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

摘要： 我们关于细胞时间是如何被控制的知识几乎完全集中在 细胞周期。细胞周期调控的长期范式认为，主要的CDK/Cyclin 振荡器(CCO)充当单元的主时钟。该主时钟的活动的增量增加 被假定为定义了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，最近的进展对教科书上的这一观点提出了质疑，因为它们揭示了 “自主时钟”：通常由CCO强制要求以核的速度运行的计时机制 部门，但已演变为自主运行，具有不同的计时角色，以驱动特定的细胞 当细胞周期突然停止、调节不当或自然沉默时的现象。尽管它们出现了 在生理学和疾病上的重要意义，自主时钟如何运作的设计原则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能够自我调整以调节其 功能，或它们如何结合在细胞内与CCO同步运行的生物物理基础 周而复始。在这里，我建议在细胞新陈代谢、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。 结合荧光蛋白设计的最新技术，我们将设计出首个此类产品 确定潜在自主时钟设计原理的振荡式双功能酶报告 细胞新陈代谢机制。通过结合分裂荧光、纳米灯笼和基于CRISPR的 重组工程技术，我们将创新一种可伸缩的酶标记来揭开 自主时钟如何自我调整以调节细胞器的生物发生，或如何失谐以扰乱有丝分裂的保真度 在疾病中。最后，我们将开发可逆光遗传学策略来测试一个由物理启发的实验 自主时钟如何与CCO相结合以与核部门同步运行 细胞周期。这些研究将(I)破译潜在的可推广的机制，即自主时钟 操作以计时并启动特定的亚细胞事件，(Ii)揭示对关系的机械性洞察 通过系统的疾病相关基因筛查，自主时钟的调节和功能之间的关系，以及(Iii) 提供有关自动时钟如何与CCO耦合的性质的未知信息，帮助生成 可用于探索在细胞分裂中调节这种耦合的分子，或调节 在终末分化细胞中CCO失活时的去偶联。总的来说，这些方法将 极大地提高了我们剖析自主时钟工作原理的能力，并承诺 有可能扩大我们对它们在健康和疾病中新出现的作用的了解。

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.