Mechanisms of Signaling on Membrane Surfaces

膜表面信号传导机制

基本信息

批准号：
10339123
负责人：
JOSEPH J FALKE
金额：
$ 43.81万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
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/10339123
关键词：
1-Phosphatidylinositol 3-Kinase 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 Malignant Neoplasms Medicine Membrane Methods Modeling Molecular Mutation Natural Immunity Output Pathway interactions Phagocytosis Phagosomes Pharmaceutical Preparations 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 base cell growth cellular imaging developmental disease human disease imaging study innovation live cell imaging macrophage pathogen programs receptor reconstitution sensor single molecule

项目摘要

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.

概括在极化巨噬细胞的前沿，基于膜的趋化途径引导细胞mi- 增加感染、炎症或组织损伤部位的引诱剂梯度。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并被摧毁。趋化和吞噬途径均受 PI3K 脂质调节激酶通过整合 Ca2+、受体、G 蛋白和其他输入信号来充当调节中心，同时磷酸化底物脂质以产生输出脂质信号。有效的脂质信号反过来激活多 ple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驱动前缘沿着引诱剂梯度上升。在吞噬作用中，脂质信号控制着吞噬过程吞噬体包括产生活性氧（ROS）以灭活病原体。密切相关 PI3K 通路调节其他细胞过程，特别是细胞生长。当失调时，PI3K 路径- 引发或加剧包括癌症和发育障碍在内的多种人类疾病的方式先天免疫、炎症或自身免疫缺陷。该研究计划针对的两类 PI3K 脂质激酶是 1 类 PI3 激酶 (PI3K1) 在极化巨噬细胞的前缘膜上产生信号脂质 PIP3，以及 3 类 PI3-激酶（PI3K3，特别是 PI3K3 复合物 II）在吞噬体表面产生 PI3P。这拟议的研究旨在通过解决基本的、广泛的问题来了解这两种途径的调节问题包括：(i) PI3K1 和 PI3K3 调节中心如何整合来自 Ca2+ 通道的多种输入，受体、G 蛋白和其他效应子，这些输入是否以相加、协同或相反的方式组合时尚？ (ii) 由此产生的 PIP3 和 PI3P 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、潜在疗法和疾病如何- 连锁突变抑制或超激活关键成分和反应步骤以产生通路扰动还是调节失调？为了回答这些问题和其他问题，PI 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 阐明通路的一个分段或信号模块中的信号机制，它在接近生理条件下在支持的脂质双层上重建。活细胞研究- 利用荧光传感器和细胞成像来测试体外机械模型的关键预测的相关性在细胞环境中。 PI 拥有良好的业绩记录并继续在其研究中发挥领导作用领域以及大学和科学界。总体而言，该研究计划非常适合继续取得对信号生物学和医学产生重大影响的根本性进展。