De Vos Winnok H, Hoebe R A, Joss G H, Haffmans W, Baatout S, Van Oostveldt P, Manders E M M
Department of Molecular Biotechnology, Faculty of Bio-engineering Sciences, Ghent University, Coupure links 653, Ghent 9000, Belgium.
Cytometry A. 2009 May;75(5):428-39. doi: 10.1002/cyto.a.20699.
Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV-304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially positioned at the interface of chromatin domains. TRF1 and TRF2 are abundantly present in these territories but not firmly bound. At the onset of mitosis, the bulk of TRF protein dissociates from telomere regions, territories disintegrate and individual telomeres become faintly visible. The combination of stable cell lines, CLEM and cytometry proved essential in providing novel insights in compartment-based nuclear organization and may serve as a model approach for investigating telomere-driven genome-instability and studying long-term nuclear dynamics.
端粒是复杂的末端结构，赋予人类染色体功能完整性和位置稳定性。尽管它们至关重要，但对于处于细胞周期中的人类细胞中端粒的组织及其动态行为，目前尚无清晰的认识。我们使用四维显微镜研究了稳定表达与mCitrine融合的端粒结合蛋白TRF1和TRF2的活人类ECV - 304细胞中端粒的时空组织。我们利用了控制光暴露显微镜（CLEM），这是一种通过限制图像中不需要完全暴露的部分的激发来大幅减少光损伤的新技术。我们发现端粒共享它们动态关联的小区域。这些区域优先定位在染色质结构域的界面处。TRF1和TRF2大量存在于这些区域，但并非紧密结合。在有丝分裂开始时，大部分TRF蛋白从端粒区域解离，区域瓦解，单个端粒变得模糊可见。稳定细胞系、CLEM和细胞计数法的结合被证明对于提供基于区室的核组织的新见解至关重要，并且可作为研究端粒驱动的基因组不稳定性和长期核动态的模型方法。
