Determining the Role of the Mannose Receptor in Macrophage Macropinocytosis

确定甘露糖受体在巨噬细胞巨胞饮作用中的作用

• 批准号: 10047139
• 负责人: Natalie Wendt Thiex
• 金额: $ 43.31万
• 依托单位: SOUTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10047139
• 关键词: Actins; Affect; Anti-Inflammatory Agents; Antigen Presentation; Autoimmune Diseases; Bacteria; Binding; Bone Marrow; Brightfield Microscopy; C-terminal; CPG-oligonucleotide; CRISPR/Cas technology; Carbohydrates; Cell membrane; Cell physiology; Cell surface; Cells; Charge; Chemicals; Chimeric Proteins; Clathrin; Collagen; Data; Dendritic Cells; Dextrans; Endocytosis; Environment; Epitopes; Evaluation; Exposure to; Extracellular Fluid; Flow Cytometry; Fluorescence Microscopy; Fluorescent Probes; Fucose; Genes; Goals; Image; Immune System Diseases; Immunologic Surveillance; Inflammation; Inflammatory; Integral Membrane Protein; Interleukin-4; Island; Knowledge; Laboratories; Ligand Binding; Ligands; Liquid substance; Malignant Neoplasms; Mannans; Mannose; Measurement; Measures; Mediating; Mediator of activation protein; Membrane; Metabolism; Microscopy; Mission; Molecular; Molecular Weight; Movement; Mus; Mutate; Organelles; Pathogenesis; Pharmacologic Substance; Phase; Phenotype; Phosphatidylinositols; Phosphotransferases; Polymers; Process; Production; Proteins; Public Health; Research; Resolution; Role; Route; Signal Transduction; Site; Specificity; Stains; Sulfate; Surface; Techniques; Testing; Therapeutic; Tumor-associated macrophages; United States National Institutes of Health; Virus; Virus Diseases; Work; cancer therapy; carbohydrate receptor; cytokine; drinking; extracellular; fluorophore; human disease; inhibitor/antagonist; innovation; live cell microscopy; lucifer yellow; macrophage; mannose receptor; novel; polymerization; receptor; solute; stem; sugar; targeted delivery; targeted treatment; therapeutic target; tool; trafficking; tripolyphosphate; uptake; whole genome; wound healing

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.

项目摘要/摘要 大型细胞增多症或“细胞饮用”是关键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。但是，机械差距很大 了解这一过程。该项目的长期目标是识别介体和细胞 大型细胞增多症的机制。总体目的是研究巨噬细胞的方式 受体（MRC1），一种细胞表面碳氢化物受体，介导了从细胞的液体和溶剂的摄取 通过大型细胞增多症的环境。中心假设是MRC1促进了分支的吸收 糖和其他配体通过结合细胞外配体并介导其随后的内在化 新形成的大型大体。此假设从初步的CRISPR/CAS9整个基因组筛选 申请人实验室产生的数据，表明MRC1基因和调节的分子 细胞表面上的MRC1蛋白丰度是大型细胞增多症的关键调节剂。假设将是 通过追求两个具体目的测试：1）确定MRC1如何促进巨噬细胞中葡聚糖的吸收， 2）在存在或不存在的情况下确定细胞表面MRC1内在化的机制 葡聚糖和其他配体。在第一个目标下，PI实验室中建立的化学共轭技术将 用于制备不同分子量，电荷和荧光团共轭的葡萄晶 评估WildType调节葡聚糖摄取效率的化学和物理参数 和MRC1缺乏巨噬细胞。第二个目标将决定MRC1吸收的路线 使用申请人建立的免疫荧光染色/显微镜方法的大斑小体 图像MACROPINOTOPOSOS期间MRC1的运动。具体而言，MRC1共定位 将测量肌动蛋白聚合，膜蛋白和3'-磷酸肌醇的产生。这 方法是创新的，因为该假设是由大型细胞增多症的新介体产生的 由CRISPR/CAS9全基因组筛选确定。此外，该策略使用针对性的基因破坏 结合新的细胞荧光探针和活细胞显微镜技术来审问 大型细胞增多症的机制。拟议的研究很重要，因为它有望扩大 理解大型细胞增多症的机械和分子机制。最终，这些知识有 确定调节大型细胞增多症并优化靶向的治疗靶标的潜力 巨噬细胞理论上使用MRC1配体治疗癌症或免疫疾病的理论。