Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells
Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells
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DOI：
10.1038/nmeth720
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发表时间：
2004-12-01
期刊：
影响因子：
48
通讯作者：
Sorkin, A
中科院分区：
文献类型：
--
作者：
Galperin, E;Verkhusha, V;Sorkin, A
细胞中几乎所有的主要过程都是由多个动态相互作用的蛋白质分子组装而成的。为了研究这种分子机制中的多蛋白质相互作用，我们开发了一种称为三发色团荧光共振能量转移（3-FRET）的荧光显微镜方法。该方法允许分析荧光标记之间的三个相互依赖的能量转移过程，例如青色、黄色和单体红色荧光蛋白。在这里，我们描述的理论和实验方法，区分平行与顺序的能量转移过程中的3-FRET系统。这些方法是在体外和培养的哺乳动物细胞中建立的，使用由两个或三个荧光蛋白连接在一起组成的嵌合蛋白。3-FRET显微镜进一步应用于分析的三个蛋白质的相互作用的组成和激活依赖的复合物在单个内体隔室。这些数据突出了3-FRET显微镜在活细胞中信号传导过程的时空调控研究中的潜力。
Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.