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; 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终端复制和终端保护问题 端粒序列到染色体的末端。正确调节端粒长度是 对于基因组完整性，细胞寿命，衰老和癌症的调节至关重要。历年， 遗传学和生化研究已经对该过程的方式揭示了大量的启示 受控。但是，拥挤的核中此过程的细胞生物学仍然很差 理解，端粒扩展的时间，动态和空间协调是 未知。为了解决这些差距，我们将利用MS2标记系统 和晕氟团团可视化活细胞中内源性端粒酶的单个分子。这里， 作为HTR流量从Cajal机构到端粒，我们将破译离散和关键步骤。 与固定细胞中早期的鱼数据相反，我们使用衍射限制的初步数据和 超分辨率成像方式与单分子鱼类相结合，表明HTR是 广泛分布在整个核中。在Telomeres，我们表明以TPP1驱动以后 招募，稳定的相互作用是通过酶及其底物之间的稳定相互作用 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体之间HTR门控和 端粒。此外，我们将设计一个简短的端粒，以描绘端粒酶动力学 需要拉长的关键端粒。最后，我们将执行基于接近度的标签 和纯化方法，以研究控制端粒酶关键步骤的因素 贩运短端粒。总而言之，我们的创新方法提供了有关 端粒扩展的精确力学在生理时间尺度上，并打开了许多未来 研究端粒维持与衰老与癌症之间联系的途径。