The Drosophila DIAP1 protein is required to prevent accumulation of a continuously generated, processed form of the apical caspase DRONC

Although loss of the inhibitor of apoptosis (LAP) 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 resulted in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc or dark largely suppressed this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibited DRICE processing following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induced DRONC processing, which occurred in two steps. The first step appeared to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK because full-length DRONC accumulated when dark was silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 resulted in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing was 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

Compensatory Proliferation Induced by Cell Death in the Drosophila Wing Disc Requires Activity of the Apical Cell Death Caspase Dronc in a Nonapoptotic Role

Achieving proper organ size requires a balance between proliferation and cell death. For example, at least 40%–60% of cells in the Drosophila wing disc can be lost, yet these discs go on to give rise to normal-looking adult wings as a result of compensatory proliferation 1, 2, 3. The signals that drive this proliferation are unknown. One intriguing possibility is that they derive, at least in part, from the dying cells. To explore this hypothesis, we activated cell death signaling in specific populations of cells in the developing wing but prevented these cells from dying through expression of the baculovirus p35 protein, which inhibits the activity of effector caspases that mediate apoptosis [4]. This allowed us to uncouple the activation steps of apoptosis from death itself. Here we report that stimulation of cell death signaling in the wing disc—in the absence of cell death—results in increased proliferation and ectopic expression of Wingless, a known mitogen in the wing. Activation of the apical cell death caspase Dronc is necessary and sufficient to drive both of these processes. Our results demonstrate an unanticipated function, the nonautonomous induction of proliferation, of an apical cell death caspase. This activity is likely to contribute to tissue homeostasis by promoting local compensatory proliferation in response to cell death. We speculate that dying cells may communicate cell fate or behavior instructions to their neighbors in other contexts as well

Expression of baculovirus P35 prevents cell death in Drosophila

The baculovirus P35 protein functions to prevent apoptotic death of infected cells. We have expressed P35 in the developing embryo and eye of the fly Drosophila melanogaster. P35 eliminates most, if not all, normally occurring cell death in these tissues, as well as X-irradiation-induced death. Excess pupal eye cells that are normally eliminated by apoptosis develop into pigment cells when their death is prevented by P35 expression. Our results suggest that one mechanism by which viruses prevent the death of the host cell is to block a cell death pathway that mediates normally occurring cell death. Identification of molecules that interact biochemically or genetically with P35 in Drosophila should provide important insights into how cell death is regulated

The Drosophila MicroRNA Mir-14 Suppresses Cell Death and Is Required for Normal Fat Metabolism

MicroRNAs (miRNAs) are small regulatory RNAs that are between 21 and 25 nucleotides in length and repress gene function through interactions with target mRNAs 1, 2. The genomes of metazoans encode on the order of several hundred miRNAs [3], but the processes they regulate have been defined for only two in C. elegans4, 5. We searched for new inhibitors of apoptotic cell death by testing existing collections of P element insertion lines for their ability to enhance a small-eye phenotype associated with eye-specific expression of the Drosophila cell death activator Reaper. Here we report the identification of the Drosophila miRNA mir-14 as a cell death suppressor. Loss of mir-14 enhances Reaper-dependent cell death, whereas ectopic expression suppresses cell death induced by multiple stimuli. Animals lacking mir-14 are viable. However, they are stress sensitive and have a reduced lifespan. Mir-14 mutants have elevated levels of the apoptotic effector caspase Drice, suggesting one potential site of action. Mir-14 also regulates fat metabolism. Deletion of mir-14 results in animals with increased levels of triacylglycerol and diacylglycerol, whereas increases in mir-14 copy number have the converse effect. We discuss possible relationships between these phenotypes

Rules of the road for insect gene drive research and testing

Approximately two years ago, two of us (E.B. and V.G.) demonstrated the first experimental application of CRISPR–Cas9 to 'drive' a desired trait throughout a population of fruit flies. In November 2015, this same team at the University of California, San Diego, joined with A.A.J. and others at the University of California, Irvine, to develop a CRISPR-based gene drive for population modification of the malaria vector mosquito Anopheles stephensi. A month later, a group in the United Kingdom applied a CRISPR-based gene drive to another malaria vector, Anopheles gambiae

