Identification of a Drosophila melanogaster ICE/CED-3-related protease, drICE.

Cysteine proteases of the ICE/CED-3 family (caspases) are required for the execution of programmed cell death (PCD) in a wide range of multicellular organisms. Caspases are implicated in the execution of apoptosis in Drosophila melanogaster by the observation that expression of baculovirus p35, a caspase inhibitor, blocks cell death in vivo in Drosophila. We report here the identification and characterization of drICE, a D. melanogaster caspase. We show that overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli and that expression of an N-terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by rpr overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. We further show that drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro and that drICE is expressed at all the stages of Drosophila development at which PCD can be induced. Taken together, these results strongly argue that drICE is an apoptotic caspase that acts downstream of rpr. drICE is therefore the first unequivocal link between the molecular machinery of Drosophila cell death and the conserved machinery of Caenorhabditis elegans and vertebrates. Identification of drICE should facilitate the elucidation of upstream regulators and downstream targets of caspases by genetic screening.     

Relationship between growth arrest and autophagy in midgut programmed cell death in Drosophila

Autophagy has been implicated in both cell survival and programmed cell death (PCD), and this may explain the apparently complex role of this catabolic process in tumourigenesis. Our previous studies have shown that caspases have little influence on Drosophila larval midgut PCD, whereas inhibition of autophagy severely delays midgut removal. To assess upstream signals that regulate autophagy and larval midgut degradation, we have examined the requirement of growth signalling pathways. Inhibition of the class I phosphoinositide-3-kinase (PI3K) pathway prevents midgut growth, whereas ectopic PI3K and Ras signalling results in larger cells with decreased autophagy and delayed midgut degradation. Furthermore, premature induction of autophagy is sufficient to induce early midgut degradation. These data indicate that autophagy and the growth regulatory pathways have an important relationship during midgut PCD. Despite the roles of autophagy in both survival and death, our findings suggest that autophagy induction occurs in response to similar signals in both scenarios.     

Lepidopteran larval midgut during prepupal instar: digestion or self-digestion?

Programmed cell death (PCD) is crucial in body restructuring during metamorphosis of holometabolous insects (those that have a pupal stage between the final larval and adult stages). Besides apoptosis, an increasing body of evidence indicates that in several insect species programmed autophagy also plays a key role in these developmental processes. We have recently characterized the midgut replacement process in Heliothis virescens larva, during the prepupal phase, responsible for the formation of a new pupal midgut. We found that the elimination of the old larval midgut epithelium is obtained by a combination of apoptotic and autophagic events. In particular, autophagic PCD completely digests decaying tissues, and provides nutrients that are rapidly absorbed by the newly formed epithelium, which is apparently functional at this early stage. The presence of both apoptosis and autophagy in the replacement of midgut cells in Lepidoptera offers the opportunity to investigate the functional peculiarities of these PCD modalities and if they share any molecular mechanism, which may account for possible cross-talk between them.     

VDAC is a conserved element of death pathways in plant and animal systems

Programmed cell death (PCD) is very much a part of plant life, although the underlying mechanisms are not so well understood as in animals. In animal cells, the voltage-dependent anion channel (VDAC), a major mitochondrial outer membrane transporter, plays an important role in apoptosis by participating in the release of intermembrane space proteins. To characterize plant PCD pathways by investigating the function of putative components in a mammalian apoptotic context, we have overexpressed a rice VDAC (osVDAC4) in the Jurkat T-cell line. Overexpression of osVDAC4 induces apoptosis, which can be blocked by Bcl-2 and the VDAC inhibitor DIDS. Modifying endogenous VDAC function by DIDS and hexokinase II (HxKII) in Jurkat cells inhibits mitochondria-mediated apoptotic pathways. Finally, we show that DIDS also abrogates heat-induced PCD in cucumber cotyledons. Our data suggest that VDAC is a conserved mitochondrial element of the death machinery in both plant and animal cells.     

