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

项目摘要 吞噬体是一种动态形成的细胞器，在吞噬细胞遇到 货物.吞噬细胞受体和其他细胞外受体在细胞凋亡之前与货物衍生的配体接合。 在细胞膜上形成吞噬杯并随后进行吞噬作用。跟随吞噬体 在形成过程中，一系列动态的步骤进行，涉及细胞器的运输和融合。最终，这些 集体分子事件影响和塑造吞噬体功能，其特征通常是通过 吞噬体生物化学的透镜（pH、金属离子丰度、氧化自由基和酶活性）。 虽然已经研究了吞噬体成熟和生物化学的许多定型特征， 从信号传导到吞噬体的综合系统水平的研究相对较少 生物化学此外，虽然该领域已经定义了一般吞噬作用的几个特征，但吞噬体 生物学非常复杂几种不同的细胞类型可以执行吞噬作用，从专业的 吞噬细胞（例如：巨噬细胞、嗜中性粒细胞、树突细胞）至非专职吞噬细胞（例如：成纤维细胞）。 增加了另一层复杂性，吞噬细胞吞噬了各种各样的货物， 凋亡小体结合吞噬体的时间成熟，这三个轴构成了一个 复杂的景观吞噬体生物。对这一景观的深入研究还没有进行， 对这个细胞器的分子控制有基本的了解。 在这里，我们提出了一个研究计划围绕的问题：“如何控制吞噬体 生物学成就？”为了解决这些问题，我的研究计划整合了遗传学，蛋白质， 工程学、系统生物学、免疫学和微生物学。我们寻求解决我们的三个知识差距， 节目最初(1)受体（吞噬细胞和可溶性配体）之间的信号传导是否存在串扰？ 吞噬作用？(2)货物和吞噬细胞的身份如何指导吞噬体的组成？(3)有哪些 控制吞噬体生物化学的分子回路在未来五年，我们将制定一项战略， 检查吞噬信号中的高阶相互作用。此外，我们将工程师的具体货物 能够在吞噬体中进行邻近标记。最后，我们将定义分子电路， 控制吞噬体生物化学。这些问题是密不可分的，我们的计划是在一个高度 合作的方式。支持我们的实验系统是一个强大的定量建模和分析 框架配备从高通量实验中获得新的见解，并提出新的实验 方向总之，这些优势使我们能够以独特的方式解决长期存在的问题， 吞噬体生物学

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.