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Cell size homeostasis is tightly controlled throughout the cell cycle [1]

['Xili Liu', 'Department Of Systems Biology', 'Harvard Medical School', 'Boston', 'Massachusetts', 'United States Of America', 'Jiawei Yan', 'Department Of Chemistry', 'Stanford University', 'Stanford']

Date: 2024-01

To achieve a stable size distribution over multiple generations, proliferating cells require a means of counteracting stochastic noise in the rate of growth, the time spent in various phases of the cell cycle, and the imprecision in the placement of the plane 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 size distribution. Furthermore, such a model would not easily account for stochastic variation in cell size during the subsequent phases of the cell cycle, which cannot be anticipated at G1/S. To address such questions, we applied computationally enhanced quantitative phase microscopy (ceQPM) to populations of cultured human cell lines, which enables highly accurate measurement of cell dry mass of individual cells throughout the cell cycle. From these measurements, we have evaluated the factors that contribute to maintaining 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 is generally not confined to regulation of the G1 length. Instead mass homeostasis is imposed throughout the cell cycle. In the cell lines examined, we find that the coefficient of variation (CV) 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 CV of cell mass is effectively constrained. We find that both mass-dependent cell cycle regulation and mass-dependent growth rate modulation contribute to reducing cell mass variation within the population. Through the interplay and coordination of these 2 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.

Cell size can be expressed either in terms of mass or volume. Cell volume tends to be a more passive response than mass to the mechanical and osmotic conditions occurring during the cell cycle and differentiation [ 22 – 25 ]. Hence, we have chosen to focus on cell mass homeostasis. There are excellent experimental means to measure cell mass in suspension culture [ 26 ], but it is much harder to measure cell mass accurately when cells are attached to a substratum, which is closer to the physiological context for most mammalian cell types. This single experimental limitation has thwarted the study of cell mass homeostasis and growth rate control in the most well-studied systems. Measuring the mass of a single cell on a culture dish accurately is surprisingly difficult. Furthermore, determining the growth rate from the time derivative of the mass is even more challenging [ 27 , 28 ]. The study of cell mass growth rate regulation in attached cells with sufficient precision to distinguish between different models of growth control required the development of new methods. To this end, we recently developed computationally enhanced quantitative phase microscopy (ceQPM), which measures cell dry mass (the cell’s mass excluding water) by the refractive index difference between cell and medium to a precision of better than 2% [ 29 ]. To describe statistically significant features of cell mass and growth rate regulation, we tracked single-cell growth and the timing of cell cycle events at a scale of thousands of cells per experiment. Using this improved technology, we could investigate the process of cell mass accumulation relative to cell cycle progression throughout the cell cycle. From these improved measurements, we could derive new understandings of cell mass homeostasis during the cell cycle in several cultured cell lines. The results challenge existing theories of cell mass (or, more colloquially, cell size) homeostasis and suggest further mechanistic experiments.

In keeping with a previous study in bacteria [ 21 ], we wish to distinguish between “size control” and “size homeostasis.” We will use the term “size control” to refer to the regulation of the mean size, such as when the mean size in a population of cells responds to a change of environment or when cells differentiate into a different cell type; whereas, we reserve the term “size homeostasis” for the control of the variance around the mean size of a population in a defined steady-state condition. Though these 2 processes may turn out to be mechanistically related, we cannot assume that they share the same mechanism. In this study, our focus is on the less well studied but perhaps more common process of size homeostasis. We used cultured cell lines because primary cells can take a very long time to reach a stable cell size in culture, whereas cell lines are much more stable and reproducible. Furthermore, cell lines have been well characterized; hence, observations from different laboratories can be readily compared and experiments can be easily replicated. Finally, we expect that size regulation would occur in all cell types, normal and transformed, embryonic and differentiated. Like other general cellular mechanisms, such as mitosis, DNA replication, and protein secretion, it is highly likely that underlying general mechanisms are conserved. To test this generality, we have studied size regulation during the cell cycle in several human cell lines of diverse origins, cultured under different conditions.

An alternative approach for regulating cell size, other than regulating it at S phase entry or in the length of other cell cycle phases, would be to regulate cell growth [ 1 , 19 ]. A few studies have suggested various types of size-dependent growth rate modulation in cultured cells. For example, Cadart and colleagues found that the slope of volume growth rate versus cell volume decreases for large cells at birth [ 7 ]; Neurohr and colleagues found that volume growth rate slows down in excessively large senescent cells [ 20 ]; and Ginzberg and colleagues found that nuclear area, an approximate proxy for cell size, is negatively correlated with growth rate at 2 points during the cell cycle [ 8 ]. Though such observations have been noted, there has been little said about their quantitative importance. Furthermore, it is hard to evaluate the various types of growth modulation, as they were discovered in different systems using different physical proxies for cell size, such as cell volume and nuclear area. Hence, little can be concluded about whether these processes coexist in the same cell, are specific to certain cell types, or are only reflected in certain types of cell measurement. Compared to studies on cell cycle control, cell growth control has received little attention.