Molecular mechanisms of cell death and phagocytosis in Drosophila

The genetic tools available in Drosophila have facilitated our understanding of how apoptosis is regulated and executed in the context of the developing organism. All embryonic apoptosis is initiated by the activity of three genes, rpr, grim and hid. Each of these genes is independently regulated, allowing developmental apoptosis to be finely controlled. These initiators in turn activate the core apoptotic machinery, including the caspases. Drosophila counterparts to other conserved components of the apoptotic machinery have been recently identified, and we discuss how these may be integrated into the process of normal developmentally regulated cell death. We also outline the role that phagocytosis plays in the final stages of apoptosis and consider the molecular mechanisms guiding the elimination of apoptotic corpses.     

Cell death and human intestinal protozoa: a brief overview

Protozoan programmed cell death or apoptosis is an important factor in the survival of the parasite and its pathogenicity. The most amazing aspect of protozoan cell death is in its molecular architecture. To date, protozoa lack most of the components of the highly complex cell death machinery studied in multicellular organisms. Hence the unique apoptotic machinery in protozoa can be exploited for the development of therapeutic drugs and diagnostic markers. This review focuses on human intestinal protozoa undergoing cell death and inducing or inhibiting host cell apoptosis. The first part of this review focuses on intestinal protozoa that undergo PCD under various stress conditions. The second part focuses on protozoa that induce or inhibit PCD in their host cell. Although these intestinal parasites differ in their mechanism of infection and intracellular localization, they may activate conserved cell death pathways within themselves and in the host cell. Understanding conserved cell death pathways in the intestinal protozoa and their host-parasite PCD relationship may lead to drug targets which can be used for a broad range of parasitic diseases.     

Yeast programmed cell death: an intricate puzzle

Yeasts as eukaryotic microorganisms with simple, well known and tractable genetics, have long been powerful model systems for studying complex biological phenomena such as the cell cycle or vesicle fusion. Until recently, yeast has been assumed as a cellular 'clean room' to study the interactions and the mechanisms of action of mammalian apoptotic regulators. However, the finding of an endogenous programmed cell death (PCD) process in yeast with an apoptotic phenotype has turned yeast into an 'unclean' but even more powerful model for apoptosis research. Yeast cells appear to possess an endogenous apoptotic machinery including its own regulators and pathway(s). Such machinery may not exactly recapitulate that of mammalian systems but it represents a simple and valuable model which will assist in the future understanding of the complex connections between apoptotic and non-apoptotic mammalian PCD pathways. Following this line of thought and in order to validate and make the most of this promising cell death model, researchers must undoubtedly address the following issues: what are the crucial yeast PCD regulators? How do they play together? What are the cell death pathways shared by yeast and mammalian PCD? Solving these questions is currently the most pressing challenge for yeast cell death researchers.     

Lepidopteran Larval Midgut During Prepupal Instar: Digestion or Self-Digestion?

Programmed cell death (PCD) is crucial in body restructuring during metamorphosis of holometabolous insects (those that have a pupal stage between the final larval and adult stages). Besides apoptosis, an increasing body of evidence indicates that in several insect species programmed autophagy also plays a key role in these developmental processes. We have recently characterized the midgut replacement process in Heliothis virescens larva, during the prepupal phase, responsible for the formation of a new pupal midgut. We found that the elimination of the old larval midgut epithelium is obtained by a combination of apoptotic and autophagic events. In particular, autophagic PCD completely digests decaying tissues, and provides nutrients that are rapidly absorbed by the newly formed epithelium, which is apparently functional at this early stage. The presence of both apoptosis and autophagy in the replacement of midgut cells in Lepidoptera offers the opportunity to investigate the functional peculiarities of these PCD modalities and if they share any molecular mechanism, which may account for possible cross-talk between them.

