A Robust and Tunable Mitotic Oscillator in Artificial Cells

[摘要] This dissertation aims to develop a droplet-based artificial cell system using cell-free extracts of Xenopus laevis eggs and understand mitotic oscillations with the proposed system. Single-cell analysis is pivotal to deciphering complex phenomena such as cellular heterogeneity, bistable switches, and oscillations, where a population ensemble cannot represent the individual behaviors. Despite having unique advantages of manipulation and characterization of biochemical networks, bulk cell-free systems lack the essential single-cell information to understand out-of-steady-state dynamics including cell cycles. In this dissertation, we present a novel artificial single-cell system for the study of mitotic dynamics by encapsulating Xenopus egg extracts in water-in-oil micro-emulsions. The artificial cells are different from real cells, i.e., their surface is formed by surfactant oil instead of the cell membrane. These ;;cells”, adjustable in sizes and periods, encapsulate cycling cytoplasmic extracts that can sustain mitotic oscillations for over 30 cycles. The artificial cells function in forms from the simplest cytoplasmic-only oscillators to the more complicated ones involving demembranated sperm chromatin that can reconstitute downstream mitotic events. The dynamic activities of cell cycle clock can be detected by fluorescent reporters such as cyclin B1-YFP and securin-mCherry. This innate flexibility makes it key to studying cell cycle clock tunability and stochasticity. Our experimental results indicate that the mitotic oscillators generated by our system are effectively tunable in frequency with cyclin B1 mRNAs and the dynamic behavior of single droplet oscillators is size-dependent. We also establish a stochastic model that highlights energy supply as an essential regulator of cell cycles. Moreover, the model explains experimental observations including the increase of baseline and amplitude of cyclin B1 time course. This dissertation study demonstrates a simple, powerful, and likely generalizable strategy of integrating single-cell approaches into conventional in vitro systems to study complex clock functions.

[发布机构] University of Michigan

[效力级别] Artificial cells

[关键词] Cell cycle clock;Artificial cells;Tunability;Biological Chemistry;Molecular;Cellular and Developmental Biology;Statistics and Numeric Data;Science;Chemistry