Cell clearance is critical for the resolution of inflammation. Defects in cell clearance can result in pathologies associated with chronic inflammation. Cells are cleared by phagocytes. These phagocytes can be either professional phagocytes whose main function is to engulf, or non-professional phagocytes, which have other tissue-resident functions but can engulf when needed. While the molecular biology of cell clearance has been heavily studied, the differences between mechanisms of engulfment by non-professional and professional phagocytes are not yet known. The study of cell clearance by non-professional phagocytes is important due to the presence of these cells in all tissues of the human body. The Drosophila ovary is a genetically tractable, in vivo model system to study engulfment by non-professional phagocytes. In response to protein deprivation, apoptosis is induced in the germline and the surrounding follicle cells engulf the dying corpses. The back and forth signaling that occurs between the dying germline and engulfing epithelial follicle cells is critical for efficient death and clearance. The germline must display eat-me signals for phagocyte recognition. The interaction of the eat-me signal and the engulfment receptor allows follicle cells to activate downstream signaling for internalization and corpse degradation. Interestingly, engulfment receptors have an additional role in driving germline death progression. The research in this dissertation focuses on the signals that drive the reciprocal signaling between non-professional phagocytes and dying cells. The results of this study demonstrate that Draper, an engulfment receptor, induces germline cell death by activating a Shark/JNK/NADPH oxidase signaling axis that is dependent on a YxxL motif. The results from this work indicate that the apoptotic machinery is activated and nurse cell nuclei are degraded in response to Draper overexpression, but Draper-induced cell death occurs independent of effector caspases, indicating that Draper induces cell death via other death pathways. An unbiased, high throughput approach to identify novel death-inducing receptors and ligands is also described. This study describes the reciprocal signaling mechanisms between engulfing and dying cells and opens up new avenues for targeting engulfment-mediated cell death. / 2021-11-08T00:00:00Z

Cell death is a fundamental aspect of development and homeostasis; its dysregulation is commonly associated with disease. Historically, apoptosis has been the most heavily studied type of cell death, but there are many other non-apoptotic forms of cell death. The Drosophila ovary provides a powerful in vivo model to study non-apoptotic cell death. Each egg chamber in the ovary contains 15 nurse cells that support an oocyte throughout development, and at the end of oogenesis the nurse cells are surrounded by stretch follicle cells and undergo non-apoptotic cell death. The work in this dissertation investigated the role of stretch follicle cells in nurse cell death. Genetic ablation of the stretch follicle cells revealed that they are required for multiple nurse cell death events including the transport of cytoplasm to the oocyte and DNA fragmentation. We found that phagocytic machinery is required in the stretch follicle cells for the acidification and elimination of nurse cells, suggesting nurse cells die by phagoptosis. Furthermore, live imaging and a transgenic engulfment detector demonstrated that nurse cells are not engulfed piece-wise despite the requirement of phagocytosis machinery, but are instead surrounded and acidified extracellularly. To determine the mechanism driving nurse cell acidification, we performed a targeted RNAi screen against lysosome-associated genes. Using tissue-specific RNAi, we demonstrated that the V-ATPase proton pump is required in the stretch follicle cells for nurse cell acidification. GFP fusion proteins and antibody staining revealed that V-ATPases become enriched and localize to the stretch follicle cell plasma membranes to acidify the nurse cells that they surround. Following acidification, the stretch follicle cells were found to release cathepsins, lysosomal proteases, to break down and degrade the nurse cell. To uncover novel pro-death proteins that mediate signaling between the stretch follicle cells and nurse cells, we utilized proximity-dependent protein labeling and identified proteins enriched in the stretch follicle cells. Altogether this work uncovers a new role for lysosomal machinery acting at the plasma membrane of stretch follicle cells to drive nurse cell death, and identifies pro-death proteins in the stretch follicle cells that promote nurse cell death.

More than 300 billion cells die in the human body every day. Although there are over a dozen different death paradigms, they all produce the same result - dead and dying cells. As they are no longer actively maintained, persistent corpses can proceed to a secondary necrotic state in which its cell membrane ruptures thus releasing its contents to the extracellular milieu. As many of the intracellular contents act as damage associated molecular patterns (DAMPs), they pose a potential danger to the rest of the surrounding tissue and organism. Excessive cell death has been correlated with diseases such as atherosclerosis, Alzheimer’s, and autoimmune disorders. To avoid damage and disease associated with cell corpses, two classes of cells evolved to clear them away – professional and nonprofessional phagocytes. A professional phagocyte's primary function is to clear away dying cells and other debris. Nonprofessional phagocytes, however, have a primary role other than clearance. When nonprofessional phagocytes encounter a cell corpse, their phagocytic machinery is engaged to clear it away. Interestingly, a recent study demonstrated that most, if not all, tissues contain nonprofessional phagocytes. To investigate nonprofessional phagocytes, the model organism Drosophila melanogaster is ideal. Drosophila is a useful model system as they have orthologs for 70% of human disease genes, a simplified immune system, and a host of genetic tools. Their ovaries have three morphologically distinct cell types – 15 nurse cells and an oocyte all surrounded by an epithelial follicle cell layer. As the ovaries are immunoprivileged, the follicle cell layer acts as the ovaries’ sole phagocytes. During late stage oogenesis, a small subsection of the follicle cell layer – the stretch follicle cells – murder the nurse cells in order to produce a fully developed oocyte. As past studies of cell corpse clearance have predominantly concentrated on the professional phagocytosis in the context of apoptotic cell corpses, there are still many gaps in our knowledge of nonprofessional phagocytosis and non-apoptotic death. This dissertation focuses on the molecular mechanisms that regulate the transition of nonprofessional phagocytes from their primary role as epithelial cells to their phagocytic role in the context of a newly characterized form of non-autonomous cell death known as phagoptosis. To gain a global view of these changes, two large scale experiments were performed – a classic genetic screen of kinases using RNAi and a high-throughput translatome study. The kinase screen identified dozens of kinase genes required for proper clearance. Of the 27 kinase genes that demonstrated a severe phenotype when knocked down, two were previously uncharacterized and six produced an “undead” phenotype, a phenotype that had only been previously witnessed when genes were perturbed in the germline. A follow up study was performed on Gprk2, one of the genes that induced a severe phenotype. By comparing the phenotypes of Gprk2 knockdowns and those of the two canonical clearance pathways, a third clearance pathway was discovered. The translatome study identified over 400 genes that were statistically significantly differentially expressed between primary state and phagocytic state follicle cells, including groups affecting calcium signaling and muscle contraction. This dissertation further describes the expansion of the molecular network of nonprofessional phagocytes driven by these large-scale experiments and their follow up studies.