Demarco Ignacio A, Periasamy Ammasi, Booker Cynthia F, Day Richard N
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Nat Methods. 2006 Jul;3(7):519-24. doi: 10.1038/nmeth889.
The mammalian cell nucleus is a dynamic and highly organized structure. Most proteins are mobile within the nuclear compartment, and this mobility reflects transient interactions with chromatin, as well as network interactions with a variety of protein partners. To study these dynamic processes in living cells, we developed an imaging method that combines the photoactivated green fluorescent protein (PA-GFP) and fluorescence resonance energy transfer (FRET) microscopy. We used this new method, photoquenching FRET (PQ-FRET), to define the dynamic interactions of the heterochromatin protein-1 alpha (HP1alpha) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) in regions of centromeric heterochromatin in mouse pituitary cells. The advantage of the PQ-FRET assay is that it provides simultaneous measurement of a protein's mobility, its exchange within macromolecular complexes and its interactions with other proteins in the living cell without the need for corrections based on reference images acquired from control cells.
哺乳动物细胞核是一个动态且高度有序的结构。大多数蛋白质在核区内是可移动的，这种移动性反映了与染色质的瞬时相互作用，以及与多种蛋白质伙伴的网络相互作用。为了在活细胞中研究这些动态过程，我们开发了一种成像方法，该方法结合了光激活绿色荧光蛋白（PA-GFP）和荧光共振能量转移（FRET）显微镜技术。我们使用这种新方法，即光猝灭FRET（PQ-FRET），来确定小鼠垂体细胞着丝粒异染色质区域中异染色质蛋白1α（HP1α）与转录因子CCAAT/增强子结合蛋白α（C/EBPα）之间的动态相互作用。PQ-FRET检测法的优点在于，它能够在活细胞中同时测量蛋白质的移动性、其在大分子复合物中的交换情况以及与其他蛋白质的相互作用，而无需基于从对照细胞获取的参考图像进行校正。
