Three-Color Spectral FRET Microscopy Localizes Three Interacting Proteins in Living Cells
Three-Color Spectral FRET Microscopy Localizes Three Interacting Proteins in Living Cells
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DOI：
10.1016/j.bpj.2010.06.004
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发表时间：
2010-08-18
影响因子：
3.4
通讯作者：
Periasamy, Ammasi
中科院分区：
文献类型：
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作者：
Sun, Yuansheng;Wallrabe, Horst;Periasamy, Ammasi
FRET技术现在通常用于建立两个蜂窝组件(A和B)之间的空间关系。添加第三个目标成分(C)增加了AB/BC/AC之间相互作用的分析的复杂性。在这里，我们描述了一种新的分析三色(ABC)FRET系统的方法，称为三色光谱FRET(3sFRET)显微镜，它完全校正了光谱穿透。该方法将FRET信号量化并计算表观能量传递效率(ES)。该方法通过测量由三种不同的荧光蛋白(FP)组成的遗传(FRET标准)结构来验证，这三种不同的荧光蛋白(FP)是MTFP、mVenus和tdTomato，它们彼此顺序相连。此外，以与3-FP构建相同的方式连接的三个2-FP参考构建物被用来表征能量转移途径。荧光寿命测量用来比较产生3-FP和2-FP融合蛋白的细胞中FP之间的相对关系。然后应用3sFRET显微镜方法研究了二聚体转录因子C/EBPα(表达MTFP或mVenus)与异染色质蛋白1α(HP1α，表达tdTomato)在活体小鼠垂体细胞中的相互作用。我们展示了3sFRET显微镜方法是如何代表一种很有前途的活细胞成像技术来监测三个标记的细胞成分之间的相互作用。
FRET technologies are now routinely used to establish the spatial relationships between two cellular components (A and B). Adding a third target component (C) increases the complexity of the analysis between interactions AB/BC/AC. Here, we describe a novel method for analyzing a three-color (ABC) FRET system called three-color spectral FRET (3sFRET) microscopy, which is fully corrected for spectral bleedthrough. The approach quantifies FRET signals and calculates the apparent energy transfer efficiencies (Es). The method was validated by measurement of a genetic (FRET standard) construct consisting of three different fluorescent proteins (FPs), mTFP, mVenus, and tdTomato, linked sequentially to one another. In addition, three 2-FP reference constructs, tethered in the same way as the 3-FP construct, were used to characterize the energy transfer pathways. Fluorescence lifetime measurements were employed to compare the relative relationships between the FPs in cells producing the 3-FP and 2-FP fusion proteins. The 3sFRET microscopy method was then applied to study the interactions of the dimeric transcription factor C/EBP alpha (expressing mTFP or mVenus) with the heterochromatin protein 1 alpha (HP1 alpha, expressing tdTomato) in live-mouse pituitary cells. We show how the 3sFRET microscopy method represents a promising live-cell imaging technique to monitor the interactions between three labeled cellular components.