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

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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.