Day R N, Periasamy A, Schaufele F
Department of Medicine, NSF Center for Biological Timing, Charlottesville, Virginia 22908, USA
Methods. 2001 Sep;25(1):4-18. doi: 10.1006/meth.2001.1211.
Cells respond to environmental cues by modifying protein complexes in the nucleus to produce a change in the pattern of gene expression. In this article, we review techniques that allow us to visualize these protein interactions as they occur in living cells. The cloning of genes from marine organisms that encode fluorescent proteins provides a way to tag and monitor the intracellular behavior of expressed fusion proteins. The genetic engineering of jellyfish green fluorescent protein (GFP) and the recent cloning of a sea anemone red fluorescent protein (RFP) have provided fluorescent tags that emit light at wavelengths ranging from the blue to the red spectrum. Several of these color variants can be readily distinguished by fluorescence microscopy, allowing them to be used in combination to monitor the behavior of two or more independent proteins in the same living cell. We describe the use of this approach to examine where transcription factors are assembled in the nucleus. To demonstrate that these labeled nuclear proteins are interacting, however, requires spatial resolution that exceeds the optical limit of the light microscope. This degree of spatial resolution can be achieved with the conventional light microscope using the technique of fluorescence resonance energy transfer (FRET). The application of FRET microscopy to detect the interactions between proteins labeled with the color variants of GFP and the limitations of the FRET approach are discussed. The use of different-color fluorescent proteins in combination with FRET offers the opportunity to study the complex behavior of key regulatory proteins in their natural environment within the living cell.
细胞通过修饰细胞核中的蛋白质复合物来响应环境信号，从而改变基因表达模式。在本文中，我们综述了一些技术，这些技术能让我们在活细胞中蛋白质相互作用发生时对其进行可视化观察。从编码荧光蛋白的海洋生物中克隆基因，为标记和监测表达的融合蛋白的细胞内行为提供了一种方法。水母绿色荧光蛋白（GFP）的基因工程以及最近海葵红色荧光蛋白（RFP）的克隆，提供了在从蓝光到红光光谱范围内发光的荧光标签。这些颜色变体中的几种可以通过荧光显微镜轻松区分，从而使它们能够组合使用，以监测同一活细胞中两种或更多种独立蛋白质的行为。我们描述了使用这种方法来研究转录因子在细胞核中组装的位置。然而，要证明这些标记的核蛋白正在相互作用，需要的空间分辨率超过了光学显微镜的光学极限。使用荧光共振能量转移（FRET）技术的传统光学显微镜可以实现这种程度的空间分辨率。本文讨论了FRET显微镜在检测用GFP颜色变体标记的蛋白质之间相互作用中的应用以及FRET方法的局限性。将不同颜色的荧光蛋白与FRET结合使用，为研究活细胞内自然环境中关键调节蛋白的复杂行为提供了机会。
