Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli.
Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli.
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DOI：
10.1098/rstb.2015.0492
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发表时间：
2016-11-05
期刊：
影响因子：
--
通讯作者：
Berry RM
中科院分区：
文献类型：
--
作者：
Di Paolo D;Afanzar O;Armitage JP;Berry RM
在过去的二十年里，基因融合荧光蛋白(FP)的使用极大地促进了对大肠杆菌趋化信号的研究，包括激活反应调节蛋白Chey及其与鞭毛马达的相互作用。然而，这种方法受到一些生物学和生物物理方面的限制：例如，当融合到一个笨重的FP上时，并不是所有的融合都是完全功能的，它的分子质量可能与其融合的对应物相似；它们可能会干扰蛋白质的天然相互作用，而FP的发色团的亮度和光稳定性较低，光漂白速度较快。最近开发的一种在活细菌中电穿孔荧光标记蛋白质的技术使我们能够绕过这些限制，在单分子水平上研究Chey的体内行为。在这里，我们展示了用有机染料标记的纯化的Chey蛋白可以以可控的浓度内化到大肠杆菌细胞中，并通过视频荧光显微镜进行成像。这种方法的使用通过显示单个Chey分子在细胞内扩散并实时与感觉簇和鞭毛马达相互作用来说明。本文是主题期刊《新细菌学》的一部分。
For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time. This article is part of the themed issue ‘The new bacteriology’.
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