Abstract
In a complex and ever-changing environment, various signal transduction pathways mediate outside signals and stress to a living cell and its intracellular responses. Eukaryotic cells utilize the DNA synthesis phase (S-phase) checkpoint to respond to DNA damage and replication stress, and the activation of the S-phase checkpoint defers the routine progression in the S phase. Through the analysis of microfluidic single-cell measurements, we find that the behavior of yeast cells exhibits bimodal distribution in the activation of the S-phase checkpoint, and the nonactivated portion of cells obeys the exponential decay law over time, the rate of which is dictated by HU dosage. Mathematical modeling and further experimental evidence from different mutant strains support the idea that the activation of the yeast S-phase checkpoint is a stochastic barrier-crossing process in a double-well system, where the barrier height is determined by both DNA replication stress and autophosphorylation of the key effector kinase Rad53. Our approach, as a novel methodology, is generally applicable to quantitative analysis of the signal transduction pathways at the single-cell level.
- Received 19 May 2020
- Revised 20 October 2020
- Accepted 17 November 2020
DOI:https://doi.org/10.1103/PhysRevX.11.011004
Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.
Published by the American Physical Society
Physics Subject Headings (PhySH)
Popular Summary
Living cells respond to changes in their environment via a complex system of chemical signals. These signals in turn trigger alterations to the cell itself via a network of internal signal pathways and genetic regulation. But not all cells respond to their environment in exactly the same way. To understand that variability, we investigate how a genetic regulation mechanism in single cells of yeast responds to chemical stress signals in the presence of noise.
Specifically, we study the S-phase checkpoint activation process in single cells of the budding yeast Saccharomyces cerevisiae. S-phase, or DNA synthesis phase, checkpoints are quality checks the cell conducts on its DNA during replication. Through analysis of time-lapse fluorescence experiments, we discover the existence of “on” and “off” cell states during the activation of S-phase checkpoints in the cell cycle. The state transition follows the off-to-on state switching with an exponentially distributed waiting time. Furthermore, we develop a theoretical model to describe the checkpoint activation as a barrier-crossing process in which the cell “hops” from one metastable state to another, triggered by noise in the chemical activation reactions. Both the chemical stress and the positive feedback of a key enzyme, known as kinase, in the pathway determine the barrier height and the switching rate.
Overall, our results provide direct evidence of in vivo stochastic cell-state transitions in single cells. The critical role of noise in triggering these transitions is key to understanding cell-to-cell variability in response to different types and levels of external stress. The single-cell trajectory analysis method that we develop is also applicable to other single-cell experiments.
