Single cell analysis of metabolism using genetically-encoded fluorescent sensors

使用基因编码荧光传感器进行代谢的单细胞分析

基本信息

批准号：
8897369
负责人：
GARY I YELLEN
金额：
$ 84.75万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2016-07-31
项目状态：
已结题

项目摘要

DESCRIPTION Abstract: Metabolic pathways provide essential energy and building blocks for the function of all cells, and dysregulation of these pathways is a central feature of cancer, diabetes, and obesity, which kill or disable millions of Americans every year. The components of core metabolic pathways such as glycolysis have been very well understood for decades, but there are still major gaps in our understanding of their integrated behavior and regulation in the context of living cells. A major challenge to understanding normal metabolism and its dysregulation in human disease is that metabolic behavior can vary dramatically from cell to cell, and over time within a single cell. For example, metabolic state can differ radically between neighboring cell types in a tissue, creating a functional segregation that is important for overall tissue function, or between a single metastatic cancer cell and the surrounding normal cells. Such spatial differences as well as dynamic changes in metabolism within a single cell are invisible to the usual biochemical methods or even modern metabolomic methods, which require disruption of the living cell and homogenization of tissue. Fluorescent sensors of metabolism, engineered by combining fluorescent proteins with metabolite binding proteins, can address this challenge by enabling us to monitor key metabolites in real time, in single living cells, or in hundreds of cells in paralle. We recently piloted the development of novel sensors for two key metabolites (ATP and NADH), in order to address specific neurobiological questions about how metabolism influences neuronal ion channels and can reduce susceptibility to epileptic seizures. But our preliminary results with these sensors have underscored the general problem of cell heterogeneity as well as the need for a much larger toolkit of fluorescent metabolite sensors. We propose to develop a suite of novel sensors for key metabolites in order to address fundamental questions of cellular me

描述抽象的：代谢途径为所有细胞的功能提供必需的能量和构建模块，而这些途径的失调是癌症、糖尿病和肥胖症的一个核心特征，这些疾病每年导致数百万美国人死亡或致残。几十年来，我们对糖酵解等核心代谢途径的组成部分已经有了很好的了解，但我们对它们在活细胞背景下的综合行为和调节的理解仍然存在重大差距。了解人类疾病中正常代谢及其失调的一个主要挑战是，细胞与细胞之间以及单个细胞内的代谢行为可能会随着时间的推移而发生巨大变化。为了例如，组织中相邻细胞类型之间的代谢状态可能存在根本差异，从而产生对整体组织功能很重要的功能分离，或者单个转移性癌细胞与周围正常细胞之间的代谢状态可能存在根本差异。这种空间差异以及单个细胞内代谢的动态变化对于通常的生化方法甚至现代代谢组学方法来说是看不见的，这需要破坏活细胞和组织均质化。通过将荧光蛋白与代谢物结合蛋白相结合而设计的代谢荧光传感器可以解决这一挑战，使我们能够实时监测单个活细胞或并行数百个细胞中的关键代谢物。我们最近试点开发了两种关键代谢物（ATP 和 NADH）的新型传感器，以解决有关代谢如何影响神经元离子通道并降低癫痫发作易感性的特定神经生物学问题。但我们对这些传感器的初步结果强调了细胞异质性的普遍问题以及对更大的荧光代谢物传感器工具包的需求。我们建议开发一套针对关键代谢物的新型传感器，以解决细胞代谢的基本问题