Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells.
ABSTRACT: Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which we name Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper-mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine-phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells.
Project description:To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, we isolated proteins that bind to the extracellular region of Draper using affinity chromatography. One of those proteins has been identified to be an uncharacterized protein called Drosophila melanogaster calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur.
Project description:The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 (ref. 1) and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. Here we show that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70, binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. We show that Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. We also show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. We propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade.
Project description:The removal of apoptotic cells by phagocytic neighbors is essential for metazoan development but remains poorly characterized. Here we report the discovery of a Drosophila phagocytosis receptor, Six-microns-under (SIMU), which is expressed in highly phagocytic cell types during development and required for efficient apoptotic cell clearance by glia in the nervous system and by macrophages elsewhere. SIMU is part of a conserved family of proteins that includes CED-1 and Draper (DRPR). Phenotypic analysis reveals that simu acts upstream of drpr in the same pathway and affects the recognition and engulfment of apoptotic cells, while drpr affects their subsequent degradation. SIMU strongly binds to apoptotic cells, presumably through its EMILIN-like domain, but requires no membrane anchoring, suggesting that it can function as a bridging molecule. Our study introduces an important factor in tissue-resident apoptotic clearance and underscores the prominent role of glia as "semiprofessional" phagocytes in the nervous system.
Project description:The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin ?PS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocyte-specific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signal-transduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells by stimulated phagocytes and is thus considered as a mechanism to prime phagocytes in innate immunity.
Project description:Phagocytosis of host cells is characteristic of tissue invasion by the intestinal ameba Entamoeba histolytica, which causes amebic dysentery and liver abscesses. Entamoeba histolytica induces host cell apoptosis and uses ligands, including C1q, on apoptotic cells to engulf them. Two mass spectrometry analyses identified calreticulin in amebic phagosome preparations, and, in addition to its function as an endoplasmic reticulum chaperone, calreticulin is believed to be the macrophage receptor for C1q. The purpose of this study was to determine if calreticulin functions as an E. histolytica C1q receptor during phagocytosis of host cells. Calreticulin was localized to the surface of E. histolytica during interaction with both Jurkat lymphocytes and erythrocytes and was present in over 75% of phagocytic cups during amebic erythrophagocytosis. Presence of calreticulin on the cell surface was further demonstrated using a method that selectively biotinylated cell surface proteins and by flow cytometry using trophozoites overexpressing epitope-tagged calreticulin. Regulated overexpression of calreticulin increased E. histolytica's ability to phagocytose apoptotic lymphocytes and calcium ionophore-treated erythrocytes but had no effect on amebic adherence to or destruction of cell monolayers or surface expression of the GalNAc lectin and serine-rich E. histolytica protein (SREHP) receptors. Finally, E. histolytica calreticulin bound specifically to apoptotic lymphocytes and to human C1q. Collectively, these data implicate cell surface calreticulin as a receptor for C1q during E. histolytica phagocytosis of host cells.
Project description:The engulfment of apoptotic cells is essential for tissue homeostasis and recovering from damage. Engulfment is mediated by receptors that recognize ligands exposed on apoptotic cells such as phosphatidylserine (PS). In this study, we convert Drosophila melanogaster S2 cells into proficient phagocytes by transfecting the Draper engulfment receptor and replacing apoptotic cells with PS-coated beads. Similar to the T cell receptor (TCR), PS-ligated Draper forms dynamic microclusters that recruit cytosolic effector proteins and exclude a bulky transmembrane phosphatase, consistent with a kinetic segregation-based triggering mechanism. However, in contrast with the TCR, localized signaling at Draper microclusters results in time-dependent depletion of actin filaments, which facilitates engulfment. The Draper-PS extracellular module can be replaced with FRB and FKBP, respectively, resulting in a rapamycin-inducible engulfment system that can be programmed toward defined targets. Collectively, our results reveal mechanistic similarities and differences between the receptors involved in apoptotic corpse clearance and mammalian immunity and demonstrate that engulfment can be reprogrammed toward nonnative targets.
Project description:Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance.
Project description:Advanced age is the greatest risk factor for neurodegenerative disorders, but the mechanisms that render the senescent brain vulnerable to disease are unclear. Glial immune responses provide neuroprotection in a variety of contexts. Thus, we explored how glial responses to neurodegeneration are altered with age. Here we show that glia-axon phagocytic interactions change dramatically in the aged Drosophila brain. Aged glia clear degenerating axons slowly due to low phosphoinositide-3-kinase (PI3K) signalling and, subsequently, reduced expression of the conserved phagocytic receptor Draper/MEGF10. Importantly, boosting PI3K/Draper activity in aged glia significantly reverses slow phagocytic responses. Moreover, several hours post axotomy, early hallmarks of Wallerian degeneration (WD) are delayed in aged flies. We propose that slow clearance of degenerating axons is mechanistically twofold, resulting from deferred initiation of axonal WD and reduced PI3K/Draper-dependent glial phagocytic function. Interventions that boost glial engulfment activity, however, can substantially reverse delayed clearance of damaged neuronal debris.
Project description:Neuronal injury triggers robust responses from glial cells, including altered gene expression and enhanced phagocytic activity to ensure prompt removal of damaged neurons. The molecular underpinnings of glial responses to trauma remain unclear. Here, we find that the evolutionarily conserved insulin-like signaling (ILS) pathway promotes glial phagocytic clearance of degenerating axons in adult Drosophila. We find that the insulin-like receptor (InR) and downstream effector Akt1 are acutely activated in local ensheathing glia after axotomy and are required for proper clearance of axonal debris. InR/Akt1 activity, it is also essential for injury-induced activation of STAT92E and its transcriptional target draper, which encodes a conserved receptor essential for glial engulfment of degenerating axons. Increasing Draper levels in adult glia partially rescues delayed clearance of severed axons in glial InR-inhibited flies. We propose that ILS functions as a key post-injury communication relay to activate glial responses, including phagocytic activity.
Project description:Neuronal injury elicits potent cellular responses from glia, but molecular pathways modulating glial activation, phagocytic function and termination of reactive responses remain poorly defined. Here we show that positive or negative regulation of glial responses to axon injury is molecularly encoded by unique isoforms of the Drosophila melanogaster engulfment receptor Draper. Draper-I promotes engulfment of axonal debris through an immunoreceptor tyrosine-based activation motif (ITAM). In contrast, Draper-II, an alternative splice variant, potently inhibits glial engulfment function. Draper-II suppresses Draper-I signaling through a previously undescribed immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain and the tyrosine phosphatase Corkscrew (Csw). Intriguingly, loss of Draper-II-Csw signaling prolongs expression of glial engulfment genes after axotomy and reduces the ability of glia to respond to secondary axotomy. Our work highlights a novel role for Draper-II in inhibiting glial responses to neurodegeneration, and indicates that a balance of opposing Draper-I and Draper-II signaling events is essential to maintain glial sensitivity to brain injury.