Mechanisms of Signaling on Membrane Surfaces

摘要 在极化的巨噬细胞的前沿，一条基于膜的趋化途径引导细胞移动。 对感染、炎症或组织损伤的部位进行吸引剂梯度的磨光。到达后，吞噬细胞- SIS途径控制着吞噬小体的形成和内化，在吞噬小体中，病原体或受损的组织和 苏被吞没并毁灭了。趋化和吞噬途径均受PI3K脂类调控 通过整合钙离子、受体、G蛋白和其他输入信号作为调节中心的激酶，而 使底物脂质磷酸化以产生输出脂质信号。强大的脂质信号反过来激活了多个- PPLE下游蛋白激酶。在趋化作用中，脂质信号控制肌动蛋白和膜重塑，以 将前缘沿吸引剂渐变向上驱动。在吞噬过程中，脂质信号控制着 吞噬小体包括产生活性氧种(ROS)以灭活病原体。密切相关 PI3K通路调节其他细胞过程，尤其是细胞生长。当失调时，PI3K通路- 引发或加剧一系列人类疾病的方式，从癌症到发育障碍 先天免疫缺陷、炎症或自身免疫缺陷。 本研究计划针对的两类PI3K脂蛋白激酶是1类PI3-激酶(PI3K1)。 在极化的巨噬细胞的前沿膜上产生信号脂质PIP3，以及Class3 在吞噬小体表面产生PI3P的PI3-激酶(PI3K3，特别是PI3K3复合体II)。这个 拟议的研究试图通过处理基本的、广泛的 问题包括：(I)PI3K1和PI3K3调控中枢如何整合来自钙离子通道的多个输入， 受体、G蛋白和其他效应器，以及这些输入是否以相加、协同或相反的方式结合 时尚？(2)由此产生的PIP3和PI3P输出脂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- 连锁突变抑制或过度激活关键成分和反应步骤，以产生途径扰动 还是监管失调？ 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来阐明通路的一部分或信号模块中的信号机制， 这是在接近生理条件下在支撑的脂质双层上重建的。活细胞研究EM- 用多重荧光传感器和细胞成像测试体外机制模型的关键预测的相关性 在蜂窝环境中。私家侦探有着良好的记录，并继续在他的研究中发挥领导作用 实地以及大学和科学界。总体而言，这项研究计划处于有利地位， 继续产生根本性的进展，对信号生物学和医学产生重大影响。

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.