Mechanisms of Signaling on Membrane Surfaces

膜表面信号传导机制

• 批准号: 10542420
• 负责人: JOSEPH J FALKE
• 金额: $ 38.04万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10542420
• 关键词: Actins; Address; Autoimmunity; Biology; Cell physiology; Cells; Chemotaxis; Communities; Complex; Defect; Disease; Enzymes; GTP-Binding Proteins; In Vitro; Infection; Inflammation; Laboratories; Leadership; Link; Lipid Bilayers; Lipids; Macrophage; Malignant Neoplasms; Medicine; Membrane; Methods; Modeling; Molecular; Mutation; Natural Immunity; Output; PIK3CG gene; Pathway interactions; Phagocytosis; Phagosomes; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; Play; Positioning Attribute; Production; Protein Kinase; Reaction; Reactive Oxygen Species; Regulation; Research; Role; Signal Pathway; Signal Transduction; Site; Surface; Targeted Research; Testing; Therapeutic; Tissues; Universities; cell growth; cell motility; cellular imaging; developmental disease; human disease; imaging study; innovation; live cell imaging; pathogen; programs; receptor; reconstitution; sensor; single molecule

SUMMARY At the leading edge of a polarized macrophage, a membrane-based chemotaxis pathway directs cell mi- gration up attractant gradients to sites of infection, inflammation, or tissue damage. Upon arrival, a phagocyto- sis pathway controls the formation and internalization of a phagosome in which the pathogens or damaged tis- sue are engulfed and destroyed. Both the chemotaxis and phagocytosis pathways are regulated by PI3K lipid kinases that serve as regulatory hubs by integrating Ca2+, receptor, G protein, and other input signals while phosphorylating substrate lipids to produce output lipid signals. The potent lipid signals, in turn, activate multi- ple downstream protein kinases. In chemotaxis, the lipid signal controls actin and membrane remodeling to drive the leading edge up the attractant gradient. In phagocytosis, the lipid signal controls processing of the phagosome including the production of reactive oxygen species (ROS) to inactivate pathogens. Closely related PI3K pathways regulate other cell processes, notably including cell growth. When dysregulated, PI3K path- ways trigger or exacerbate a wide array of human diseases ranging from cancer and developmental disorders to defects in innate immunity, inflammation or autoimmunity. The two classes of PI3K lipid kinases targeted by this research program are Class 1 PI3-Kinases (PI3K1) that generate the signaling lipid PIP3 at the leading edge membrane of polarized macrophages, and Class 3 PI3-Kinases (PI3K3, specifically PI3K3 Complex II) that produce PI3P on the surface of the phagosome. The proposed research seeks to understand the regulation of both pathways by addressing fundamental, broad questions including: (i) How do PI3K1 and PI3K3 regulatory hubs integrate multiple inputs from Ca2+ channels, receptors, G proteins and other effectors, and do these inputs combine in additive, synergistic, or opposing fashions? (ii) How do the resulting PIP3 and PI3P output lipids activate downstream protein kinases, including some of the most important master kinases in the cell? (iii) How do drugs, potential therapeutics, and disease- linked mutations inhibit or superactivate key components and reaction steps to generate pathway perturbation or dysregulation? To answer these and other questions, the PI's laboratory has developed a unique, two-pronged approach combining innovative, in vitro single molecule methods with live cell imaging studies. The in vitro studies utilize single molecule TIRF to elucidate signaling mechanisms in a subsection of the pathway, or signaling module, that is reconstituted on a supported lipid bilayer under near physiological conditions. The live cell studies em- ploy fluorescent sensors and cell imaging to test key predictions of the in vitro mechanistic model for relevance in the cellular context. The PI has a strong track record and continues to play leadership roles in his research field, as well as the university and scientific communities. Overall, this research program is well positioned to continue generating fundamental advances with significant impacts on signaling biology and medicine.

总结 在极化的巨噬细胞的前沿，一种基于膜的趋化性途径指导细胞迁移， 将引诱剂梯度梯度增加到感染、炎症或组织损伤的部位。到达后，吞噬细胞- SIS途径控制吞噬体的形成和内化，其中病原体或受损组织- 苏被吞没和摧毁。趋化和吞噬途径均受PI 3 K脂质的调节 激酶作为调节中心，通过整合Ca 2+、受体、G蛋白和其他输入信号， 磷酸化底物脂质以产生输出脂质信号。这些强有力的脂质信号反过来又激活了多- 下游蛋白激酶。在趋化性中，脂质信号控制肌动蛋白和膜重塑， 将前缘推向引诱剂梯度。在吞噬作用中，脂质信号控制细胞的加工， 吞噬体包括产生活性氧（ROS）来抵抗病原体。密切相关 PI 3 K途径调节其他细胞过程，特别是包括细胞生长。当失调时，PI 3 K通路- 引发或加剧多种人类疾病的方式， 先天免疫缺陷、炎症或自身免疫。 本研究计划靶向的两类PI 3 K脂质激酶是1类PI 3激酶（PI 3 K1） 在极化巨噬细胞的前缘膜产生信号脂质PIP 3， 在吞噬体表面产生PI 3 P的PI 3-激酶（PI 3 K3，特别是PI 3 K3复合物II）。的 拟议的研究旨在通过解决基本的，广泛的， 问题包括：（i）PI 3 K1和PI 3 K3调节中心如何整合来自Ca 2+通道的多种输入， 受体、G蛋白和其他效应物，以及这些输入联合收割机是以相加、协同还是相反的方式结合 时尚？(ii)产生的PIP 3和PI 3 P输出脂质如何激活下游蛋白激酶，包括 细胞中一些最重要的主激酶(iii)药物，潜在的治疗方法和疾病- 连锁突变抑制或超活化关键组分和反应步骤，以产生途径干扰 还是失调 为了回答这些和其他问题，PI的实验室开发了一种独特的双管齐下的方法 将创新的体外单分子方法与活细胞成像研究相结合。体外研究利用 单分子TIRF，以阐明途径的一个子部分中的信号传导机制，或信号传导模块， 其在接近生理条件下在支持的脂质双层上重构。活细胞研究em- 使用荧光传感器和细胞成像来测试体外机制模型的关键预测的相关性 in the cellular细胞context上下文. PI拥有良好的业绩记录，并继续在其研究中发挥领导作用 该领域，以及大学和科学界。总的来说，这项研究计划是很好的定位， 继续产生对信号生物学和医学产生重大影响的根本性进展。