Addendum to:

Programmed Cell Death and Stem Cell Differentiation are Responsible for Midgut Replacement in Heliothis virescens During Prepupal Instar

G. Tettamanti, A. Grimaldi, M. Casartelli, E. Ambrosetti, B. Ponti, T. Congiu, R. Ferrarese, M.L. Rivas-Pena, F. Pennacchio and M.D. Eguileor

Cell Tissue Res 2007; In press     

Constitutive caspase-like machinery executes programmed cell death in plant cells

The morphological features of programmed cell death (PCD) and the molecular machinery involved in the death program in animal cells have been intensively studied. In plants, cell death has been widely observed in predictable patterns throughout differentiation processes and in defense responses. Several lines of evidence argue that plant PCD shares some characteristic features with animal PCD. However, the molecular components of the plant PCD machinery remain obscure. We have shown that plant cells undergo PCD by constitutively expressed molecular machinery upon induction with the fungal elicitor EIX or by staurosporine in the presence of cycloheximide. The permeable peptide caspase inhibitors, zVAD-fmk and zBocD-fmk, blocked PCD induced by EIX or staurosporine. Using labeled VAD-fmk, active caspase-like proteases were detected within intact cells and in cell extracts of the PCD-induced cells. These findings suggest that caspase-like proteases are responsible for the execution of PCD in plant cells.     

Programmed cell death in salivary glands of Drosophila arizonae and Drosophila mulleri

Programmed cell death (PCD) in insect metamorphosis assumes a great diversity of morphology and controlling processes that are still not well understood. With the objective of obtaining information about the PCD process, salivary glands of Drosophila arizonae and D. mulleri were studied during larval-pupal development. From the results, it can be concluded that the type of the PCD that occurs in these organs is morphologically typical of apoptosis (formation of apoptotic nuclei, followed by fragmentation into apoptotic bodies). Histolysis happens in both species, between 22 and 23 h after pupation. There were no significant differences between the species studied. Apoptosis does not occur simultaneously in all cells. Cytoplasmic acid phosphatase activity gradually increases during development, suggesting the existence of acid phosphatases that are only expressed during the apoptotic stage. Twenty hours after pupation, salivary glands already show biochemical alterations relative to nuclear permeability such as acidification, possibly due to the fusion of lysosomes with the nucleus a few hours before apoptosis. Autophagy seems to act together with apoptosis and has a secondary role in cell death.     

Promoter analysis of the Drosophila melanogaster gene encoding the pterin 4alpha-carbinolamine dehydratase.

Pterin-4alpha-carbinolamine dehydratase (PCD) is a key enzyme in the regeneration pathway of tetrahydrobiopterin. Previously, we isolated and reported the Drosophila melanogaster gene encoding PCD. In the present study, we isolated and characterized the Drosophila virilis gene encoding PCD. The Drosophila virilis PCD gene has two introns and an open reading frame to encode a protein of 101 amino acids. The amino acid sequence of Drosophila virilis PCD shows a 83% homology to that of the Drosophila melanogaster PCD protein. From the alignment of the nucleotide sequence in the 5'-flanking region of the Drosophila melanogaster and Drosophila virilis PCD genes, we found four conserved sequences. Using a transient transfection assay, we showed that one of the conserved sequences (-127 to approximately -115) is critical for expression, also the minimal promoter region between -127 and +51 is necessary for the efficient expression of Drosophila melanogaster PCD.     

果蝇变态过程中的细胞凋亡和细胞自噬

程序化细胞死亡(programmed cell death,PCD)分为I型PCD细胞凋亡(apoptosis)和II型PCD细胞自噬(autophagy)。果蝇等完全变态昆虫有2种类型的器官:即细胞内分裂器官(如脂肪体、表皮、唾液腺、中肠、马氏管等)和有丝分裂器官(复眼、翅膀、足、神经系统等)。在昆虫变态过程中,细胞内分裂器官进行器官重建,幼虫器官大量发生细胞凋亡和细胞自噬到最后完全消亡,同时成虫器官由干细胞从新生成;而有丝分裂器官则由幼虫器官直接发育为成虫器官。在果蝇等昆虫的变态过程中,细胞凋亡和细胞自噬在幼虫器官的死亡和成虫器官的生成中发挥了非常重要的作用。文章简要介绍细胞凋亡和细胞自噬在果蝇变态过程中的生理功能和分子调控机制。     

