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

在单分子水平上研究活细胞中的端粒酶动力学

• 批准号: 10753326
• 负责人: Pascal Chartrand
• 金额: $ 42.36万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10753326
• 关键词: Address; Aging; Bacteriophages; Base Pairing; Biochemical; Biology; Catalytic Domain; Cell Aging; Cell Nucleus; Cell Proliferation; Cells; Cellular biology; Chromosomal Stability; Chromosomes; Crowding; Cytoplasmic Granules; DNA; Data; Development; Engineering; Ensure; Enzymes; Eukaryotic Cell; Future; Genetic; Goals; Human; Image; Label; Length; Ligation; Light; Link; Longevity; MS2 coat protein; Maintenance; Malignant Neoplasms; Mechanics; Membrane; Methodology; Organelles; Phase; Photobleaching; Physiological; Prevention; Process; Proteome; Proteomics; RNA; RNA-Directed DNA Polymerase; Regulation; Ribonucleoproteins; Role; System; TEL1 Gene; Telomerase; Telomerase RNA Component; Telomere Maintenance; Testing; Tumor Suppression; Untranslated RNA; Visualization; Work; Yeasts; cancer cell; cancer therapy; cell fixing; cell growth regulation; design; experimental study; fluorophore; genetic approach; genome integrity; image visualization; imaging modality; innovation; molecular imaging; novel; photoactivation; prevent; recruit; single molecule; stem; superresolution imaging; telomere; trafficking

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.

抽象的： 真核细胞通过添加解决末端复制和末端保护问题 端粒序列到染色体末端。端粒长度的正确调节是 对于基因组完整性、细胞寿命调节、衰老和癌症至关重要。历年， 遗传和生化研究为这一过程的发生提供了大量线索 受控。然而，在拥挤的细胞核中这一过程的细胞生物学仍然很差 端粒延伸的时间、动态和空间协调是 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 Halo 荧光团以可视化活细胞中内源端粒酶的单分子。这里， 我们将破译 hTR 从卡哈尔体到端粒的离散且关键的步骤。 与早期固定细胞中的 FISH 数据相反，我们使用衍射极限和 超分辨率成像方式结合单分子 FISH 表明 hTR 广泛分布于整个细胞核。在端粒方面，我们表明遵循 TPP1 驱动 通过招募，酶与其底物之间建立稳定的相互作用 RNA：DNA 碱基配对。我们的目标是将光活化和光漂白实验应用于 测试催化亚基 hTERT 在 Cajal 小体和 hTR 门控中的作用 端粒。此外，我们将设计一个短端粒来描述端粒酶动力学 需要延长的关键端粒。最后，我们将执行基于邻近度的标记 和纯化方法来研究控制端粒酶关键步骤的因素 贩运到短端粒。总而言之，我们的创新方法提供了详细的视图 端粒在生理时间尺度上延伸的精确机制，开辟了许多未来 研究端粒维持与衰老以及癌症之间联系的途径。