High-resolution mapping of the period landscape reveals polymorphism in cell cycle frequency tuning
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Evaluation Summary:
This paper uses microfluidics and Xenopus extracts to investigate the effects of mitotic feedbacks on the cell cycle period. It is shown that the inhibition of G2 regulatory positive feedback loops does not reduce the tunability of the cell cycle oscillations, while interference with PP2A phosphatase can completely block of the cell cycle. The experiments are well-conducted and the results should be of interest to researchers interested in the cell cycle, specifically in the regulation of mitosis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Many biological oscillators exhibit widely tunable frequency in adapting to environmental changes. Although theoretical studies have proposed positive feedback as a mechanism underlying an oscillator’s large tunability, there have been no experiments to test it. Here, applying droplet microfluidics, we created a population of synthetic cells, each containing a cell-cycle oscillator and varying concentrations of cyclin B mRNAs for speed-tuning and positive-feedback inhibitors for modulating network interactions, allowing a continuous mapping of the cell-cycle period landscape in response to network perturbation. We found that although the cell cycle’s high tunability to cyclin B can reduce with Wee1 inhibition, the reduction is not as great as theoretically predicted, and another positive-feedback regulator, PP2A, may provide additional machinery to ensure the robustness of cell cycle period tunability. Remarkably, we discovered polymorphic responses of cell cycles to the PP2A inhibition. Droplet cells display a monomodal distribution of oscillations peaking at either low or high PP2A activity or a bimodal distribution with both low and high PP2A peaks. We explain such polymorphism by a model of two interlinked bistable switches of Cdk1 and PP2A where cell cycles exhibit two different oscillatory modes in the absence or presence of PP2A bistability.
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Evaluation Summary:
This paper uses microfluidics and Xenopus extracts to investigate the effects of mitotic feedbacks on the cell cycle period. It is shown that the inhibition of G2 regulatory positive feedback loops does not reduce the tunability of the cell cycle oscillations, while interference with PP2A phosphatase can completely block of the cell cycle. The experiments are well-conducted and the results should be of interest to researchers interested in the cell cycle, specifically in the regulation of mitosis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This paper uses microfluidics and Xenopus extracts to show the effects of mitotic feedbacks on the cell cycle period. The authors show that perturbing both the activity of Wee1 and mitotic phosphatases can alter the cell cycle period. These are well-conducted and original experiments. A weakness of the paper is that it is framed very generally as a mechanism of controlling the cell cycle period. However, there are many additional mechanisms that likely contribute to control of cell cycle duration. This paper should be of interest to researchers working on the cell cycle, specifically on the regulation of mitosis. The microfluidic methodology should be generally applicable and significantly extends the ability to analyze multiple extracts at the same time.
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Reviewer #2 (Public Review):
This is a study employing an innovative approach of microencapsulation of Xenopus cycling extract to address the role of the positive feedback branches in the cell cycle oscillation.
Xenopus extract is an established system to study the mechanism of cell division. At the core of the Xenopus cell cycle is a negative feedback oscillator of cyclinB-CDK-APC/C. Recently, the importance of phosphatases, such as pp2a, in regulating cell cycle was proposed. In addition to the central negative feedback circuit, the role of various positive feedback branches in a broader cell cycle network has been discussed, mostly through simulation.
The method created in this study is a quantitative and high throughput platform to address mechanistic questions in Xenopus cell cycle. Their study explored the important question of …
Reviewer #2 (Public Review):
This is a study employing an innovative approach of microencapsulation of Xenopus cycling extract to address the role of the positive feedback branches in the cell cycle oscillation.
Xenopus extract is an established system to study the mechanism of cell division. At the core of the Xenopus cell cycle is a negative feedback oscillator of cyclinB-CDK-APC/C. Recently, the importance of phosphatases, such as pp2a, in regulating cell cycle was proposed. In addition to the central negative feedback circuit, the role of various positive feedback branches in a broader cell cycle network has been discussed, mostly through simulation.
The method created in this study is a quantitative and high throughput platform to address mechanistic questions in Xenopus cell cycle. Their study explored the important question of the functions of different feedback modules in the cell cycle.
I find their results generally interesting and valuable to the cell cycle community, though some appear incomplete.
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Reviewer #3 (Public Review):
The authors developed a microfluidic device and a reporter molecule to study cell cycle progression in micro size droplets of in frog egg extracts. They have investigated how interference with regulators of the G2/M transition or mitotic exit control the oscillations in the activity of the main cell cycle controller, Cdk1. They found that the inhibition of the G2 controller Wee1 did not reduce the responsiveness of the system for changes in Cyclin B levels. On the contrary, inhibition of PP2A leads to diverse responses, all having more severe effect on the cell cycle, some cases a complete block of Cdk1 activity changes. They update a published mathematical model to explain the observations of the diverse responses to PP2A inhibition.
Technically and methodologically a great paper, where several new tools …
Reviewer #3 (Public Review):
The authors developed a microfluidic device and a reporter molecule to study cell cycle progression in micro size droplets of in frog egg extracts. They have investigated how interference with regulators of the G2/M transition or mitotic exit control the oscillations in the activity of the main cell cycle controller, Cdk1. They found that the inhibition of the G2 controller Wee1 did not reduce the responsiveness of the system for changes in Cyclin B levels. On the contrary, inhibition of PP2A leads to diverse responses, all having more severe effect on the cell cycle, some cases a complete block of Cdk1 activity changes. They update a published mathematical model to explain the observations of the diverse responses to PP2A inhibition.
Technically and methodologically a great paper, where several new tools were developed, but the biological conclusions are somewhat limited, given earlier work in the field.
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