The effector caspases drICE and dcp-1 have partially overlapping functions in the apoptotic pathway in Drosophila

Caspases are essential components of the apoptotic machinery in both vertebrates and invertebrates. Here, we report the isolation of a mutant allele of the Drosophila effector caspase drICE as a strong suppressor of hid- (head involution defective-) induced apoptosis. This mutant was used to determine the apoptotic role of drICE. Our data are consistent with an important function of drICE for developmental and irradiation-induced cell death. Epistatic analysis suggests that drICE acts genetically downstream of Drosophila inhibitor of apoptosis protein 1 (Diap1). However, although cell death is significantly reduced in drICE mutants in all assays, it is not completely blocked. A double-mutant analysis between drICE and death caspase-1 (dcp-1), another effector caspase, reveals that some cells (type I) strictly require drICE for apoptosis, whereas other cells (type II) require either drICE or dcp-1. Thus, these data demonstrate a barely appreciated complexity in the apoptotic pathway, and are consistent with current models about effector caspase regulation in both vertebrates and invertebrates.     

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, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster 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 was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.     

Altered cytochrome c display precedes apoptotic cell death in Drosophila

Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.     

Drosophila Bcl-2 proteins participate in stress-induced apoptosis, but are not required for normal development

Many developing tissues require programmed cell death (PCD) for proper formation. In mice and C. elegans, developmental PCD is regulated by the Bcl-2 family of proteins. Two bcl-2 genes are encoded in the Drosophila genome (debcl/dBorg1/Drob-1/dBok and buffy/dBorg2) and previous RNAi-based studies suggested a requirement for these in embryonic development. However, we report here that, despite the fact that many tissues in fruit flies are shaped by PCD, deletion of the bcl-2 genes does not perturb normal development. We investigated whether the fly bcl-2 genes regulate non-apoptotic processes that require caspases, but found these to be bcl-2 gene-independent. However, irradiation of the mutants demonstrates that DNA damage-induced apoptosis, mediated by Reaper, is blocked by buffy and that debcl is required to inhibit buffy. Our results demonstrate that developmental PCD regulation in the fly does not rely upon the Bcl-2 proteins, but that they provide an added layer of protection in the apoptotic response to stress.     

Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.     

Comparative Genomics of Phylogenetically Diverse Unicellular Eukaryotes Provide New Insights into the Genetic Basis for the Evolution of the Programmed Cell Death Machinery

Programmed cell death (PCD) represents a significant component of normal growth and development in multicellular organisms. Recently, PCD-like processes have been reported in single-celled eukaryotes, implying that some components of the PCD machinery existed early in eukaryotic evolution. This study provides a comparative analysis of PCD-related sequences across more than 50 unicellular genera from four eukaryotic supergroups: Unikonts, Excavata, Chromalveolata, and Plantae. A complex set of PCD-related sequences that correspond to domains or proteins associated with all main functional classes—from ligands and receptors to executors of PCD—was found in many unicellular lineages. Several PCD domains and proteins previously thought to be restricted to animals or land plants are also present in unicellular species. Noteworthy, the yeast, Saccharomyces cerevisiae—used as an experimental model system for PCD research, has a rather reduced set of PCD-related sequences relative to other unicellular species. The phylogenetic distribution of the PCD-related sequences identified in unicellular lineages suggests that the genetic basis for the evolution of the complex PCD machinery present in extant multicellular lineages has been established early in the evolution of eukaryotes. The shaping of the PCD machinery in multicellular lineages involved the duplication, co-option, recruitment, and shuffling of domains already present in their unicellular ancestors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.