Single cell analysis of metabolism using genetically-encoded fluorescent sensors

描述 摘要： 代谢途径为所有细胞的功能提供必要的能量和构建模块，这些途径的失调是癌症，糖尿病和肥胖症的核心特征，每年有数百万美国人死亡或致残。几十年来，核心代谢途径（如糖酵解）的组成部分已经得到了很好的理解，但我们在活细胞背景下对其整合行为和调控的理解仍然存在重大差距。 理解人类疾病中的正常代谢及其失调的一个主要挑战是，代谢行为可以在细胞与细胞之间以及在单个细胞内随着时间的推移而发生显着变化。为 例如，代谢状态可以在组织中的相邻细胞类型之间根本不同，从而产生对整体组织功能重要的功能分离，或者在单个转移性癌细胞和周围正常细胞之间根本不同。这种空间差异以及单细胞内代谢的动态变化对于通常的生物化学方法甚至现代代谢组学方法是不可见的，这些方法需要破坏活细胞并使组织均质化。 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通过使我们能够在单个活细胞或数百个细胞中真实的实时监测关键代谢物来解决这一挑战。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。但是我们对这些传感器的初步研究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包。 我们建议开发一套新的关键代谢物传感器，以解决细胞代谢的基本问题。

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