Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor
Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor
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DOI：
10.1016/j.bios.2016.06.049
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发表时间：
2016-12-15
影响因子：
12.6
通讯作者：
Ahmad, Altaf
中科院分区：
文献类型：
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作者：
Ahmad, Mohammad;Ameen, Seema;Ahmad, Altaf
甘氨酸甜菜碱(GB)是在高盐度条件下细胞内积累的极高水平的关键相容溶质之一。它在维持细胞渗透压而不影响生理过程中起着至关重要的作用。因此，分析活细胞中应激诱导的生理状态需要实时监测细胞内的GB水平。甘氨酸甜菜碱光传感器(GBOS)是本研究中开发的一种基于遗传编码的FRET纳米传感器，可以实时监测活细胞内的GB水平。这种纳米传感器是通过将GB结合蛋白(PROX)夹在Forster共振能量转移(FRET)对、青色荧光蛋白(CFP)和黄色荧光蛋白(YFP)之间而研制出来的。作为感觉域的ProX的构象变化报告了在体外和体内条件下这种相容溶质水平的变化。Gb与感觉域的结合接近于导致FRET比率增加的荧光部分。因此，GB浓度的任何变化都与FRET比率的变化相关。该传感器还实时报告了在添加其前体胆碱后，在大肠杆菌细胞中的GB细胞动力学。在酵母和哺乳动物细胞中也表达了GBOS，以监测细胞内的GB。因此，GBOS代表了一种独特的基于FRET的纳米传感器，它允许对活细胞中的GB进行非侵入性比率分析。(C)2016爱思唯尔B.V.保留所有权利。
Glycine betaine (GB) is one of the key compatible solutes that accumulate in the cell at exceedingly high level under the conditions of high salinity. It plays a crucial role in the maintenance of osmolarity of the cell without affecting the physiological processes. Analysis of stress-induced physiological conditions in living cells, therefore, requires real-time monitoring of cellular GB level. Glycine Betaine Optical Sensor (GBOS), a genetically-encoded FRET-based nanosensor developed in this study, allows the real-time monitoring of GB levels inside living cells. This nanosensor has been developed by sandwiching GB binding protein (ProX) between the Forster resonance energy transfer (FRET) pair, the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Conformational change in ProX, which was used as sensory domain, reported the change in the level of this compatible solute in in vitro and in vivo conditions. Binding of the GB to the sensory domain fetches close to both the fluorescent moieties that result in the form of increased FRET ratio. So, any change in the concentration of GB is correlated with change in FRET ratio. This sensor also reported the GB cellular dynamics in real-time in Escherichia coli cells after the addition of its precursor, choline. The GBOS was also expressed in yeast and mammalian cells to monitor the intracellular GB. Therefore, the GBOS represents a unique FRET-based nanosensor which allows the non-invasive ratiometric analysis of the GB in living cells. (C) 2016 Elsevier B.V. All rights reserved.