Multiplex Imaging of Rho GTPase Activities in Living Cells.
Multiplex Imaging of Rho GTPase Activities in Living Cells.
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DOI：
10.1007/978-1-0716-1593-5_4
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Hodgson L
中科院分区：
文献类型：
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作者：
Bhalla RM;Hülsemann M;Verkhusha PV;Walker MG;Shcherbakova DM;Hodgson L
Förster共振能量转移（FRET）生物传感器是流行的，可用于直接观察活细胞中的细胞信号通路。直到最近，多重成像的基因编码的FRET生物传感器，同时监测几种蛋白质的活动在一个细胞中是有限的，由于缺乏光谱兼容的FRET对荧光蛋白。随着miRFP系列近红外（NIR）荧光蛋白的最新发展，我们现在能够将FRET生物传感器的光谱从蓝-绿-黄扩展到NIR。这些新的NIR FRET生物传感器能够与常用的青黄色FRET生物传感器一起直接多重成像。我们在本文中描述了一种方法，以生产细胞系窝藏两个兼容的FRET生物传感器。然后，我们将讨论如何在活细胞中直接多重成像这些FRET生物传感器。本文所述的方法通常适用于利用青黄色和NIR荧光的遗传编码的比率FRET生物传感器的任何组合。
Förster resonance energy transfer (FRET) biosensors are popular and useful for directly observing cellular signaling pathways in living cells. Until recently, multiplex imaging of genetically encoded FRET biosensors to simultaneously monitor several protein activities in one cell was limited due to a lack of spectrally compatible FRET pair of fluorescent proteins. With the recent development of miRFP series of near-infrared (NIR) fluorescent proteins, we are now able to extend the spectrum of FRET biosensors beyond blue-green-yellow into NIR. These new NIR FRET biosensors enable direct multiplex imaging together with commonly used cyan-yellow FRET biosensors. We describe herein a method to produce cell lines harboring two compatible FRET biosensors. We will then discuss how to directly multiplex-image these FRET biosensors in living cells. The approaches described herein are generally applicable to any combinations of genetically encoded, ratiometric FRET biosensors utilizing the cyan-yellow and NIR fluorescence.