High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein
High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein
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DOI：
10.1016/j.bpc.2007.01.008
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发表时间：
2007-05-01
影响因子：
3.8
通讯作者：
Magennis, Steven W.
中科院分区：
文献类型：
--
作者：
Millington, Michael;Grindlay, G. Joan;Magennis, Steven W.
我们用广域光子计数膜研究了固定细胞和活细胞中的FRET，并使用对照FRET对。我们研究了表达青色荧光蛋白(CFP)或CFP与黄色荧光蛋白(YFP)融合的固定哺乳动物细胞，以及表达Cerulean或Cerulean-Venus融合蛋白的活真菌细胞。我们发现哺乳动物和真菌细胞的荧光行为基本上是相同的。重要的是，高精度的薄膜数据能够重复地解析多个荧光衰变，从而揭示了关于经历FRET的蛋白质群体部分的新信息，并减少了在测量供体或受体距离时的误差。我们对这个简单控制系统的结果表明，对更复杂的蛋白质-蛋白质相互作用的活体薄膜研究将从这种定量测量中受益匪浅。(C)2007 Elsevier B.V.保留所有权利。
We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donoracceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements. (c) 2007 Elsevier B.V. All rights reserved.