SHP1 Phosphatase/SYK Kinase Balance Controls the Actin Economy and Macropinocytosis in Macrophages

SHP1 磷酸酶/SYK 激酶平衡控制巨噬细胞中的肌动蛋白经济和巨胞饮作用

• 批准号: 10593084
• 负责人: Natalie Wendt Thiex
• 金额: $ 38.92万
• 依托单位: SOUTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-20 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593084
• 关键词: 3-Dimensional; Actins; Animals; Anti-Inflammatory Agents; Antigen Presentation; Autoimmune Diseases; Biological Response Modifiers; CRISPR imaging; CRISPR/Cas technology; Cell membrane; Cell physiology; Cells; Centers of Research Excellence; Chimeric Proteins; Data; Disease; Endotoxins; Equilibrium; Extracellular Fluid; F-Actin; Fluorescent Probes; Frequencies; G Actin; Gene Expression; Genes; Goals; ITAM; ITIM; Image; Immunoassay; Immunologic Receptors; Immunologic Surveillance; Inflammation; Inflammatory; Knockout Mice; Knowledge; Laboratories; Ligands; Macrophage; Macrophage Activation; Measures; Mediating; Mediator; Membrane; Metabolism; Microscopy; Modeling; Mus; NR0B2 gene; Pathway Analysis; Phagocytosis; Phenotype; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphotransferases; Polymers; Process; Regulation; Research; Resolution; Rest; Role; SYK gene; Shapes; Signal Transduction; Stimulus; TLR4 gene; TYROBP gene; Techniques; Testing; Translational Research; Virus Diseases; Work; cell motility; chronic wound; drinking; extracellular; imaging approach; inhibitor; innovation; insight; live cell imaging; live cell microscopy; novel; polymerization; promoter; recruit; response; solute; spatiotemporal; stem; therapeutic target; tool; tool development; transcriptome; uptake; whole genome; wound healing

Project Summary/Abstract Macropinocytosis, or “cell drinking,” is central to several macrophage functions including wound healing, antigen presentation, and 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 novel mediators and cellular mechanisms of macropinocytosis. The overall objective is to investigate how macrophages regulate actin dynamics, phosphoinositide signaling and macropinocytotic efficiency in response to pro- and anti- inflammatory stimuli. The central hypothesis is that the balance of SHP1 phosphatase and SYK kinase activity optimizes the macrophage “actin economy” for macropinocytosis or other actin-dependent processes depending on the activation state of the cell. This hypothesis stems from preliminary CRISPR/Cas9 whole genome screen data produced in the applicant's laboratory indicating that SHP1 and SYK are key regulators of macropinocytosis. The hypothesis will be tested by pursuing two specific aims: 1) Determine the effect of SYK/SHP1 balance on actin dynamics and phosphoinositide signaling at forming macropinosomes; and 2) Determine how ITAM and ITIM containing immune receptors modulate macropinocytosis via the SHP1/SYK balance in resting and activated macrophages. Under the first aim, an already proven live-cell imaging approach established by the applicant will be used to image actin and phosphoinositide dynamics in wildtype or CRISPR/Cas9 gene-disrupted primary murine macrophages with resting, inflammatory, or anti-inflammatory activation states. Under the second aim, mechanisms of SHP1 and SYK activation by recruitment to immune receptors will be tested using fluorescent protein chimeras of SHP1 or SYK in wild type or gene-disrupted macrophages in response to inflammatory or anti-inflammatory stimuli. This approach is innovative because the hypothesis was generated from novel mediators of macropinocytosis identified by a CRISPR/Cas9 whole genome screen. Furthermore, this strategy uses targeted gene disruptions in combination with cellular fluorescent probes and advanced live-cell microscopy techniques for three-dimensional live-cell imaging of the spatiotemporal dynamics of actin polymerization and phosphoinositide signaling during macropinocytosis. The proposed research is significant because it is expected to expand the understanding of novel mechanisms macropinocytosis and the role of macropinocytosis in inflammation. Ultimately, such knowledge has the potential to identify therapeutic targets for modulation of macropinocytosis efficiency and the treatment of inflammatory diseases such as chronic wounds or auto-immune disorders.

项目摘要/摘要 大型细胞增多症或“细胞饮用”是几种巨噬细胞功能的核心，包括伤口愈合， 抗原表现和炎症的分辨率。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新型介体和蜂窝 大型细胞增多症的机制。总体目的是研究巨噬细胞如何调节肌动蛋白 动力学，磷酸肌醇信号传导和大型诱导效率，以响应促疾病和抗 炎症刺激。中心假设是SHP1磷酸酶和SYK激酶活性的平衡 优化巨噬细胞的“肌动蛋白经济”用于大型细胞增多症或其他依赖肌动蛋白的过程 取决于细胞的激活状态。此假设从初步的CRISPR/CAS9整个 申请人实验室中产生的基因组筛查数据，表明SHP1和SYK是 大型细胞增多症。该假设将通过追求两个具体目的来检验：1）确定的影响 SYK/SHP1在形成大型体体时肌动蛋白动力学和磷酸肌醇信号传导的平衡；和2） 确定ITAM和ITIM如何包含免疫受体通过SHP1/SYK调节大型细胞增多症 平衡静止和激活的巨噬细胞。在第一个目标下，已经经过验证的活细胞成像 申请人建立的方法将用于对野生型中的肌动蛋白和磷酸肌醇动力学进行图像图像 或CRISPR/CAS9基因破坏的主要鼠巨噬细胞，带有静止，炎症或抗炎 激活状态。在第二个目标下，SHP1和SYK激活的机制通过募集到免疫 将使用SHP1或SYK的荧光蛋白嵌合在野生型或基因dimed的荧光蛋白嵌合进行测试 巨噬细胞响应炎症或抗炎刺激。这种方法是创新的，因为 该假设是由由CRISPR/CAS9整体鉴定的新型大型细胞增多症的新型介体产生的 基因组屏幕。此外，该策略使用靶向基因破坏与细胞结合 荧光问题和高级活细胞显微镜技术，用于三维活细胞成像 肌动蛋白聚合和磷酸肌醇信号传导的时空动力学。这 拟议的研究很重要，因为有望扩大对新机制的理解 大型细胞增多症和大型细胞增多症在炎症中的作用。最终，这样的知识具有 鉴定调节大型细胞增多症效率的治疗靶标的潜力 炎症性疾病，例如慢性逻辑或自身免疫性疾病。