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.

项目总结/摘要 巨胞饮作用，或“细胞饮水”，是几种巨噬细胞功能的核心，包括伤口愈合， 抗原呈递和炎症消退。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新的介质和细胞 巨胞饮作用的机制。总体目标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巨胞饮效率， 炎症刺激。中心假设是SHP 1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”，用于巨胞饮或其他肌动蛋白依赖性过程 这取决于电池的激活状态。这一假设源于初步的CRISPR/Cas9整体 在申请人的实验室中产生的基因组筛选数据表明SHP 1和SYK是 巨胞饮作用该假设将通过追求两个具体目标进行测试：1）确定 SYK/SHP 1在形成大胞饮体时对肌动蛋白动力学和磷酸肌醇信号传导的平衡;以及2） 确定含有ITAM和ITIM的免疫受体如何通过SHP 1/SYK调节巨胞饮作用 在静止和激活的巨噬细胞中的平衡。在第一个目标下，一个已经被证明的活细胞成像 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成像 或CRISPR/Cas9基因破坏的原代鼠巨噬细胞与静息的、炎性的或抗炎的巨噬细胞联合使用。 激活状态。在第二个目标下，SHP 1和SYK通过募集免疫细胞激活的机制是： 受体将使用野生型或基因破坏的SHP 1或SYK的荧光蛋白嵌合体进行测试。 巨噬细胞对炎症或抗炎刺激的反应。这种方法是创新的，因为 该假设是由CRISPR/Cas9整体鉴定的巨胞饮新介质产生的， 基因组筛选此外，该策略结合细胞技术使用靶向基因破坏 荧光探针和先进的活细胞显微镜技术的三维活细胞成像的 巨胞饮作用期间肌动蛋白聚合和磷酸肌醇信号传导的时空动力学。的 拟议的研究意义重大，因为它有望扩大对新机制的理解 巨胞饮和巨胞饮在炎症中的作用。最终，这些知识 鉴定用于调节巨胞饮效率和治疗 炎症性疾病，如慢性伤口或自身免疫性疾病。