Spatiotemporal control of reactive oxygen species in T cells

描述（由申请人提供）：活性氧（ROS）在不同的细胞位置产生-通过氧化酶和线粒体的细胞器位置-并在距离产生部位仅几纳米的地方发挥作用。关于细胞如何使用和区分质膜产生的、线粒体的或细胞外来源的活性氧来控制信号转导，知之甚少。本申请的目的是通过开发位点特异性ROS染料、高通量微流体系统和计算模型来研究T细胞信号传导过程中ROS的时空动态。我们推测ROS的亚细胞来源在线粒体、内质网和质膜氧化酶之间形成了一个紧密连接的网络，以调节T细胞信号传导。这项研究的基本原理是，通过了解ROS在抗原识别过程中何时何地用于靶向蛋白质氧化，细胞氧化可以从现象学观察转变为疾病状态的相关诊断生物标志物。在这个项目中，我们将开发两种使能技术，以促进研究位点特异性活性氧对T细胞活化的影响。首先，我们将创建一种新的膜特异性染料，用于检测NAPDH氧化酶产生的超氧化物。其次，我们将设计用于单细胞操作和高通量成像分析的微流控平台，能够产生时间可调（即振荡）刺激递送外源分子。这些技术将用于确定局部ROS源对T细胞信号传导的贡献，并研究ROS生成与钙之间的时空关系。单细胞分析和控制系统理论将用于生成钙水平和亚细胞ROS区室之间的反馈控制的计算模型。这项研究具有创新性，因为它融合了新成像探针和微流体平台的技术发展，以解决在T细胞信号传导过程中分析局部（而不是全局）氧化应激的挑战。这项工作的结果有望从根本上推进我们对细胞如何使用空间上不同的ROS源来调节受体启动的信号传导的理解。这些知识将对最终通过用于疾病诊断和治疗的更生物相关的指标重新定义细胞内氧化产生重大影响。 公共卫生相关性：细胞氧化，即无法补偿活性氧的产生，是许多免疫相关疾病的标志。由于全球测量细胞氧化不提供任何时空信息的活性氧产生，与氧化应激相关的疾病状态被过于简化。该项目通过分子探针和单细胞分析来提高我们对亚细胞活性氧如何控制T细胞功能的理解。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) are produced in distinct cellular locations - by the organelle location of oxidases and mitochondria - and exert their effects only nanometers from the site of production. Little is known about how cells use and discriminate between plasma membrane generated, mitochondrial, or extracellular sources of reactive oxygen species to control signal transduction. The objective of this application is to investigate ROS spatiotemporal dynamics during T cell signaling through the development of site-specific ROS dyes, high-throughput microfluidic systems, and computational models. We hypothesize that the subcellular sources of ROS create a tightly connected network between mitochondria, endoplasmic reticulum and plasma membrane oxidases to regulate T cell signaling. The rationale for this research is that by understanding when and where ROS is used to target protein oxidation during antigen recognition, cellular oxidation can move from phenomenological observation to a relevant diagnostic biomarker for disease state. In this project we will develop two enabling technologies to facilitate the investigation of site- specific ROS on T cell activation. First, we will create a new membrane-specific dye for detection of superoxide production by NAPDH oxidases. Secondly, we will design microfluidic platforms for single cell manipulation and high-throughput imaging analysis, capable of generating temporally tunable (i.e. oscillatory) stimulations delivering exogenous molecules. These technologies will be used to determine the contributions of localized ROS sources to T cell signaling and investigate spatiotemporal relationships between ROS generation and calcium. Single cell analysis and control systems theory will be used to generate computational models of feedback control between calcium levels and subcellular ROS compartments. The proposed research is innovative because it merges the technological developments of new imaging probes and microfluidic platforms to address the challenge of analyzing local (rather than global) oxidative stress during T cell signaling. The outcomes of this work are expected to fundamentally advance our understanding of how cells use spatially distinct ROS sources to regulate receptor-initiated signaling. This knowledge will have large impact in ultimately redefining intracellular oxidation by more biologically relevant metrics for diagnosis and treatment of diseases. PUBLIC HEALTH RELEVANCE: Cellular oxidation, an inability to compensate against production of reactive oxygen species, is a hallmark of numerous immune-related diseases. Because global measurements of cellular oxidation do not provide any spatiotemporal information about reactive oxygen species production, disease states associated with oxidative stress are oversimplified. This project advances methods molecular probes and single cell analysis to improve our understanding of how subcellular reactive oxygen species control the functioning of T cells.