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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

基本信息

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
项目类别：
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

项目摘要

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.

项目总结/文摘