Spying on Neuronal Membrane Potential with Genetically Targetable Voltage Indicators
Spying on Neuronal Membrane Potential with Genetically Targetable Voltage Indicators
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DOI：
10.1021/jacs.8b11997
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发表时间：
2019-01-23
影响因子：
15
通讯作者：
Miller, Evan W.
中科院分区：
文献类型：
--
作者：
Grenier, Vincent;Daws, Brittany R.;Miller, Evan W.
活细胞中电压动态的光学测量方法很有吸引力，因为它们提供了超过传统电极测量的空间分辨率和超过广泛使用的Ca2+成像的时间分辨率。化学合成的电压敏感染料使用光诱导电子转移作为电压感应触发器，具有高电压灵敏度和快速响应动力学，但针对特定细胞的化学指标仍然是一个突出的挑战。在这里，我们提出了一个易于功能化的新家族，基于荧光素的电压敏感荧光染料（肌氨酸-电压荧光），它可以共价附着在基因编码的细胞表面受体上，以实现基因定义的神经元的电压成像。我们合成了四种新的VoltageFluor衍生物，它们具有羧酸功能，可以简单地偶联到柔性系链上。这组新染料中的最佳染料通过聚乙二醇（PEG）连接到一个小肽（SpyTag， 13个氨基酸）上，该肽指导其结合并与其结合伙伴SpyCatcher （15 kDa）形成共价键。新的VoltageSpy染料有效地标记表达细胞表面SpyCatcher的细胞，显示出良好的电压敏感性，并保持快速响应动力学。在培养的神经元中，VoltageSpy染料能够对神经元体细胞的动作电位进行稳健的单次光学检测，灵敏度超过遗传编码的电压指标。重要的是，化学合成染料的遗传靶向使VoltageSpy能够在距离细胞体数十至数百微米的单次试验中报告轴突和树突的动作电位。利用VoltageSpy对合成电压指标进行遗传靶向，可以在低纳摩尔染料浓度下进行电压成像，并为合成指标的速度和灵敏度与基因编码探针的细胞分辨率增强相结合提供了一种有前途的方法。
Methods for optical measurement of voltage dynamics in living cells are attractive because they provide spatial resolution surpassing traditional electrode-based measurements and temporal resolution exceeding that of widely used Ca2+ imaging. Chemically synthesized voltage sensitive dyes that use photoinduced electron transfer as a voltage-sensing trigger offer high voltage sensitivity and fast response kinetics, but targeting chemical indicators to specific cells remains an outstanding challenge. Here, we present a new family of readily functionalizable, fluorescein-based voltage-sensitive fluorescent dyes (sarcosine-VoltageFluors) that can be covalently attached to a genetically encoded cell surface receptor to achieve voltage imaging from genetically defined neurons. We synthesized four new VoltageFluor derivatives that possess carboxylic acid functionality for simple conjugation to flexible tethers. The best of this new group of dyes was conjugated via a polyethylene glycol (PEG) linker to a small peptide (SpyTag, 13 amino acids) that directs binding and formation of a covalent bond with its binding partner, SpyCatcher (15 kDa). The new VoltageSpy dyes effectively label cells expressing cell-surface SpyCatcher, display good voltage sensitivity, and maintain fast-response kinetics. In cultured neurons, VoltageSpy dyes enable robust, single-trial optical detection of action potentials at neuronal soma with sensitivity exceeding genetically encoded voltage indicators. Importantly, genetic targeting of chemically synthesized dyes enables VoltageSpy to report on action potentials in axons and dendrites in single trials, tens to hundreds of micrometers away from the cell body. Genetic targeting of synthetic voltage indicators with VoltageSpy enables voltage imaging with low nanomolar dye concentration and offers a promising method for allying the speed and sensitivity of synthetic indicators with the enhanced cellular resolution of genetically encoded probes.