Spatiotemporal control of reactive oxygen species in T cells

说明(申请人提供)：活性氧物种(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--并在离产生地点只有几纳米的地方发挥作用。关于细胞如何利用和区分质膜产生的、线粒体的或细胞外的活性氧源来控制信号转导，人们知之甚少。这项应用的目的是通过开发特定部位的ROS染料、高通量微流控系统和计算模型来研究T细胞信号转导过程中的ROS时空动力学。我们假设，ROS的亚细胞来源在线粒体、内质网和质膜氧化酶之间建立了一个紧密联系的网络，以调节T细胞信号。这项研究的基本原理是，通过了解在抗原识别过程中何时何地使用ROS来靶向蛋白质氧化，细胞氧化可以从现象学观察转移到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，以便于研究特定部位的ROS对T细胞激活的影响。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，能够产生能够传递外源分子的时间可调(即振荡)刺激。这些技术将被用来确定局部ROS来源对T细胞信号的贡献，并研究ROS生成和钙之间的时空关系。单细胞分析和控制系统理论将被用来生成钙水平和亚细胞ROS隔间之间反馈控制的计算模型。这项拟议的研究具有创新性，因为它融合了新的成像探针和微流控平台的技术发展，以应对在T细胞信号传递过程中分析局部(而不是全球)氧化应激的挑战。这项工作的结果有望从根本上促进我们对细胞如何利用空间上不同的ROS来源来调节受体启动的信号的理解。这一知识将对最终通过更多与疾病诊断和治疗的生物学相关指标重新定义细胞内氧化产生重大影响。

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.