Investigating the Regulatory Pathways of the Mitotic Oscillator Via a High-Throughput Droplet-Based System

Abstract

This dissertation aims to investigate the properties of the mitotic oscillator and its complex regulatory mechanism. Responsible for the progression of the cell cycle through its various stages, the mitotic oscillator is highly conserved in organisms and is essential for life, however the design principles of the biochemical network behind the oscillator and the significance of its topology are not fully understood. Falling into the “activator-inhibitor” category, the mitotic circuit has a negative-plus-positive feedback structure. To untangle the complex interactions of this circuit, in this work I present a novel droplet-based platform combined with microfluidic techniques where Xenopus egg extracts were encapsulated in water-in-oil microemulsions. Our system allows for the fine tuning of all kinds of variables including droplet size and extract content and can reliably reconstitute the cell cycle dynamics. Primary tests using this system indicate that the mitotic oscillators are to a certain extent robust to perturbations while tunable in bi-stability, speed, and accuracy. Experimentations on partial inhibition of the network revealed shortcomings of current cell cycle models, which cannot explain the oscillator’s multimodal behavior and thus suggests other essential regulatory pathways. Taking advantage of the setup. I also investigated the oscillator behavior at different energy levels and discovered a non-monotonic response in oscillator speed and accuracy, which points to interesting mechanisms designed to sense and respond to a cell’s energy budget.