Determining the Role of the Mannose Receptor in Macrophage Macropinocytosis

项目摘要/摘要 巨噬细胞吞噬，或“细胞饮水”，是巨噬细胞关键功能的中心，包括伤口愈合、抗原 呈现，以及炎症的消退。然而，在机械方面有很大的差距。 对这个过程的理解。这个项目的长期目标是确定介体和细胞 巨噬细胞吞噬的机制。总体目标是研究巨噬细胞甘露糖是如何 受体(MRC1)是一种细胞表面碳水化合物受体，介导细胞对液体和溶质的摄取。 通过巨噬细胞吞噬作用的环境。中心假说是MRC1促进了对分支的摄取 糖和其他配体通过结合细胞外配体并介导它们随后的内化 新形成的大管胞小体。这一假说源于CRISPR/Cas9全基因组初步筛查 申请人实验室产生的数据表明，mrc1基因和调节 细胞表面MRC1蛋白的丰富是巨噬细胞吞噬的关键调节因子。假设将是 通过追求两个特定目标进行测试：1)确定MRC1如何促进巨噬细胞对葡聚糖的摄取， 以及2)确定在存在或不存在的情况下MRC1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，在PI的实验室中建立的化学共轭技术将 用于制备不同分子量、电荷和荧光团的葡聚糖偶联物 影响野生型葡聚糖摄取效率的化学和物理参数的评价 和MRC1缺乏的巨噬细胞。第二个目标将确定MRC1被吸收到 使用由申请人建立的免疫荧光染色/显微镜方法来 想象一下巨噬细胞吞噬过程中MRc1的运动。具体地说，MRC1与以下站点共本地化 将测量肌动蛋白聚合、膜突出和3‘-磷脂酰肌醇的产量。这 方法是创新的，因为这一假说是由巨饮细胞增多的新介质产生的 经CRISPR/Cas9全基因组筛选鉴定。此外，这一策略使用了定向基因中断。 结合新的细胞荧光探针和活细胞显微镜技术来询问 巨噬细胞吞噬的机制。这项拟议的研究具有重要意义，因为它有望扩大 了解巨噬细胞增多的机制和分子机制。归根结底，这些知识已经 确定调节巨噬细胞增多的治疗靶点和优化靶向的可能性 利用MRC1配体向巨噬细胞提供治疗药物，用于治疗癌症或免疫疾病。

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.