Decoding the fundamental principles of autonomous clocks: mechanism, design and function

解读自主时钟的基本原理：机制、设计和功能

• 批准号: 10685116
• 负责人: Mustafa Gonenc Aydogan
• 金额: $ 145.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-20 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685116
• 关键词: Address; Biogenesis; Biological; Biophysics; Cell Cycle; Cell Differentiation process; Cell division; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Coupling; Crystallization; Cyclins; Disease; Enzymes; Event; Fluorescence; Genetic; Genetic Screening; Health; Knowledge; Maintenance; Mediating; Metabolism; Mitosis; Mitotic; Molecular; Nature; Organelles; Outcome; Pathology; Phenotype; Physics; Physiology; Protein Engineering; Quality Control; Regulation; Reporter; Role; Running; Techniques; Technology; Testing; Textbooks; Time; Work; design; innovation; insight; nuclear division; optogenetics

Abstract: Our knowledge of how cellular time is controlled has been centered almost exclusively within the realms of the cell cycle. The long-standing paradigm of how the cell cycle is regulated holds that the principal Cdk/Cyclin oscillator (CCO) acts a master clock for the cell. Incremental increase in the activity of this master clock has been postulated to define a set of thresholds to time and execute different cellular events that lead to mitosis. Recent advances, however, have called this textbook view into question, as they reveal the existence of `autonomous clocks': timing mechanisms that are normally entrained by the CCO to run at the pace of nuclear divisions, but have evolved to run autonomously with distinct timekeeping roles, so as to drive specific cellular phenomena when the cell cycle is abruptly halted, mis-regulated or naturally silenced. Despite their emerging significance in physiology and disease, the design principles of how autonomous clocks operate remain largely unknown. Similarly, we still do not know whether and how autonomous clocks can self-tune to regulate their function, or the biophysical underpinnings of how they couple to run in synchrony with the CCO during the cell cycle. Here I propose to address these questions in the context of cellular metabolism, organelle biogenesis and the maintenance of mitotic fidelity – three pivotal aspects of the cell cycle that enable successful cell divisions. Bringing together a palette of latest techniques in fluorescent protein design, we will design a first-of-its-kind oscillatory bifunctional enzyme reporter to identify the design principle of a potential autonomous clock mechanism in cellular metabolism. By combining split-fluorescence, nanolanterns and CRISPR-based recombineering technologies, we will innovate a scalable enzyme marker to unravel the genetic landscape of how an autonomous clock can self-tune to regulate organelle biogenesis, or mis-tune to perturb mitotic fidelity in disease. Finally, we will develop reversible optogenetics strategies to test a physics-inspired experimental framework on how autonomous clocks can couple with the CCO to run at the pace of nuclear divisions during the cell cycle. These studies will (i) decipher potentially generalizable mechanisms by which autonomous clocks operate to time and initiate specific sub-cellular events, (ii) reveal mechanistic insights into the relationship between the tuning and function of autonomous clocks via systematic disease-relevant genetic screens, and (iii) yield uncharted information on the nature of how autonomous clocks couple to the CCO, helping to generate scorable phenotypes for exploring molecules that mediate such coupling in dividing cells, or regulate a decoupling when the CCO is inactivated in terminally differentiated cells. Broadly, these approaches will significantly advance our ability to dissect the working principles of autonomous clocks, and promise the exciting possibility of expanding our knowledge on their emerging roles in health and disease.

抽象的： 我们对如何控制细胞时间的知识几乎集中在专有领域内。 细胞周期如何调节的长期范例认为主要的 Cdk/Cyclin。 振荡器（CCO）充当单元的主时钟，该主时钟的活动增量增加。 已被假定定义一组阈值来计时和执行导致有丝分裂的不同细胞事件。 然而，最近的进展使教科书的观点受到质疑，因为它们揭示了 “自主时钟”：通常由 CCO 控制以核速度运行的计时机制 部门，但已经发展到具有不同计时角色的自主运行，从而驱动特定的细胞 尽管出现了细胞周期突然停止、失调或自然沉默的现象。 在生理学和疾病方面具有重要意义，自主时钟的设计原理在很大程度上仍然存在 同样，我们仍然不知道自主时钟是否以及如何自我调整来调节它们。 功能，或它们如何在细胞期间与 CCO 同步运行的生物物理基础 在这里，我建议在细胞代谢、细胞器生物发生和循环的背景下解决这些问题。 维持有丝分裂保真度——细胞周期的三个关键方面，使细胞能够成功分裂。 汇集荧光蛋白设计中的最新技术，我们将设计出一种首个 振荡双功能酶报告基因确定潜在自主时钟的设计原理 通过结合分裂荧光、纳米灯和基于 CRISPR 的细胞代谢机制。 重组工程技术，我们将创新可扩展的酶标记物，以揭开基因图谱 自主时钟如何自我调节以调节细胞器的生物发生，或错误调节以扰乱有丝分裂的保真度 最后，我们将开发可逆光遗传学策略来测试受物理启发的实验。 关于自主时钟如何与 CCO 结合以在期间以核分裂的速度运行的框架 这些研究将（i）破译自主时钟的潜在普遍机制。 操作来计时并启动特定的亚细胞事件，（ii）揭示对这种关系的机制见解 通过系统的疾病相关遗传筛选来调节自主时钟和功能之间的关系，以及（iii） 产生有关自主时钟如何与 CCO 耦合的性质的未知信息，有助于生成 可评分的表型，用于探索介导分裂细胞中这种偶联的分子，或调节 当 CCO 在终末分化细胞中失活时，这些方法将发生解偶联。 显着提高我们剖析自主时钟工作原理的能力，并承诺令人兴奋的成果 扩大我们对它们在健康和疾病中新兴作用的认识的可能性。