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

摘要： 我们关于细胞时间是如何控制的知识几乎完全集中在细胞周期的领域。 细胞周期细胞周期如何调控的长期范式认为，主要的Cdk/Cyclin 振荡器（CCO）充当小区的主时钟。主时钟活动的逐步增加 被假定为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，最近的进展对这一教科书观点提出了质疑，因为它们揭示了 “自主时钟”：通常由CCO控制的定时机制， 分裂，但已经进化到自主运行，具有不同的计时作用，以便驱动特定的细胞 当细胞周期突然停止，错误调节或自然沉默时的现象。尽管他们的崛起 尽管在生理学和疾病中具有重要意义，但自主时钟如何运作的设计原理在很大程度上仍然存在， 未知同样，我们仍然不知道自主时钟是否以及如何自我调节以调节它们的频率。 功能，或生物物理基础，他们如何耦合运行在同步与CCO在细胞 周期在这里，我建议解决这些问题的背景下，细胞代谢，细胞器生物发生和 维持有丝分裂的保真度--细胞周期的三个关键方面，使细胞能够成功分裂。 汇集了荧光蛋白设计的最新技术，我们将设计出一种 振荡双功能酶报告，以确定潜在的自主时钟的设计原理 细胞代谢机制。通过结合分裂荧光，纳米荧光和CRISPR 重组工程技术，我们将创新一种可扩展的酶标记，以解开遗传景观， 自主时钟如何自我调节以调节细胞器的生物发生，或失调以干扰有丝分裂的保真度 疾病。最后，我们将开发可逆的光遗传学策略来测试一个物理启发的实验。 关于自主时钟如何与CCO耦合以在核分裂期间以核分裂的速度运行的框架 细胞周期这些研究将（i）破译自主时钟可能普遍适用的机制， 操作时间和启动特定的亚细胞事件，（ii）揭示了对关系的机械见解 通过系统性疾病相关遗传筛查，自主时钟的调谐和功能之间的关系，以及（iii） 产生关于自主时钟如何耦合到CCO的性质的未知信息， 可评分的表型，用于探索在分裂细胞中介导这种偶联的分子，或调节 当CCO在终末分化的细胞中失活时解偶联。总的来说，这些方法将 大大提高了我们剖析自主时钟工作原理的能力，并承诺令人兴奋的 扩大我们对它们在健康和疾病中新出现的作用的认识的可能性。

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.