Phagocytosis In Drosophila Research Articles

The Drosophila chemokine–like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons

Phagocytic clearance of degenerating neurons is triggered by "eat-me" signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes invivo remains poorly understood. Using multiple models of dendrite degeneration, we show that the Drosophila chemokine-like protein Orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of Orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the Orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between Orion and human immunomodulatory proteins are important for Orion function. Thus, our results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons.

Phagocytosis in Drosophila: From molecules and cellular machinery to physiology

Phagocytosis is an evolutionarily conserved mechanism that plays a key role in both host defence and tissue homeostasis in multicellular organisms. A range of surface receptors expressed on different cell types allow discriminating between self and non-self (or altered) material, thus enabling phagocytosis of pathogens and apoptotic cells. The phagocytosis process can be divided into four main steps: 1) binding of the phagocyte to the target particle, 2) particle internalization and phagosome formation, through remodelling of the plasma membrane, 3) phagosome maturation, and 4) particle destruction in the phagolysosome. In this review, we describe our present knowledge on phagocytosis in the fruit fly Drosophila melanogaster, assessing each of the key steps involved in engulfment of both apoptotic cells and bacteria. We also assess the physiological role of phagocytosis in host defence, development and tissue homeostasis.

Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila

BackgroundThe regulatory mechanisms of hematopoiesis and cellular immunity show a high degree of similarity between insects and mammals, and Drosophila has become a good model for investigating cellular immune responses. Jumeau (Jumu) is a member of the winged-helix/forkhead (FKH) transcription factor family and is required for Drosophila development. Adult jumu mutant flies show defective hemocyte phagocytosis and a weaker defense capability against pathogen infection. Here, we further investigated the role of jumu in the regulation of larval hemocyte development and phagocytosis.MethodsIn vivo phagocytosis assays, immunohistochemistry, Real-time quantitative PCR and immunoblotting were performed to investigate the effect of Jumu on hemocyte phagocytosis. 5-Bromo-2-deoxyUridine (BrdU) labeling, phospho-histone H3 (PH3) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were performed to analyze the proliferation and apoptosis of hemocyte; immunohistochemistry and Mosaic analysis with a repressible cell marker (MARCM) clone analysis were performed to investigate the role of Jumu in the activation of Toll pathway.ResultsJumu indirectly controls hemocyte phagocytosis by regulating the expression of NimC1 and cytoskeleton reorganization. The loss of jumu also causes abnormal proliferation and differentiation in circulating hemocytes. Our results suggest that a severe deficiency of jumu leads to the generation of enlarged multinucleate hemocytes by affecting the normal cell mitosis process and induces numerous lamellocytes by activating the Toll pathway.ConclusionsJumu regulates circulating hemocyte differentiation and phagocytosis in Drosophila. Our findings provide new insight into the mechanistic roles of cytoskeleton regulatory proteins in phagocytosis and establish a basis for further analyses of the regulatory mechanism of the mammalian ortholog of Jumu in mammalian innate immunity.

The Splicing Factor RNA-Binding Fox Protein 1 Mediates the Cellular Immune Response in Drosophila melanogaster.

The uptake and destruction of bacteria by phagocytic cells is an essential defense mechanism in metazoans. To identify novel genes involved in the phagocytosis of Staphylococcus aureus, a major human pathogen, we assessed the phagocytic capacity of adult blood cells (hemocytes) of the fruit fly, Drosophila melanogaster, by testing several lines of the Drosophila Genetic Reference Panel. Natural genetic variation in the gene RNA-binding Fox protein 1 (Rbfox1) correlated with low phagocytic capacity in hemocytes, pointing to Rbfox1 as a candidate regulator of phagocytosis. Loss of Rbfox1 resulted in increased expression of the Ig superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of Dscam4 in Rbfox1-depleted blood cells rescued the fly's cellular immune response to S. aureus, indicating that downregulation of Dscam4 by Rbfox1 is critical for S. aureus phagocytosis in Drosophila To our knowledge, this study is the first to demonstrate a link between Rbfox1, Dscam4, and host defense against S. aureus.

