Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy.
Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy.
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DOI：
10.1210/mend.12.9.0168
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发表时间：
1998-09
影响因子：
--
通讯作者：
R. Day
中科院分区：
文献类型：
--
作者：
R. Day
垂体特异性转录因子Pit-1在与特定DNA元件相互作用时形成二聚体，并已显示与其他几种核蛋白相关。最近，技术已经变得可用，允许可视化蛋白质-蛋白质相互作用，因为它们发生在单个活细胞中。在这项研究中，荧光共振能量转移（FRET）显微镜的技术被用来可视化的Pit-1蛋白融合到水母绿色荧光蛋白（GFP），发射绿色或蓝色光[蓝色荧光蛋白（BFP）]的光谱变体的物理相互作用。使用优化的成像系统来区分来自共表达BFP和GFP融合蛋白的单细胞的荧光信号，并确定光谱重叠对FRET图像中检测到的背景荧光的贡献。使用来自表达融合蛋白的活细胞的能量转移信号来证明FRET信号的获得，其中GFP通过短蛋白质接头连接到BFP。制备了编码GFP-和BFP-Pit-1蛋白的遗传载体，并证实了融合蛋白的生物学功能。共表达GFP-和BFP-Pit-1的HeLa细胞的FRET显微镜显示了能量转移，这需要两个荧光团分开小于100 A。生物化学研究表明，Pit-1与c-Ets-1和雌激素受体都有物理相互作用。共表达BFP-Pit-1和GFP-Ets-1的细胞的FRET成像证明了这些融合蛋白之间的能量转移，这一结果与它们在这些活细胞的细胞核中的结合一致。相反，没有证据表明BFP-Pit-1和雌激素受体-GFP融合蛋白之间存在能量转移。这里描述的FRET成像方法很可能可以应用于各种细胞环境中的许多不同蛋白质-伴侣对。
The pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 A. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.