General principles of single-construct chromosomal gene drive

Gene drive systems are genetic elements capable of spreading into a population even if they confer a fitness cost to their host. We consider a class of drive systems consisting of a chromosomally located, linked cluster of genes, the presence of which renders specific classes of offspring arising from specific parental crosses unviable. Under permissive conditions, a number of these elements are capable of distorting the offspring ratio in their favor. We use a population genetic framework to derive conditions under which these elements spread to fixation in a population or induce a population crash. Many of these systems can be engineered using combinations of toxin and antidote genes, analogous to Medea, which consists of a maternal toxin and zygotic antidote. The majority of toxin–antidote drive systems require a critical frequency to be exceeded before they spread into a population. Of particular interest, a Z-linked Medea construct with a recessive antidote is expected to induce an all-male population crash for release frequencies above 50%. We suggest molecular tools that may be used to build these systems, and discuss their relevance to the control of a variety of insect pest species, including mosquito vectors of diseases such as malaria and dengue fever

A reporter for amyloid precursor protein γ-secretase activity in Drosophila

A key event in the pathogenesis of Alzheimer's disease (AD) is the deposition of senile plaques consisting largely of a peptide known as β-amyloid (Aβ) that is derived from the amyloid precursor protein (APP). A proteolytic activity called γ-secretase cleaves APP in the transmembrane domain and is required for Aβ generation. Aberrant γ-secretase cleavage of APP underlies the majority of early onset, familial AD. γ-Secretase resides in a large multi-protein complex, of which Presenilin, Nicastrin, APH-1 and PEN-2 are four essential components. Thus, identifying components and pathways by which the γ-secretase activity is regulated is crucial to understanding the mechanisms underlying AD pathogenesis, and may provide new diagnostic tools and therapeutic targets. Here we describe the generation of Drosophila that act as living reporters of γ-secretase activity in the fly eye. In these reporter flies the size of the eye correlates with the level of endogenous γ-secretase activity, and is very sensitive to the levels of three genes required for APP γ-secretase activity, presenilin, nicastrin and aph-1. Thus, these flies provide a sensitized system with which to identify other components of the γ-secretase complex and regulators of its activity. We have used these flies to carry out a screen for mutations that suppress γ-secretase activity and have identified a small chromosomal region that contains a gene or genes whose products may promote γ-secretase activity

Identification of a component of Drosophila polar granules

Information necessary for the formation of pole cells, precursors of the germ line, is provided maternally and localized to the posterior pole of the Drosophila egg. The maternal origin and posterior localization of polar granules suggest that they may be associated with pole cell determinants. We have generated an antibody (Mab46F11) against polar granules. In oocytes and early embryos, the Mab46F11 antigen is sharply localized to the posterior embryonic pole. In pole cells, it becomes associated with nuclear bodies within, and nuage around, the nucleus. Immunoreactivity remains associated with cells of the germ line throughout the life cycle of both males and females. This antibody recognizes a 72-74 X 10^(3) Mr protein and is useful both as a pole lineage marker and in biochemical studies of polar granules

Cleave and Rescue, a novel selfish genetic element and general strategy for gene drive

There is great interest in being able to spread beneficial traits throughout wild populations in ways that are self-sustaining. Here, we describe a chromosomal selfish genetic element, CleaveR [Cleave and Rescue (ClvR)], able to achieve this goal. ClvR comprises two linked chromosomal components. One, germline-expressed Cas9 and guide RNAs (gRNAs)—the Cleaver—cleaves and thereby disrupts endogenous copies of a gene whose product is essential. The other, a recoded version of the essential gene resistant to cleavage and gene conversion with cleaved copies—the Rescue—provides essential gene function. ClvRenhances its transmission, and that of linked genes, by creating conditions in which progeny lacking ClvR die because they have no functional copies of the essential gene. In contrast, those who inherit ClvR survive, resulting in an increase in ClvR frequency. ClvR is predicted to spread to fixation under diverse conditions. To test these predictions, we generated a ClvR element in Drosophila melanogaster. ClvR^(tko) is located on chromosome 3 and uses Cas9 and four gRNAs to disrupt melanogaster technical knockout (tko), an X-linked essential gene. Rescue activity is provided by tko from Drosophila virilis. ClvR^(tko) results in germline and maternal carryover-dependent inactivation of melanogaster tko(>99% per generation); lethality caused by this loss is rescued by the virilis transgene; ClvR^(tko) activities are robust to genetic diversity in strains from five continents; and uncleavable but functional melanogaster tko alleles were not observed. Finally, ClvR^(tko) spreads to transgene fixation. The simplicity of ClvR suggests it may be useful for altering populations in diverse species

The role of cytochrome c in caspase activation in Drosophila melanogaster cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals