Fluorogenic Targeting of Voltage-Sensitive Dyes to Neurons.
Fluorogenic Targeting of Voltage-Sensitive Dyes to Neurons.
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DOI：
10.1021/jacs.7b07047
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发表时间：
2017-12-06
影响因子：
15
通讯作者：
Miller EW
中科院分区：
文献类型：
--
作者：
Liu P;Grenier V;Hong W;Muller VR;Miller EW
我们提出了一种方法，通过在细胞表面进行酶催化的荧光反应，将电压敏感的荧光染料靶向特定的细胞。染料/酶杂交物是由基于光致电子转移(PET)的荧光电压指示剂和细胞表面表达的互补酶组成的。外源性酶对染料的作用导致染料的荧光激活，使定义的神经元能够进行快速电压成像，灵敏度超过纯粹的遗传编码方法。我们使用体积庞大的环丙基甲基乙酰氧甲酯来减弱一种基于宠物的电压敏感染料或VoltageFluor的荧光。在猪肝酯酶(PLE)的作用下，水解性稳定的酯可以被去除，从而显示出明亮的、未经修饰的VoltageFluor。我们在体外和活细胞中证实了化学修饰的VoltageFluor是PLE的底物。当PLE靶向细胞膜的外表面时，它控制细胞的外观染色。使用神经元特异性启动子可以直接对哺乳动物神经元进行染色，从而在单次试验中提供清晰的神经元动作电位检测。所有以酯酶表达为靶点的新电压荧光都报告了培养的哺乳动物神经元中的单峰。最好的VF-EX2，信噪比(SNR)几乎是可比的遗传编码电压记录器的两倍。通过将PLE靶向神经元，VF-EX2可以询问5-羟色胺对培养的海马神经元的神经调制作用。总而言之，我们展示了合成化学和生物化学的结合使培养中遗传定义的神经元能够进行明亮和快速的电压成像。
We present a method to target voltage-sensitive fluorescent dyes to specified cells using an enzyme-catalyzed fluorogenic reaction on cell surfaces. The dye/enzyme hybrids are composed of a photoinduced electron transfer (PeT)-based fluorescent voltage indicator and complementary enzyme expressed on the cell surface. Action of the exogenous enzyme on the dye results in fluorogenic activation of the dye, enabling fast voltage imaging in defined neurons, with sensitivity surpassing purely genetically-encoded approaches. We employ a bulky cyclopropylmethyl acetoxymethyl ester to diminish the fluorescence of a PeT-based voltage-sensitive dye, or VoltageFluor. The hydrolytically stable ester can be removed by the action of porcine liver esterase (PLE) to reveal the bright, unmodified VoltageFluor. We established that the chemically modified VoltageFluor is a substrate for PLE in vitro and in live cells. When PLE is targeted to the external face of cell membranes, it controls the apparent staining of cells. Use of neuron-specific promoters can direct staining to mammalian neurons to provide clear detection of neuronal action potentials in single trials. All of the new VoltageFluors targeted by esterase expression report single spikes in cultured mammalian neurons. The best, VF-EX2, does so with a signal-to-noise ratio (SNR) nearly double that of comparable genetically-encoded voltage reporters. By targeting PLE to neurons, VF-EX2 can interrogate the neuromodulatory effects of serotonin in cultured hippocampal neurons. Together, we show that a combination of synthetic chemistry and biochemistry enables bright and fast voltage imaging from genetically-defined neurons in culture.
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影响因子：
25
作者：
St-Pierre, Francois;Marshall, Jesse D.;Yang, Ying;Gong, Yiyang;Schnitzer, Mark J.;Lin, Michael Z.
通讯作者：
Lin, Michael Z.
DOI：
10.1073/pnas.1111943109
发表时间：
2012-03-27
影响因子：
11.1
作者：
Tian, Lin;Yang, Yunlei;Lavis, Luke D.
通讯作者：
Lavis, Luke D.
影响因子：
16.2
作者：
Jin L;Han Z;Platisa J;Wooltorton JR;Cohen LB;Pieribone VA
通讯作者：
Pieribone VA
影响因子：
15
作者：
Urano, Y;Kamiya, M;Nagano, T
通讯作者：
Nagano, T
影响因子：
7.8
作者：
Miller EW
通讯作者：
Miller EW