Oscilloscopes Spatio-Temporal Metabolomics (RMI)

描述（由申请人提供）：该项目的目标是开发一个系统，用于在细胞水平上对体内代谢波动进行实时时空量化。为了实现这一目标，我们结合了四项关键技术：i)报告特定代谢物的遗传编码荧光纳米传感器蛋白（GENAs）；Ii)蛋白质工程技术，允许对大量感兴趣的代谢物系统地构建纳米传感器；Iii)定量实时荧光显微镜可视化技术，允许这些纳米传感器在活细胞中使用；iv)通过功能获得（蛋白质过表达）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。我们的方法是通用的，可扩展的，敏感的和动态的，可以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个信号状态扰动的代谢变化。预计许多信号通路的扰动和代谢谱的改变将为疾病状态的相关变化、重述和诊断潜力以及信号网络的新生物学发现提供一个庞大的数据库。我们组建了一个由四组组成的研究小组，每组都为项目带来了部分所需的技能。Meyer小组（斯坦福大学）长期参与GENAs的开发，并且也积极参与了用于代谢可视化的荧光显微镜仪器的开发。此外，迈耶小组还参与了代谢扰动试剂集的开发。Frommer小组（华盛顿卡内基研究所）已经开发出用于糖传感的GENAs，并且已经证明这些基因可以用于体内代谢成像。约克小组（杜克大学）一直深入参与基因操作，代谢和发现细胞内信号通路。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方面发挥了重要作用，这些工具用于自由基操纵配体结合特异性。在项目结束时，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)代谢波动随遗传扰动和细胞环境外源变化的时空成像数据集。

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.