Oscilloscopes Spatio-Temporal Metabolomics (RMI)

描述（由申请人提供）：本项目的目标是开发一种用于在细胞水平上实时体内时空定量代谢波动的系统。为了实现这一目标，我们结合了四项关键技术：i）基因编码的荧光纳米传感器蛋白（热纳斯），报告特定的代谢物; ii）蛋白质工程技术，允许系统构建大量感兴趣的代谢物的纳米传感器; 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）干扰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，使时间分辨的测量在毫秒范围内。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。预计代谢谱中的许多信号通路扰动和改变将提供相关变化的大型数据库、疾病状态的重演和诊断潜力以及信号网络的新生物学发现。我们组建了一个由四个小组组成的研究团队，每个小组都为项目带来了所需的部分技能。Meyer小组（斯坦福大学）长期参与热纳斯的开发，并且还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，Meyer小组还参与了代谢扰动试剂组的开发。Frommer小组（华盛顿卡内基研究所）已经开发出用于感测糖的热纳斯，并且已经证明这些可以用于体内代谢成像。约克小组（杜克大学）一直深入参与遗传操纵、代谢和细胞内信号通路的发现。Hellinga小组（杜克大学）一直致力于开发和实验验证计算蛋白质设计工具，以彻底操纵配体结合特异性。在项目期结束时，我们的目标是提供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性变化的函数。

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.