Expression of MEGF10 in cholinergic and glutamatergic neurons

Multiple-EGF like domains 10 (MEGF10) is the mammalian homologue of Draper, a Drosophila phagocytosis receptor that plays an important role in synapse elimination and cell type-specific recognition. However, the expression and function of MEGF10 in the brain remain to be elucidated. Therefore, we aimed to clarify the regions and types of neurons that express MEGF10 in the brain, and to determine whether cells expressing MEGF10 possess phagocytic abilities. Our results indicated that MEGF10 is expressed in cholinergic and glutamatergic neurons of the cortex, hippocampus, and substantia nigra. Furthermore, the ratio of neurons expressing MEGF10 was higher in the cortex and hippocampus of hAPP transgenic mice than in those of wild-type mice. Phagocytic activity was also observed in neuronal cells expressing MEGF10. Thus, our results indicate that MEGF10 may be responsible for phagocytic activity targeted toward unwanted substances such as amyloid in cholinergic and glutamatergic neurons.

JNK pathway activation is able to synchronize neuronal death and glial phagocytosis in Drosophila.

Glial phagocytosis of superfluous neurons and damaged or aberrant neuronal material is crucial for normal development and maintenance of the CNS. However, the molecular mechanisms underlying the relationship between neuronal death and glial phagocytosis are poorly understood. We describe a novel mechanism that is able to synchronize neuronal cell death and glial phagocytosis of dying neurons in the Drosophila embryonic CNS. This mechanism involves c-Jun N-terminal kinase (JNK) signaling, which is required for developmental apoptosis of specific neurons during embryogenesis. We demonstrate that the dJNK pathway gain-of-function in neurons leads to dJNK signaling in glia, which results in upregulation of glial phagocytosis. Importantly, this promotion of phagocytosis is not mediated by upregulation of the glial phagocytic receptors SIMU and DRPR, but by increasing glial capacity to degrade apoptotic particles inside phagosomes. The proposed mechanism may be important for removal of damaged neurons in the developing and mature CNS.

Independent Recognition of Staphylococcus aureus by Two Receptors for Phagocytosis in Drosophila

Integrin βν, one of two β subunits of Drosophila integrin, acts as a receptor in the phagocytosis of apoptotic cells. We here examined the involvement of this receptor in defense against infection by Staphylococcus aureus. Flies lacking integrin βν died earlier than control flies upon a septic but not oral infection with this bacterium. A loss of integrin βν reduced the phagocytosis of S. aureus and increased bacterial growth in flies. In contrast, the level of mRNA of an antimicrobial peptide produced upon infection was unchanged in integrin βν-lacking flies. The simultaneous loss of integrin βν and Draper, another receptor involved in the phagocytosis of S. aureus, brought about a further decrease in the level of phagocytosis and accelerated death of flies compared with the loss of either receptor alone. A strain of S. aureus lacking lipoteichoic acid, a cell wall component serving as a ligand for Draper, was susceptible to integrin βν-mediated phagocytosis. In contrast, a S. aureus mutant strain that produces small amounts of peptidoglycan was less efficiently phagocytosed by larval hemocytes, and a loss of integrin βν in hemocytes reduced a difference in the susceptibility to phagocytosis between parental and mutant strains. Furthermore, a series of experiments revealed the binding of integrin βν to peptidoglycan of S. aureus. Taken together, these results suggested that Draper and integrin βν cooperate in the phagocytic elimination of S. aureus by recognizing distinct cell wall components, and that this dual recognition system is necessary for the host organism to survive infection.

Transcriptional regulation of eater gene expression in Drosophila blood cells

Eater is a transmembrane protein that mediates phagocytosis in Drosophila. eater was identified in a microarray analysis of genes downregulated in S2 cells, in which Serpent had been knocked down by RNAi. The gene was shown to be expressed predominantly in plasmatocytes after embryonic development. We have extensively analyzed the transcriptional enhancer controlling eater expression with the following findings: the enhancer reproduces the plasmatocyte expression pattern of the gene as verified by anti-P1 antibody staining and a 526-basepair DNA region is active in lymph gland and hemolymph plasmatocytes. This DNA contains several GATA elements that serve as putative-binding sites for Serpent. Site-directed mutagenesis of two of these GATA sites abolishes eater expression in both lymph gland and hemolymph plasmatocytes. This suggests that Serpent regulates eater expression by binding these GATA sites, which was confirmed by gel shift analysis. These analyses allowed us to use eater-Gal4 to force plasmatocyte to lamellocyte differentiation.

