Phosphoinositide metabolism and signaling during late phagosome maturation and phagolysosome resolution

Phagocytosis, the regulated uptake of particulate matter by cells, is essential to immunity and homeostasis. Phagocytes are tasked with the immune surveillance that prevents infection by invading pathogens. Additionally, these cells clear accumulated apoptotic debris that results from the daily turnover of billions of cells. In order to fulfill these paramount functions, phagocytes recognize targets and internalize them into membrane-bound compartments termed phagosomes. Following internalization, cells degrade the phagosomal contents and dispose of them resuming the immune response. Phagocytosis is therefore divided into three stages: formation, maturation and resolution. Formation refers to the recognition and engulfment of prey. The transformation that enables degradation of the target is termed maturation. Finally, the elimination and often re-utilization of degraded contents is known as resolution. Most of the phenomena that govern each of these stages are orchestrated by acute signaling events. Specialized signaling phospholipids known as phosphoinositides mediate the recruitment of effector proteins to phagosomes. While the roles of diverse phosphoinositides during formation and the early maturation have been studied and partially elucidated, little is known about them during the late maturation and resolution stages. These late stages are essential for degradation of the phagocytic targets, processing of phagosomal contents and return to homeostasis by phagocytes in order to resume their responses. The work described in this dissertation was conducted with the aim of characterizing phosphatidylinositol 4-phosphate (PtdIns4P)—a phosphoinositide present in the plasma membrane, Golgi apparatus and late compartments—during phagocytosis. Additionally, bacterial species such as Legionella, have been shown to commandeer PtdIns4P metabolism during infection, emphasizing the potential importance of this phosphoinositide during the innate immune response. For this work, I specifically focused on elucidating the dynamics, metabolism and functions of PtdIns4P during each stage of phagocytosis. Chapter I, consists of relevant background on the cell biology of phagocytosis and the involvement of phosphoinositides during the process. The aims and hypothesis of my work are described in Chapter 2. Chapter 3 describes the methodology used throughout this work. In Chapter 4, I detail PtdIns4P dynamics and metabolic mechanisms during phagosome formation and maturation. Additionally, I define the functional implications of PtdIns4P in maturing phagosomes. In Chapter 5 I detail how the endoplasmic reticulum regulates both phagosomal PtdIns4P levels and recruitment of its effectors to the compartment. Finally, I explore the relevance of these events during resolution. This work establishes the importance of PtdIns4P during phagocytosis emphasizing the necessity of an exquisite coordination of lipid metabolism during the process.Ph.D.2019-11-19 00:00:0