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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：
9116838
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 2018-07-31
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

描述摘要：代谢途径为所有细胞的功能提供必要的能量和基石，而这些途径的失调是癌症、糖尿病和肥胖症的核心特征，这些疾病每年导致数百万美国人死亡或残疾。糖酵解等核心代谢途径的组成已经被很好地理解了几十年，但在活细胞的背景下，我们对它们的整合行为和调控的理解仍然存在重大差距。理解人类疾病中正常代谢及其失调的一个主要挑战是，代谢行为在不同细胞之间以及随着时间的推移在单个细胞内可能会有很大差异。为例如，组织中相邻的细胞类型之间的代谢状态可能会完全不同，从而造成对整个组织功能很重要的功能隔离，或者在单个转移癌细胞和周围正常细胞之间造成功能隔离。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方法是看不见的，这些方法需要破坏活细胞和组织均质化。通过将荧光蛋白和代谢物结合蛋白相结合而设计的代谢荧光传感器，可以通过使我们能够实时监测单个活细胞中的关键代谢物，或者并行在数百个细胞中监测关键代谢物来应对这一挑战。我们最近尝试开发两种关键代谢物(ATP和NADH)的新型传感器，以解决特定的神经生物学问题，即代谢如何影响神经元离子通道，并可以降低癫痫发作的易感性。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，以及需要更大的荧光代谢物传感器工具箱。我们建议开发一套用于关键代谢物的新型传感器，以解决细胞代谢的基本问题。