atg rescued the eye phenotype caused by dcp- gf. paradoxically, we found that over-expression of full-length dcp- induced autophagy, as atg8b-gfp, an indicator of autophagy, was increased in the eye imaginal discs and in the s cell line. taken together, these data suggest that autophagy suppresses dcp-1-mediated apoptotic cell death, whereas dcp- positively regulates autophagy, possibly through feedback regulation.  we identified a number of dcp- modifiers that genetically interact with dcp-1-induced cell death. our results showing that dcp- and autophagy-related genes influence each other will aid future investigations of the complicated relationships between apoptosis and autophagy. 