Spatio-temporal synchronization of biological oscillators

The circadian clock is a cell-autonomous and self-sustained oscillator with a period of about 24 hours that controls many aspects of cellular physiology. The cell cycle is also a fundamental periodic process, with a period in the range of one day in mammals. A large number of studies showed that these two cycles interact at the molecular level. Mathematical theory of coupled oscillators predicts that in the presence of coupling, two cycles with similar periods can synchronize. Consequently, the molecular interactions between the cell cycle and the circadian clock could lead to their synchronization. Such synchrony is consistent with a number of studies showing that cells divide more at specific circadian times. Moreover, a few works also report effects of cell divisions on the circadian oscillator. More detailed mathematical models of the molecular networks involved also predict that the two cycles can synchronize. To better characterize this potential synchronization in mammalian cells, we monitored the mutual interactions between the two oscillators by time-lapse imaging of single NIH3T3 fibroblasts during several days. To do so, we used a fluorescent reporter under the control of a circadian promoter and monitored the cell cycle progression by detecting the time of mitosis. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 hours before the peak in circadian Rev-Erbα -YFP reporter expression. In principle, such synchrony may be caused by coupling from one oscillator onto the other, or in both directions. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling strongly suggest that reverse coupling is predominant in NIH3T3 cells. In order to further test the direction of the coupling, we performed several perturbation experiments; we changed the cell cycle period by acquiring recordings at different temperatures, we analyzed cell lines containing genetic knock-down of circadian genes and used drugs that target specific proteins of the cell cycle and the circadian clock. These experiments showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. Moreover, the set of perturbations on the periods of either oscillator induced shifts in the relative phases of circadian and cell cycles that were consistent with a scenario in which the circadian cycle resonates to the cell cycle. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.