Determining the Role of the Mannose Receptor in Macrophage Macropinocytosis

项目总结/摘要 巨胞饮作用，或“细胞饮水”，是关键的巨噬细胞功能的核心，包括伤口愈合，抗原 呈现，和炎症的解决。然而，在机制方面存在很大差距， 理解这个过程。该项目的长期目标是确定介质和细胞 巨胞饮作用的机制。总体目标是研究巨噬细胞甘露糖 受体（MRC 1），一种细胞表面碳水化合物受体，介导从细胞中摄取液体和溶质， 环境通过macropinocytosis中心假设是MRC 1促进了分支的摄取。 糖和其他配体通过结合细胞外配体并介导其随后的内化， 新形成的大胞饮体。这一假设源于初步的CRISPR/Cas9全基因组筛选 在申请人的实验室中产生的数据，证明Mrc 1基因和调节Mrc 1基因的分子， 细胞表面的MRC 1蛋白丰度是巨胞饮的关键调节因子。假设是 通过追求两个特定目标进行测试：1）确定MRC 1如何促进巨噬细胞中葡聚糖的摄取， 和2）确定在存在或不存在以下物质的情况下，MRC 1从细胞表面内化的机制： 葡聚糖和其他配体。在第一个目标下，PI实验室建立的化学缀合技术将 用于制备不同分子量、电荷和荧光团缀合的葡聚糖， 调节野生型葡聚糖摄取效率的化学和物理参数的评价 和MRC 1缺陷型巨噬细胞。第二个目标将确定MRC 1摄取进入细胞的途径。 使用由申请人建立的免疫荧光染色/显微镜方法， 在巨胞饮作用期间对MRC 1的运动进行成像。具体而言，MRC 1与以下位点的共定位 将测量肌动蛋白聚合、膜突出和3 ′-磷酸肌醇的产生。这 方法是创新的，因为该假设是从新的巨胞饮介质产生的 通过CRISPR/Cas9全基因组筛选鉴定。此外，该策略使用靶向基因破坏 结合新的细胞荧光探针和活细胞显微镜技术， 巨胞饮作用的机制。拟议的研究意义重大，因为它有望扩大 理解巨胞饮作用的机制和分子机制。最终，这些知识 确定调节巨胞饮作用的治疗靶点和优化靶向的潜力 用于使用MRC 1配体治疗癌症或免疫疾病的巨噬细胞治疗剂。

Project Summary/Abstract Macropinocytosis, or “cell drinking,” is central to critical macrophage functions including wound healing, antigen presentation, and the resolution of inflammation. However, there are large gaps in the mechanistic understanding of this process. The long-term goal of this project is to identify the mediators and the cellular mechanisms of macropinocytosis. The overall objective is to investigate how the macrophage mannose receptor (MRC1), a cell surface carbohydrate receptor, mediates uptake of fluids and solutes from the cellular environment via macropinocytosis. The central hypothesis is that MRC1 promotes the uptake of branched sugars and other ligands by binding extracellular ligands and mediating their subsequent internalization on newly forming macropinosomes. This hypothesis stems from preliminary CRISPR/Cas9 whole genome screen data produced in the applicant's laboratory, demonstrating that the Mrc1 gene and molecules that regulate MRC1 protein abundance on the cell surface are key regulators of macropinocytosis. The hypothesis will be tested by pursuing two specific aims: 1) Determine how MRC1 promotes uptake of dextran in macrophages, and 2) Determine the mechanisms of MRC1 internalization from the cell surface in the presence or absence of dextran and other ligands. Under the first aim, a chemical conjugation technique established in the PI's lab will be used to prepare dextrans of different molecular weights, charges and fluorophore conjugations for evaluation of the chemical and physical parameters that modulate the uptake efficiency of dextrans by wildtype and MRC1-deficient macrophages. The second aim will determine the route of MRC1 uptake into macropinosomes using an immunofluorescent staining/microscopy approach established by the applicant to image the movements of MRC1 during macropinocytosis. Specifically, co-localization of MRC1 with sites of actin polymerization, membrane protrusion, and 3'-phosphoinositide production will be measured. This approach is innovative because the hypothesis was generated from new mediators of macropinocytosis identified by a CRISPR/Cas9 whole genome screen. Furthermore, this strategy uses targeted gene disruptions in combination with new cellular fluorescent probes and live-cell microscopy techniques to interrogate the mechanisms of macropinocytosis. The proposed research is significant because it is expected to expand the understanding of machinery and molecular mechanisms of macropinocytosis. Ultimately, such knowledge has the potential to identify therapeutic targets for the modulation of macropinocytosis and to optimize the targeting of therapeutics to macrophages for treatment of cancer or immune diseases using MRC1 ligands.