Oscilloscopes Spatio-Temporal Metabolomics (RMI)

描述(由申请人提供)：该项目的目标是开发一种在细胞水平上实时在体内对代谢波动进行时空量化的系统。为了实现这一目标，我们结合了四项关键技术：i)报告特定代谢物的基因编码的荧光纳米传感器蛋白(GENA)；ii)允许针对大量感兴趣的代谢物系统构建纳米传感器的蛋白质工程技术；iii)允许在活细胞中使用这些纳米传感器的定量实时荧光显微镜可视化技术；iv)通过功能获得(蛋白质过度表达)和功能丧失(缺失文库，RNAi)扰乱信号转导途径的基因工程细胞系。我们的方法是可推广的、可扩展的、敏感的和动态的，能够在毫秒范围内进行时间分辨测量。使用这些试剂，预计将分析超过10,000个信号状态扰动以了解代谢变化。预计，许多信号通路的扰动和代谢谱的改变将提供一个大型数据库，包括相关变化、疾病状态的概括和诊断潜力以及信号网络的新的生物学发现。我们组建了一个由四个小组组成的研究小组，每个小组都为项目带来了所需的部分技能。迈耶集团(斯坦福大学)长期参与GENAs的开发，并大力参与代谢可视化荧光显微镜仪器的开发。此外，迈耶团队还参与了针对代谢扰动的试剂集的开发。弗罗默小组(华盛顿卡内基研究所)已经开发出用于感知糖分的GENA，并证明了它们可以用于体内的代谢成像。约克小组(杜克大学)一直深入参与遗传操作、新陈代谢和细胞内信号通路的发现。Hellinga小组(杜克大学)在计算蛋白质设计工具的开发和实验验证中发挥了重要作用，这些工具用于自由基操作配体结合的特异性。在项目期结束时，我们的目标是交付i)纳米传感器工具包；ii)工程细胞系工具包；iii)代谢波动的时空成像数据集，作为遗传扰动和细胞环境外源变化的函数。

DESCRIPTION (provided by applicant): The goal of this project is develop a system for real-time in vivo spatiotemporal quantification of metabolic fluctuations at the cellular level. To achieve this, we combines four key technologies: i) genetically encoded fluorescent nanosensor proteins (GENAs) that report on specific metabolites; ii) protein engineering techniques that permit the systematic construction of nanosensors for a large number of metabolites of interest; iii) quantitative real-time fluorescent microscopy visualization techniques that permit these nanosensors to be used in living cells; iv) genetically engineered cell lines that perturb signal transduction pathways by gain-of-function (protein over-expression) and loss-of-function (deletion libraries, RNAi). Our approach is generalizable, scalable, sensitive and dynamic enabling time-resolved measurements in the millisecond range. Using these reagents, it is anticipated that over 10,000 signaling state perturbations will be analyzed for metabolic changes. It is anticipated that many of the signal pathway perturbations and alterations in metabolic profiles will provide a large database of correlative changes, recapitulation and diagnostic potential of disease states and novel biological discovery of signaling networks. We have assembled a research team consisting of four groups that each bring part of the required skills to the project. The Meyer group (Stanford University) has a long-standing involvement in the development of GENAs, and has also been heavily involved in the development of the fluorescence microscopy instrumentation for metabolic visualization. Furthermore, the Meyer group is also involved in development of reagent sets for metabolic perturbations. The Frommer group (Carnegie Institute of Washington) has developed GENAs for sensing of sugars, and has demonstrated that these can be used for in vivo metabolic imaging. The York group (Duke University) has been deeply involved in the genetic manipulation, metabolism and discovery of intracellular signaling pathways. The Hellinga group (Duke University) has been instrumental in the development and experimental validation of computational protein design tools for the radical manipulation of ligand-binding specificities. At the end of the project period, we aim to deliver i) nanosensor toolkits; ii) engineered cell line toolkits; iii) spatiotemporal imaging datasets of metabolic fluctuations as a function of genetic perturbations and exogenous changes in cellular environment.