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.

Circadian rhythm and cell cycle

Rosamaria Cannavo

Circadian rhythms are biological processes found in most living organisms, displaying a roughly 24-hour period, responding primarily to light darkness cycles in an organism's environment. At the cellular level, the circa-24h rhythmicity is generated by a molecular clock based on a transcription-translation feedback network and consists of a cell-autonomous and self-sustained oscillator. In conditions where cells proliferate, the cell division cycle can be also considered as an oscillator. Since both processes run with similar periods in several mammalian cells, it is reasonable to expect that interactions between these two cycles may cause synchronization. Many studies reported evidences of interactions between the circadian and the cell cycles in different organisms. In particular, it appeared that in several systems, specific cell cycle phases occur in distinct temporal windows rather than being randomly distributed in time. These findings led to the concept of circadian gating of the cell cycle, through which the circadian clock can favor or forbid cell cycle transitions at specific circadian phases. However, it was also reported the converse, namely an effect of cell division on the circadian oscillator. Even though interactions between the circadian clock and the cell cycle have been identified in both directions, the dynamical consequences and the directionality of the coupling at the single-cell level were not extensively investigated. In order to better characterize the potential synchronization in mammalian cells, we estimated the mutual interactions between circadian clock and cell cycle in NIH3T3 mouse fibroblasts by the use of time-lapse fluorescent microscopy in combination with statistical analysis and mathematical modeling. NIH3T3 cells, harboring a fluorescent reporter under the control of the circadian RevErb-a gene promoter, were imaged for several days allowing the simultaneous detection of circadian oscillations and time of divisions. The analysis of thousands of circadian cycles in dividing cells indicated that both oscillators are synchronized, with cell divisions occurring about 5 h before the peak of the circadian RevErb-a reporter. We tested several perturbations such as different serum concentrations, different temperatures, treatment with pharmacological compounds and shRNA-mediated knockdown of circadian regulators. Surprisingly, this showed that circadian rhythm and cell cycle remain synchronized over the wide range of conditions probed. Our data showed that this synchronization state reflects an unexpected predominant influence of the cell cycle on the circadian oscillator, and did not support the leading hypothesis about a circadian gating of the cell cycle. The stochastic modeling of two interacting phase oscillators allowed us to identify the parameters of the coupling functions, revealing an acceleration of circadian phase after the division. The work presented in this thesis sheds light on the interaction between two fundamentally recurrent cellular processes in mammalian cells and provides a deeper understanding of the role of the circadian clock in proliferating cells and tissues. These findings might have significant implications for chronobiology and chronotherapeutics.

EPFL2016

Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells

Jonathan Bieler, Rosamaria Cannavo, Cédric Gobet, Kyle Gustafson, Felix Naef

Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. 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 h before the peak in circadian Rev-Erba-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

Nature / European Molecular Biology Organization2014

Correlation between cell cycle and calcium phosphate transient transfection of CHO cells

Frédéric Grosjean

Calcium phosphate transfection is a widely used method to produce recombinant proteins in mammalian cells. However, the mechanisms involved in plasmid DNA transfer to the nucleus of the transfected cells remain poorly understood and result in great variation of transfection efficiency. The role of the cell cycle in transfection by the calcium phosphate method was studied using Chinese Hamster Ovary (CHO) cells as a model system. Adherent cell cultures were synchronized at the G1/S boundary using mimosine. At various time points after the removal of the drug the cells were transfected with reporter plasmids that expressed either the enhanced green fluorescent protein (EGFP) or a red fluorescent protein (DsRedExpress). Fluorescent protein expression was monitored by fluorometry and live imaging. Cell cycle was monitored by flow cytometry. It was shown that the cell cycle had major implications on at least two levels. The correlation between the percentage of cells in S phase at the time of transfection complex addition and the resulting reporter gene expression demonstrated that efficient transfer of exogenous DNA in mammalian cells was dependent on the cell cycle. A faster transfection protocol, developed within this work, showed that the optimal timing for the transfection complex addition was different depending on the transfection set-ups used. Further investigations demonstrated that mitosis played a key role, probably due to the nuclear membrane disruption, to provide access to the nuclear environment for the plasmid DNA molecules. The glycerol shock performed at the end of the transfection was also shown to play an important role for efficient transfer of reporter gene in nuclei. This strong osmotic shock resulted in a cellular volume decrease of approximately 55%. As no intracellular plasmids were lost during the process, this resulted in a significant increase in intracellular plasmid concentration. Glycerol shock has to be performed while cells proceed through mitosis. The nuclear membrane disruption and the increasing intracellular plasmid concentration probably facilitate the access to the nuclear environment. The synergy of glycerol shock and cells proceeding through mitosis was a requirement for efficient reporter gene transfer in the nuclei. If one of those two was omitted, or if they did not happened at the same time, very low reporter gene expression levels were achieved. As it was found that plasmid DNA was driving reporter gene expression even after a delay of 45 minutes post cellular uptake, it was possible to efficiently target additional sub-populations of cells as they proceeded through mitosis with additional glycerol shocks. Those repetitive glycerol shocks resulted in an increase of positive cells among the transfected populations. In conclusion, the results obtained in this work permitted to define a model explaining the importance of the cell cycle in the calcium phosphate transfection, highlighting mitosis and glycerol shock as key events in plasmid DNA molecules transfer to the nucleus.