Beyond G1/S regulation: How cell size homeostasis is tightly controlled throughout the cell cycle?

To achieve a stable mass distribution over multiple generations, proliferating cells require some means of counteracting stochastic noise in the rate of growth, the time spent in the cell cycle, and the imprecision of the equality of cell division. In the most widely accepted model, cell size is thought to be regulated at the G1/S transition, such that cells smaller than a critical size pause at the end of G1 phase until they have accumulated mass to a predetermined size threshold, at which point the cells proceed through the rest of the cell cycle. However, a model, based solely on a specific size checkpoint at G1/S, cannot readily explain why cells with deficient G1/S control mechanisms are still able to maintain a very stable cell mass distribution. Furthermore, such a model would not easily account for how stochastic variation in cell mass during the subsequent phases of the cell cycle can be anticipated at G1/S. To address such questions, we applied computationally enhanced Quantitative Phase Microscopy (ceQPM) to populations of proliferating cells, which enables highly accurate measurement of cell dry mass of individual cells throughout the cell cycle. From these measurements we can evaluate the factors that contribute to cell mass homeostasis at any point in the cell cycle. Our findings reveal that cell mass homeostasis is accurately maintained, despite disruptions to the normal G1/S machinery or perturbations in the rate of cell growth. Control of cell mass accumulation is clearly not confined to the G1/S transition but is instead exerted throughout the cell cycle. Using several mammalian cell types, we find that the coefficient of variation in dry mass of cells in the population begins to decline well before the G1/S transition and continues to decline throughout S and G2 phases. Among the different cell types tested, the detailed response of cell growth rate to cell mass differs. However, in general, when it falls below that for exponential growth, the natural increase in the coefficient of variation of cell mass is effectively constrained. We find that both size-dependent cell cycle regulation and size-dependent growth rate modulation contribute to reducing cell mass variation within the population. Through the interplay and coordination of these two processes, accurate cell mass homeostasis emerges. Such findings reveal previously unappreciated and very general principles of cell size control in proliferating cells. These same regulatory processes might also be operative in terminally differentiated cells. Further quantitative dynamical studies should lead to a better understanding of the underlying molecular mechanisms of cell size control. ### Competing Interest Statement The authors have declared no competing interest.