In 1985, Zetterberg and colleagues reported that the variation of G1 length in mouse fibroblast cells accounted for most of the variation in cell cycle length when cells switched from quiescence to proliferation [ 14 ]. However, a later study in several cell lines found the G1, S, and G2 phase lengths had comparable variability and were all positively correlated with the cell cycle length in normal cycling populations [ 15 ], implying a dependency of cell cycle phase lengths on cell size outside of G1. Furthermore, regulation of the S and G2 lengths is known to make a contribution to size homeostasis in lower eukaryotic organisms, such as budding and fission yeasts [ 16 – 18 ]. However, evidence of size-dependent regulation outside of G1 has seldom been reported in mammalian cells [ 4 , 7 ]. Little is known about whether the nonG1 phases play an appreciable role in maintaining mammalian cell size homeostasis or whether variation in cell size introduced in the nonG1 phases is somehow fully compensated at the next G1/S transition.

The size distribution of a population of proliferating cells is accurately maintained over many generations, despite variability in the growth rate and the duration of the cell cycle in individual cells, as well as the imprecision in the equipartition of daughter cells at mitosis. Each of these factors is known to contribute to a dispersion in cell size within a population [ 1 ]. It has long been evident that there must be some “correction” mechanism that would act within individual cells to counteract the combined effects of all the sources of random variation and thereby ensure a stable size distribution in the population over many generations [ 2 ]. Studies on mammalian and yeast cell size up to now have focused on 1 attractive and plausible mechanism for size homeostasis: a dependence of the G1 length inversely with cell size. Theoretically, such a mechanism should allow small cells to “catch up” with larger cells by spending a longer time growing in the G1 phase. Such a process would be expected to reduce cell size variation by normalizing size at the point of S phase entry [ 2 – 9 ]. Several molecular players in this process have been suggested, such as the dilution of retinoblastoma (Rb) protein [ 6 , 9 , 10 ] and the activation of p38 MAPK kinase [ 11 , 12 ]. However, such a mechanism, while attractive for its simplicity, cannot in principle fully explain the constancy in the cell size distribution over many generations. Specifically, if G1 length regulation were the only operative mechanism, cells would have no way to anticipate the random variation introduced during the subsequent nonG1 cell cycle phases, a period longer than G1 in most proliferating cell types. Nevertheless, most proliferating cell populations, regardless of their surrounding environment and genetic background, manage to achieve highly accurate size homeostasis [ 13 ].

Results

Other explanations for how a population of cells might reduce its cell mass variation We evaluated additional processes that could potentially contribute to the reduction in cell mass CV but were not accounted for in our stochastic model. In principle, any process that affects the likelihood of cell division or cell viability differentially in large and small cells could influence the distribution of cell mass within a population. To estimate the importance of such effects, we examined the rate of cell death and cell cycle arrest through long-term measurements of cell growth and proliferation. During the 48 to 72-h duration of our cell measurements, we defined cell cycle arrest events as instances where a cell remained in the same cell cycle phase while its mass continued to increase throughout the experiment. Furthermore, cell death was identified by a sudden and drastic decrease in cell dry mass, suggesting cell membrane permeabilization. We found events of cell cycle arrest or cell death in the culture affected no more than 2% of cells in all the conditions that were studied (S9 Table). In particular, neither cell cycle arrest nor cell death occurred frequently enough to contribute significantly to cell mass homeostasis in any of the experiments that we have described. It is worth noting that the remarkably low frequency of cell cycle arrest in cells treated with rapamycin and palbociclib at the drug concentrations used in this study suggests that these drugs at low concentrations do not induce quiescence or senescence at the population level (S9 Table). Furthermore, the concentrations of these drugs did not appear to be toxic enough to cause significant cell death (S9 Table). One intriguing observation was that some large RPE-1 cells treated with palbociclib experienced a partial loss of cytoplasm during mitosis (S9 Table and S1 Movie). This cytoplasmic loss could be attributed to incomplete cortical contraction during mitotic rounding [63]. The amount of mass loss appeared to be random. Notably, these rare events, accounting for approximately 0.5% of cells, did not have a significant impact at the population level on cell mass homeostasis in the presence of palbociclib. It is worth noting that although these mechanisms were of negligible importance in the specific experimental setting of our study, they might still play a significant role in a tissue setting, for example, during wound healing, regeneration, aging, and/or disease.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002453

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