High-dimensional characterization of phagosome composition, control and phagocytic receptor redundancy

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Project Summary The phagosome is a dynamically formed organelle that is generated upon phagocyte encounter with cargo. Phagocytic receptors and other extracellular receptors engage with cargo-derived ligands prior to the formation of the phagocytic cup at the cell membrane and subsequent phagocytosis. Following phagosome formation, a dynamic series of steps proceed involving organelle trafficking and fusion. Ultimately, these collective molecular events influence and shape phagosome function which is often characterized through the lens of phagosome biochemistry (pH, metal ion abundance, oxidative radicals, and enzyme activity). While many of the stereotyped features of phagosome maturation and biochemistry have been studied, there has been relatively fewer studies that take an integrated systems-level view from signaling to phagosome biochemistry. Furthermore, while the field has defined several features of general phagocytosis, phagosome biology is incredibly complex. Several distinct cell types can perform phagocytosis ranging from professional phagocytes (ex: macrophages, neutrophils, dendritic cells) to non-professional phagocytes (ex: fibroblasts). Adding another layer of complexity, phagocytes engulf a diverse array of cargo ranging from pathogens to apoptotic bodies. Combined with the temporal maturation of the phagosome, these three axes construct a complex landscape for phagosome biology. In depth study of this landscape has not been performed limiting our fundamental understanding of molecular control of this organelle. Here, we propose a research program centered around the question: “how is control of phagosome biology achieved?” To address these questions, my research program integrates approaches in genetics, protein engineering, systems biology, immunology, and microbiology. We seek to address three knowledge gaps in our program initially. (1) Is there crosstalk in signaling among receptors (phagocytic and soluble ligand) during phagocytosis? (2) How do cargo and phagocyte identity instruct phagosome composition? (3) What are the molecular circuits that control phagosome biochemistry? Over the next five years, we will develop a strategy to examine higher-order interactions in phagocytosis signaling. Furthermore, we will engineer specific cargo capable of performing proximity labeling in the phagosome. Lastly, we will define the molecular circuits that control phagosome biochemistry. These questions are inextricably coupled, and our program operates in a highly collaborative manner. Supporting our experimental systems is a strong quantitative modeling and analytical framework equipped to derive novel insights from high-throughput experiments and propose new experimental directions. Together, these strengths position us in a unique manner to address longstanding questions in phagosome biology.