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 As they are no longer actively maintained, persistent corpses can proceed to a secondary necrotic state in which its cell membrane ruptures thus releasing its contents to the extracellular milieu.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.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/46918 |
| Date | 15 September 2023 |
| Creators | Lebo, Diane Patricia Vig |
| Contributors | McCall, Kimberly |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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