Regulation of Phagocyte Immunity by Lipid Signals

Phagocytosis, the process by which relatively large particles are internalized into a membrane-bound compartment known as the phagosome, is paramount to both fighting infection and maintaining tissue homeostasis. Professional phagocytes routinely contend with a large assortment of targets, ranging from small pathogenic bacteria to large fungal networks. Non-professional and professional phagocytes also cooperate in the daunting yet critical task of clearing hundreds of billions of apoptotic cells on a daily basis. By promptly removing effete cells, phagocytes prevent secondary necrosis and the onset of autoimmunity, aid in tissue remodelling during wound healing, and orchestrate morphogenesis throughout development. To carry out these functions, phagocytes must be able to effectively find, engulf and dispose of their prey. In this dissertation, I focus on the phagocytic response from the conceptual stages of: 1) capture, 2) uptake and 3) degradation-all presented as self-contained chapters-while placing particular emphasis on the mechanisms by which glycerophospholipids and diacylglycerols regulate these processes. I provide evidence that phosphatidic acid, which is uniquely enriched in the plasmalemma of professional phagocytes, facilitates the continuous membrane ruffling that underlies the uptake of particulate and fluid-phase antigen. Similarly, I argue that phosphatidylinositol-3,4,5-trisphosphate biosynthesis signals actin remodelling during the uptake of large particles, thereby mediating the orderly progression of pseudopodia around large targets. I then discuss the role of diacylglycerol kinases in controlling deployment of the NADPH oxidase, thus fine-tuning the compromise between eliciting effective antimicrobial responses and preventing oxidative damage to the host. Notably, phosphoinositide signalling is often subverted by invading microorganisms in order to avoid phagocyte recognition. The final study in this thesis, also a self-contained chapter, describes a phosphoinositide-centric mechanism by which gliotoxin, an Aspergillus fumigatus mycotoxin, precludes macrophage immune defences.Ph.D

Chemokine Signaling Enhances CD36 Responsiveness toward Oxidized Low-Density Lipoproteins and Accelerates Foam Cell Formation

Excessive uptake of oxidized low-density lipoproteins (oxLDL) by macrophages is a fundamental characteristic of atherosclerosis. However, signals regulating the engagement of these ligands remain elusive. Using single-molecule imaging, we discovered a mechanism whereby chemokine signaling enhanced binding of oxLDL to the scavenger receptor, CD36. By activating the Rap1-GTPase, chemokines promoted integrin-mediated adhesion of macrophages to the substratum. As a result, cells exhibited pronounced remodeling of the cortical actin cytoskeleton that increased CD36 clustering. Remarkably, CD36 clusters formed predominantly within actin-poor regions of the cortex, and these regions were primed to engage oxLDL. In accordance with enhanced ligand engagement, prolonged exposure of macrophages to chemokines amplified the accumulation of esterified cholesterol, thereby accentuating the foam cell phenotype. These findings imply that the activation of integrins by chemokine signaling exerts feedforward control over receptor clustering and effectively alters the threshold for cells to engage ligands

Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes

Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking