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：
8703697
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 2017-07-31
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了必要的能量和构建基础，这些途径的失调是癌症，糖尿病和肥胖症的主要特征，每年杀死或禁用数百万美国人。数十年来，人们对核代谢途径（例如糖酵解）的组成部分进行了充分的理解，但是在我们对生物细胞背景下它们的综合行为和调节的理解仍然存在重大差距。理解正常代谢及其在人类疾病中的失调的主要挑战是，新陈代谢行为在细胞之间以及随着时间的流逝会在单个细胞中发生巨大变化。为了例如，代谢状态在组织中相邻细胞类型之间可能会差异，从而产生功能分离，对于整体组织功能很重要，或者在单个转移性癌细胞和周围正常细胞之间很重要。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方法都是看不见的，这些方法需要破坏活细胞和组织的均质化。代谢的荧光传感器通过将荧光蛋白与代谢物结合蛋白结合而设计的代谢传感器可以通过使我们能够实时监测关键代谢物，单个活细胞或Paralle中数百个细胞中来解决这一挑战。我们最近试图开发两个关键代谢产物（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的特定神经生物学问题，并可以减少对癫痫发作的易感性。但是，这些传感器的初步结果强调了细胞异质性的一般问题，以及需要更大的荧光代谢物传感器工具包。我们建议为关键代谢产物开发一套新型传感器，以解决细胞ME的基本问题