Quantitative imaging of protein interactions in the cell nucleus.
Quantitative imaging of protein interactions in the cell nucleus.
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DOI：
10.2144/05383rv01
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发表时间：
2005-03
期刊：
影响因子：
2.7
通讯作者：
T. Voss;Ignacio A. Demarco;R. Day
中科院分区：
文献类型：
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作者：
T. Voss;Ignacio A. Demarco;R. Day
在过去的十年中，基因编码的荧光蛋白已被广泛用作活细胞中的非侵入性标记。荧光蛋白的发展，加上数字成像的进步，导致了活细胞成像方法的快速发展。这些方法正被应用于解决特定亚细胞区室内特定蛋白质的募集、共定位和相互作用的生物学问题。然而，在这种快速发展之后，出现了与越来越大和更复杂的数字成像数据集的获取和分析相关的重要问题。以哺乳动物细胞核中的蛋白质定位为例，我们将回顾定量成像应用于分析活细胞群体中蛋白质的亚细胞分布和共定位的一些最新进展。在这份报告中，我们审查的原则，获得荧光共振能量转移（FRET）显微镜测量，以确定蛋白质之间的空间关系。然后，我们讨论荧光寿命成像显微镜（FLIM）提供了一种方法，是独立的基于强度的测量，以检测本地化的蛋白质相互作用的空间分辨率。最后，我们认为潜在的问题与融合到荧光蛋白的蛋白质的表达从活细胞的FRET为基础的测量。
Over the past decade, genetically encoded fluorescent proteins have become widely used as noninvasive markers in living cells. The development of fluorescent proteins, coupled with advances in digital imaging, has led to the rapid evolution of live-cell imaging methods. These approaches are being applied to address biological questions of the recruitment, co-localization, and interactions of specific proteins within particular subcellular compartments. In the wake of this rapid progress, however, come important issues associated with the acquisition and analysis of ever larger and more complex digital imaging data sets. Using protein localization in the mammalian cell nucleus as an example, we will review some recent developments in the application of quantitative imaging to analyze subcellular distribution and co-localization of proteins in populations of living cells. In this report, we review the principles of acquiring fluorescence resonance energy transfer (FRET) microscopy measurements to define the spatial relationships between proteins. We then discuss how fluorescence lifetime imaging microscopy (FLIM) provides a method that is independent of intensity-based measurements to detect localized protein interactions with spatial resolution. Finally, we consider potential problems associated with the expression of proteins fused to fluorescent proteins for FRET-based measurements from living cells.