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

项目摘要 吞噬小体是一种动态形成的细胞器，当吞噬细胞与 货物。吞噬细胞受体和其他细胞外受体与货物衍生的配体在 在细胞膜上形成吞噬杯并随后吞噬。跟随吞噬小体 形成，是涉及细胞器贩运和融合的一系列动态步骤。最终，这些 集体分子事件影响和塑造吞噬小体功能，这通常是通过 吞噬小体生物化学(pH、金属离子丰度、氧化自由基和酶活性)的透镜。 虽然已经研究了吞噬小体成熟和生物化学的许多刻板特征， 从信号转导到吞噬体集成系统水平观点的研究相对较少 生物化学。此外，尽管该领域已经定义了一般吞噬作用的几个特征，但吞噬小体 生物学是极其复杂的。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、中性粒细胞、树突状细胞)到非专业吞噬细胞(例如：成纤维细胞)。 吞噬细胞又增加了一层复杂性，吞噬了从病原体到 凋亡小体。结合吞噬小体的时间成熟，这三个轴构成了一个 噬菌体生物学的复杂景观。对这一景观的深入研究还没有进行，这限制了我们 对这种细胞器的分子控制有基本的了解。 在这里，我们提出了一个围绕这样一个问题的研究计划：吞噬小体是如何控制的 生物学成就了吗？为了解决这些问题，我的研究项目整合了遗传学、蛋白质等领域的方法 工程学、系统生物学、免疫学和微生物学。我们寻求解决以下三个方面的知识差距 最初的计划。(1)受体(吞噬细胞和可溶性配体)之间的信号传递是否存在串扰 吞噬作用？(2)货物和吞噬细胞的特性如何指示吞噬小体的组成？(3)什么是 控制吞噬小体生物化学的分子回路？在未来五年，我们将制定一项战略，以 研究吞噬信号中的高阶相互作用。此外，我们还将设计特定的货物 能够在吞噬小体中进行邻近标记。最后，我们将定义分子电路 控制吞噬小体生化。这些问题是千丝万缕的，我们的节目在高度的 合作的态度。支持我们的实验系统的是强大的定量建模和分析 具备从高通量实验中获得新见解并提出新实验的框架 方向。总而言之，这些优势使我们以独特的方式解决长期存在的问题 噬菌体生物学。

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.