Study on the flux and role of calcium during efferocytosis

Abstract

Efferocytosis is a process by which phagocytes ingest and eliminate apoptotic cells. During efferocytosis, calcium flux is carefully regulated, and finally the intracellular calcium level of phagocytes increases. Calcium plays an essential part in the effective and timely removal of apoptotic cells, but its specific function is not well understood. In part I, I report that Mertk-mediated intracellular calcium elevation is necessary for myosin II contraction and F-actin disassembly, both of which are essential for phagocytic cup closure. This was the result of a disturbed interaction between Calmodulin (CaM) and myosin light chain kinase (MLCK) in the absence of calcium, which led to decreased phosphorylation of myosin light chain (MLC) and a delay in F-actin disassembly. In part II, I investigated proteins interacting with Tim-4, a receptor that binds to an “eat me signal” phosphatidylserine (PS) of apoptotic cells, in phagocytes. Tim-4 is incapable of signal transduction and collaborates with other engulfment receptors to facilitate internalization. Nevertheless, the identity of these cooperating receptors is still poorly defined. In my study, I used in silico techniques to look for a transmembrane protein with a FN3 domain that could interact with Tim-4. EphA2, which has an extracellular FN3 domain, was discovered as a protein that interacts with Tim-4 via the IgV domain of Tim-4 and the FN3 domain of EphA2. However, the expression of EphA2 had no effect on the ability of Tim-4 to mediate the phagocytosis of apoptotic cells or polystyrene beads, suggesting the interaction may have a role in other cellular activity. In part III, I explored a novel binding partner of Elmo1. This adaptor protein, known for binding to Dock180 to activate Rac and subsequently modulating actin dynamics during efferocytosis, raised intriguing questions about its role in different cellular processes. I investigated biochemical interaction between Elmo1 and Cyclin I, which is a member of the Cyclin family, and the potential roles of this newly identified partnership, particularly within the context of myogenesis. Recognizing the parallels between efferocytosis and myogenesis, I considered the factors that contribute to cell fate decisions, acknowledging that the ultimate destiny of a cell hinges on specific signals, receptors, and the context in which these interactions take place. Additionally, CD47, which is conventionally known as a “don't eat me signal” in phagocytosis, was shown to be involved in myogenesis. Blocking CD47 with anti-CD47 antibodies significantly decreased myoblast fusion. In addition to these findings, I developed a pipeline for automating the analysis of myotube fusion. This tool, created with the user-friendly CellProfiler software, streamlines the examination of both the fusion index and the morphological characteristics of myotubes.