Investigating telomerase dynamics in live cells at a single-molecule level

摘要： 真核细胞通过添加来解决末端复制和末端保护问题 端粒序列延伸到染色体末端。端粒长度的适当调节是 对基因组完整性、调节细胞寿命、衰老和癌症至关重要。多年来， 遗传和生化研究揭示了这一过程是如何发生的。 控制住了。然而，在拥挤的细胞核中这一过程的细胞生物学仍然很差。 了解端粒延伸的时间、动力学和空间协调性 未知。为了解决我们理解中的这些差距，我们将利用MS2标记系统 和Halo-荧光团在活细胞中显示内源性端粒酶的单分子。这里, 我们将破译HTR从Cajal小体到端粒的离散和关键步骤。 与早期固定细胞中的FISH数据相反，我们的初步数据使用了衍射限制和 结合单分子FISH的超分辨率成像模式表明，HTR是 广泛分布于整个细胞核。在端粒上，我们显示了以下由TPP1驱动的 募集时，酶与其底物之间建立了稳定的相互作用 RNA：DNA碱基配对。我们的目标是将光活化和光漂白实验应用于 测试催化亚单位hTERT在Cajal小体和HTR门控中的作用 端粒。此外，我们将设计一个短的端粒来描述端粒酶的动力学。 需要延长的关键端粒。最后，我们将执行基于邻近度的标注 和纯化方法学研究控制端粒酶关键步骤的因素 贩卖短端粒。总而言之，我们的创新方法提供了一个详细的视图 生理时间的端粒延伸的精确机制和打开许多未来 研究端粒维持与衰老和癌症之间联系的途径。

Abstract: Eukaryotic cells solve the end-replication and end-protection problems through the addition of telomere sequences to the ends of chromosomes. Proper regulation of telomere length is critical for genome integrity, regulation of cellular lifespan, aging, and cancer. Over the years, genetic and biochemical studies have shed an enormous amount of light on how this process is controlled. However, the cell biology of this process in the crowded nucleus remains poorly understood, and the timing, dynamics, and spatial coordination of telomere extension are unknown. To address these gaps in our understanding, we will exploit the MS2 tagging system and Halo-fluorophore to visualize single molecules of endogenous telomerase in live cells. Here, we will decipher discrete and critical steps as hTR traffics from Cajal bodies to telomeres. Contrary to earlier FISH data in fixed cells, our preliminary data using diffraction-limited and super-resolution imaging modalities combined with single-molecule FISH show that hTR is broadly distributed throughout the nucleus. At telomeres, we show that following TPP1-driven recruitment, stable interactions are established between the enzyme and its substrate by RNA:DNA base pairing. Our goal is to apply photoactivation and photobleaching experiments to test the role of the catalytic subunit, hTERT, in the gating of hTR between the Cajal bodies and telomeres. In addition, we will engineer a short telomere to depict telomerase dynamics at critical telomeres that need to be elongated. Lastly, we will perform a proximity-based labeling and purification methodology to investigate the factors that control key steps of telomerase trafficking to short telomeres. All in all, our innovative approach offers a detailed view of the precise mechanics of telomere extension at physiological timescales and opens many future avenues for the study of the link between telomere maintenance and aging as well as cancer.