Drosophila phagocytosis - still many unknowns under the surface

In mammals, phagocytosis coordinates host defence on two levels: It acts both as an effector of the innate immunity, as well as an initiator of the adaptive immunity. The fruit fly Drosophila melanogaster (D. melanogaster) lacks the adaptive immune response, and the role of Drosophila plasmatocytes, cells that resemble phagocytosing mammalian macrophages, is limited to innate immune responses. During the past years, several studies have shed light on the role of phagocytosis in the Drosophila host defence. At least in some infection models, the systemic production of potent antimicrobial peptides (AMPs) does not completely compensate for the need for cellular immune responses. As a model, Drosophila offers powerful tools for studying phagocytosis including, large-scale RNA interference (RNAi) based in vitro screens that can be combined with classical Drosophila genetics. These kinds of approaches have led to important discoveries related especially to microbial recognition by Drosophila plasmatocytes. Events following initial recognition, however, have remained more elusive. This review summarizes the current knowledge on Drosophila phagocytosis focusing on the most recent advancements in the field, and highlighting the benefits the Drosophila system has to offer for research on phagocytosis.

Six-Microns-Under Acts Upstream of Draper in the Glial Phagocytosis of Apoptotic Neurons

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.

Identification of calreticulin as a marker for phagocytosis of apoptotic cells in Drosophila

Apoptotic cell phagocytosis is initiated through the specific interaction between markers for phagocytosis present at the surface of targets and their receptors of phagocytes. Although many molecules have been proposed to be phagocytosis markers and receptors in mammals, information as to the identity of those molecules is limited for invertebrate animals. Calreticulin, a molecular chaperone that functions in the lumen of the endoplasmic reticulum, was recently reported to be the second general marker, the membrane phospholipid phosphatidylserine being the first, for mammalian apoptotic cells to be recognized by phagocytes. We here asked whether or not calreticulin serves as a marker for phagocytosis in Drosophila. Phagocytosis of apoptotic S2 cells by Drosophila hemocyte-derived l(2)mbn cells, which we previously showed to occur independent of phosphatidylserine, was inhibited by the addition of anti-calreticulin antibody. This inhibition was observed when the target cells, but not phagocytes, were pre-incubated with the antibody. In addition, RNA interference-mediated reduction of calreticulin expression in apoptotic S2 cells, but not in l(2)mbn cells, reduced the level of phagocytosis. An immunocytochemical analysis revealed that calreticulin is widely distributed at the surface of viable S2 cells. After the induction of apoptosis, cell surface calreticulin seemed to form aggregates, with no change in its amount. Furthermore, in embryos of a mutant Drosophila strain that expresses calreticulin at a reduced level, the level of phagocytosis of apoptotic cells was about a half of that observed in embryos of a wild-type strain. These results collectively indicate that calreticulin is the first molecule to be identified as a marker for phagocytosis of apoptotic cells by Drosophila phagocytes.

Eater, a Transmembrane Protein Mediating Phagocytosis of Bacterial Pathogens in Drosophila

Phagocytosis is a complex, evolutionarily conserved process that plays a central role in host defense against infection. We have identified a predicted transmembrane protein, Eater, which is involved in phagocytosis in Drosophila. Transcriptional silencing of the eater gene in a macrophage cell line led to a significant reduction in the binding and internalization of bacteria. Moreover, the N terminus of the Eater protein mediated direct microbial binding which could be inhibited with scavenger receptor ligands, acetylated, and oxidized low-density lipoprotein. In vivo, eater expression was restricted to blood cells. Flies lacking the eater gene displayed normal responses in NF-kappaB-like Toll and IMD signaling pathways but showed impaired phagocytosis and decreased survival after bacterial infection. Our results suggest that Eater is a major phagocytic receptor for a broad range of bacterial pathogens in Drosophila and provide a powerful model to address the role of phagocytosis in vivo.

Identification of cytoskeletal regulatory proteins required for efficient phagocytosis in Drosophila

Phagocytosis is a complex and apparently evolutionarily conserved process that plays a central role in the immune response to infection. By ultrastructural and functional criteria, Drosophila hemocyte (macrophage) phagocytosis resembles mammalian phagocytosis. Using a non-saturated forward genetic screen for larval hemocyte phagocytosis mutants, D-SCAR and profilin were identified as important regulators of phagocytosis in Drosophila. In both hemocytes ex vivo and the macrophage-like S2 cell line, lack of D-SCAR significantly decreased phagocytosis of Escherichia coli and Staphylococcus aureus. In contrast, profilin mutant hemocytes exhibited increased phagocytic activity. Analysis of double mutants suggests that D-SCAR and profilin interact during phagocytosis. Finally, RNA interference studies in S2 cells indicated that the D-SCAR homolog D-WASp also participates in phagocytosis. This study demonstrates that Drosophila provides a viable model system in which to dissect the complex interactions that regulate phagocytosis.

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.