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

摘要： 真核细胞通过添加 染色体末端的端粒序列。端粒长度的适当调节是 对基因组完整性、细胞寿命调节、衰老和癌症至关重要。多年来， 遗传和生物化学研究已经揭示了这个过程是如何发生的， 控制。然而，在拥挤的细胞核中这一过程的细胞生物学仍然很差 理解，端粒延伸的时间，动力学和空间协调是 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 和卤素荧光团，以可视化活细胞中内源性端粒酶的单分子。在这里， 我们将破译hTR从卡哈尔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，我们使用衍射限制和 超分辨率成像模式结合单分子FISH显示，hTR是 广泛分布在整个细胞核。在端粒，我们表明，以下TPP1驱动 在酶与其底物之间建立稳定的相互作用， RNA：DNA碱基配对。我们的目标是应用光活化和光漂白实验， 测试催化亚基hTERT在Cajal小体之间的hTR门控中的作用， 端粒此外，我们将设计一个短端粒来描绘端粒酶动力学， 关键的端粒需要被拉长。最后，我们将执行基于邻近度的标记 研究端粒酶关键步骤的控制因素 短端粒的运输。总而言之，我们的创新方法提供了一个详细的视图， 在生理时间尺度上端粒延伸的精确机制，并开辟了许多未来 研究端粒维持与衰老以及癌症之间的联系的途径。

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.