Optical sensing of voltage, pH, and small molecules using microbial rhodopsins

使用微生物视紫红质对电压、pH 和小分子进行光学传感

• 批准号: 7981713
• 负责人: Adam Ezra Cohen
• 金额: $ 252万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7981713
• 关键词: Biology; Cardiac; Cells; Chemicals; Chloride Ion; Chlorides; Dreams; Environment; Goals; Image; Lasers; Life; Membrane; Membrane Potentials; Methodology; Microbial Rhodopsins; Mitochondria; Modality; Neuroglia; Neurons; Optics; Positioning Attribute; Protein Engineering; Proteins; Signal Transduction; System; base; design and construction; directed evolution; in vivo; optical sensor; response; sensor; small molecule; voltage

DESCRIPTION (Provided by the applicant) Abstract: Our goal is to introduce a new class of genetically encoded optical indicators, based on the huge diversity of environmentally sensitive spectral shifts that occur naturally in microbial rhodopsin proteins. We recently created a microbial rhodopsin-based fluorescent indicator of pH, with a sensitive range from pH 6.8 to 8.8. We have preliminary results on a microbial rhodopsin-based indicator of membrane potential, which shows greater sensitivity than any existing optical sensor of membrane potential. Just as GFP revolutionized biology through its ability to track the positions of proteins in cells, we believe that microbial rhodopsins will have a broad impact through their ability to transduce the physical and chemical environment into an optical signal. Sensing voltage is our first target. Neuroscientists have long dreamed of a genetically encoded sensor that gives an optical signal in response to a change in membrane potential, with the goal of imaging electrical activity of neurons in vivo. Such a molecule could also be used to probe membrane potentials in mitochondria, cardiac cells, or in other non-neuronal cells. Our strategy is completely different from previous approaches to optical voltage sensing, and has already shown promising results. The technical implementation involves a) protein engineering and directed evolution to optimize an electrochromic response, and b) design and construction of an ultrasensitive laser imaging system capable of detecting this response in living cells. The methodology developed for sensing pH and voltage will later be applied to other sensing modalities, such as chloride and membrane tension. Public Health Relevance: We are working to develop a new class of molecules that allow us to see changes in voltage or pH inside of single cells. Neurons use voltage to communicate, so the ability to see neuronal activity will provide insights into brain function.

描述（由申请人提供） 摘要：我们的目标是引入一类新的遗传编码的光学指示剂，基于微生物视紫红质蛋白中自然发生的环境敏感光谱变化的巨大多样性。我们最近创建了一种基于微生物视紫红质的pH荧光指示剂，其敏感范围为pH 6.8至8.8。我们有一个基于微生物视紫红质的膜电位指示剂，这表明比任何现有的膜电位的光学传感器更高的灵敏度的初步结果。正如绿色荧光蛋白通过其跟踪细胞中蛋白质位置的能力彻底改变了生物学一样，我们相信微生物视紫红质将通过其将物理和化学环境转化为光学信号的能力产生广泛的影响。感应电压是我们的第一个目标。神经科学家长期以来一直梦想着一种基因编码的传感器，它可以响应膜电位的变化而发出光学信号，其目标是对体内神经元的电活动进行成像。这种分子也可用于探测线粒体、心肌细胞或其他非神经元细胞中的膜电位。我们的策略与以前的光学电压传感方法完全不同，并且已经显示出有希望的结果。该技术实现涉及a）蛋白质工程和定向进化以优化电致变色响应，以及B）能够检测活细胞中的该响应的超灵敏激光成像系统的设计和构建。为感测pH和电压而开发的方法稍后将应用于其他感测模式，例如氯化物和膜张力。 公共卫生相关性：我们正在努力开发一类新的分子，使我们能够看到单细胞内电压或pH值的变化。神经元使用电压进行交流，因此观察神经元活动的能力将提供对大脑功能的见解。

项目成果

Screening fluorescent voltage indicators with spontaneously spiking HEK cells.

筛选荧光电压指示器自发尖峰HEK细胞。

• DOI: 10.1371/journal.pone.0085221
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Park J;Werley CA;Venkatachalam V;Kralj JM;Dib-Hajj SD;Waxman SG;Cohen AE
• 通讯作者: Cohen AE

Photostick: a method for selective isolation of target cells from culture.

• DOI: 10.1039/c4sc03676j
• 发表时间: 2015-03
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Chien MP;Werley CA;Farhi SL;Cohen AE
• 通讯作者: Cohen AE

Convex lens-induced confinement for imaging single molecules.

• DOI: 10.1021/ac101041s
• 发表时间: 2010-07-15
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Leslie, Sabrina R.;Fields, Alexander P.;Cohen, Adam E.
• 通讯作者: Cohen, Adam E.

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond.

• DOI: 10.1016/j.tibtech.2017.04.002
• 发表时间: 2017-07
• 期刊: Trends in biotechnology
• 影响因子: 17.3
• 作者: Zhang H;Cohen AE
• 通讯作者: Cohen AE

All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS.

• DOI: 10.1016/j.stemcr.2018.04.020
• 发表时间: 2018-06-05
• 期刊: Stem cell reports
• 影响因子: 5.9
• 作者: Kiskinis E;Kralj JM;Zou P;Weinstein EN;Zhang H;Tsioras K;Wiskow O;Ortega JA;Eggan K;Cohen AE
• 通讯作者: Cohen AE