Saori Nonaka

Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper.

The membrane phospholipid phosphatidylserine is exposed on the cell surface during apoptosis and acts as an eat-me signal in the phagocytosis of apoptotic cells in mammals and nematodes. However, whether this is also true in insects was unclear. When milk fat globule-epidermal growth factor 8, a phosphatidylserine-binding protein of mammals, was ectopically expressed in Drosophila, the level of phagocytosis was reduced, whereas this was not the case for the same protein lacking a domain responsible for the binding to phosphatidylserine. We found that the extracellular region of Draper, an engulfment receptor of Drosophila, binds to phosphatidylserine in an enzyme-linked immunosorbent assay-like solid-phase assay and in an assay for surface plasmon resonance. A portion of Draper containing domains EMI and NIM located close to the N-terminus was required for binding to phosphatidylserine, and a Draper protein lacking this region was not active in Drosophila. Finally, the level of tyrosine-phosphorylated Draper, indicative of the activation of Draper, in a hemocyte-derived cell line was increased after treatment with phosphatidylserine-containing liposome. These results indicated that phosphatidylserine serves as an eat-me signal in the phagocytic removal of apoptotic cells in Drosophila and that Draper is a phosphatidylserine-binding receptor for phagocytosis.

Integrin βν-mediated Phagocytosis of Apoptotic Cells in Drosophila Embryos

To identify molecules that play roles in the clearance of apoptotic cells by Drosophila phagocytes, we examined a series of monoclonal antibodies raised against larval hemocytes for effects on phagocytosis in vitro. One antibody that inhibited phagocytosis recognized terribly reduced optic lobes (Trol), a core protein of the perlecan-type proteoglycan, and the level of phagocytosis in embryos of a Trol-lacking fly line was lower than in a control line. The treatment of a hemocyte cell line with a recombinant Trol protein containing the amino acid sequence RGD augmented the phosphorylation of focal adhesion kinase, a hallmark of integrin activation. A loss of integrin βν, one of the two β subunits of Drosophila integrin, brought about a reduction in the level of apoptotic cell clearance in embryos. The presence of integrin βν at the surface of embryonic hemocytes was confirmed, and forced expression of integrin βν in hemocytes of an integrin βν-lacking fly line recovered the defective phenotype of phagocytosis. Finally, the level of phagocytosis in a fly line that lacks both integrin βν and Draper, another receptor required for the phagocytosis of apoptotic cells, was lower than that in a fly line lacking either protein. We suggest that integrin βν serves as a phagocytosis receptor responsible for the clearance of apoptotic cells in Drosophila, independent of Draper.

Integrin αPS3/βν-mediated Phagocytosis of Apoptotic Cells and Bacteria in Drosophila

Background: Drosophila integrin βν plays a role in the phagocytosis of apoptotic cells and bacteria, but its partner α-subunit remains to be identified. Results: Of 5 α-subunits, αPS3 was physically and functionally associated with βν. Conclusion: αPS3/βν serves as a receptor for phagocytosis in Drosophila. Significance: The heterodimeric structure of Drosophila integrin has been genetically and biochemically solved. Integrins exert a variety of cellular functions as heterodimers of two transmembrane subunits named α and β. Integrin βν, a β-subunit of Drosophila integrin, is involved in the phagocytosis of apoptotic cells and bacteria. Here, we searched for an α-subunit that forms a complex and cooperates with βν. Examinations of RNAi-treated animals suggested that αPS3, but not any of four other α-subunits, is required for the effective phagocytosis of apoptotic cells in Drosophila embryos. The mutation of αPS3-encoding scb, deficiency, insertion of P-element, or alteration of nucleotide sequences, brought about a reduction in the level of phagocytosis. The defect in phagocytosis by deficiency was reverted by the forced expression of scb. Furthermore, flies in which the expression of both αPS3 and βν was inhibited by RNAi showed a level of phagocytosis almost equal to that observed in flies with RNAi for either subunit alone. A loss of αPS3 also decreased the activity of larval hemocytes in the phagocytosis of Staphylococcus aureus. Finally, a co-immunoprecipitation analysis using a Drosophila cell line treated with a chemical cross-linker suggested a physical association between αPS3 and βν. These results collectively indicated that integrin αPS3/βν serves as a receptor in the phagocytosis of apoptotic cells and bacteria by Drosophila phagocytes.

Signaling pathway for phagocyte priming upon encounter with apoptotic cells

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 αPS3. 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.

Characterization of Spz5 as a novel ligand for Drosophila Toll-1 receptor

The Drosophila Toll-1 receptor is involved in embryonic development, innate immunity, and tissue homeostasis. Currently, as a ligand for the Toll-1 receptor, only Spätzle (Spz) has been identified and characterized. We previously reported that Drosophila larva-derived tissue extract contains ligand activity for the Toll-1 receptor, which differs from Spz based on the observation that larval extract prepared from spz mutants possessed full ligand activity. Here, we demonstrate that Spz5, a member of the Spz family of proteins, functions as a ligand for the Toll-1 receptor. Processing of Spz5 by Furin protease, which is known to be important for ligand activity of Spz5 to Toll-6, is not required for its function to the Toll-1 receptor. By generating a spz5 null mutant, we further showed that the Toll-1 ligand activity of larva-derived extract is mainly derived from Spz5. Finally, we found a genetic interaction between spz and spz5 in terms of developmental processes. This study identified a novel ligand for the Drosophila Toll-1 receptor, providing evidence that Toll-1 is a multi-ligand receptor, similar to the mammalian Toll-like receptor.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Mechanisms and Significance of Phagocytic Elimination of Cells Undergoing Apoptotic Death

Cells that have become unwanted by the body need to be selectively, rapidly, and safely removed. The removal of these cells is achieved by apoptosis-dependent phagocytosis: unwanted cells are induced to undergo apoptosis and given susceptibility to phagocytosis. Phagocytes recognize these cells using engulfment receptors that bind substances expressed on the surface of target cells during the apoptotic process. The phagocytic elimination of cells undergoing apoptosis is a mechanism that is conserved among multicellular organisms. Malfunctions in this process may lead to structural and functional defects in morphogenesis and tissue homeostasis. Therefore, molecules involved in this phenomenon may be targeted in medical treatments. The mechanisms responsible for the apoptosis-dependent phagocytosis of unwanted cells as well as its physiological and pathological consequences are described herein.