The durations of cell cycle phases for HeLa, RPE-1, RPE-1 in 100 nM rapamycin or 50 nM palbociclib at cell mass homeostasis.

MAD is the median absolute deviation, and nMAD is MAD normalized by the median in robust statistics. (DOCX)</p

Impact of growth rate variability on division mass CV, <i>CV</i>(<i>m</i>_<i>i</i>(<i>T</i>_<i>i</i>)), in the stochastic model.

The stochastic model is described in Section 4, Scenario IX in S1 Text. All parameter values used in this simulation are listed in the table at the end of Section 4, with the exception of CVgr, which is varied in this simulation. Solid blue lines indicate the simulation results. Filled blue circles are the division mass CV when simulated with the CVgr estimated from experimental data. Dashed black lines represent the division mass CV measured in experiments. The data underlying this figure and the scripts used to generate the plots are available on the Open Science Framework at osf.io/3kyvw. (EPS)</p

The values of <i>λ</i>′ and <i>α</i>′ used in Fig 5L, for untreated HeLa and RPE-1 cells, as well as RPE-1 cells treated with 50 nM palbociclib or 100 nM rapamycin.

The values of λ′ and α′ used in Fig 5L, for untreated HeLa and RPE-1 cells, as well as RPE-1 cells treated with 50 nM palbociclib or 100 nM rapamycin.</p

Cell mass variation is tightly controlled in mammalian cell lines and is robust to perturbations in G1/S regulation or growth rate.

(A–C) An abstract model of cell mass homeostasis at different G1 regulation strengths, represented by the slope of G1 length vs. birth mass correlation. The corresponding model and simulation parameters are in the Section 1 in S1 Text. In the model, we assume cells grow exponentially, and the G1 length control is the only mechanism to reduce cell mass variation. (A) Correlations between G1 length and birth mass. Blue: no G1 length control; red: with strong G1 length control; yellow: with weak G1 length control. (B) Cell mass CV changes with cell cycle progression during 1 cell cycle with the corresponding G1 length regulation in (A). (C) Birth mass CV changes across generations with the corresponding G1 length regulation in (A). (D–G) The mean birth mass (D), birth mass CV (E), division mass CV (F), and DA std. (G) for different cell lines. (H–K) The mean birth mass (H), birth mass CV (I), division mass CV (J), and DA std. (K) for RPE-1 and U2OS cells in normal culture medium, medium with 50 nM palbociclib, and medium with 100 nM rapamycin at cell mass homeostasis. Error bars in (D–K) indicate the standard deviation of 3 or more measurements. The data underlying this figure and the scripts used to generate the plots are available on the Open Science Framework at osf.io/3kyvw. CV, coefficient of variation; DA std., standard deviation of Division Asymmetry.</p

The normalized fitting parameters for the cell mass vs. growth rate correlations for different cell lines.

For correlations fitted better by the linear model, , the normalized parameters α′ and β′ are listed in the table, with α′ = αT>, , where T> and mb> are the means of cell cycle length and cell birth mass, respectively. For exponential growth, α′ = ln2 ~= 0.693. For correlations fitted better by the bilinear model, , the normalized parameters a′, b′, γ′, and are listed in the table, with a′ = aT>, . The correlation slopes, α′, a′, and γ′, lower than 0.75 or higher than 1.25-fold (arbitrarily chosen thresholds) of ln2 were highlighted. SE and BI denote the type of growth rate modulation, where SE stands for sub-exponential and BI stands for bilinear. (DOCX)</p

Comparison of the linear and bilinear fits for the cell mass vs. cell cycle phase length correlations.

The significantly better fits (p_bilinear or p_linear p (DOCX)</p

Characteristics of the human cell lines used in this study.

Characteristics of the human cell lines used in this study.</p

The teeter-totter model of cell mass homeostasis.

Cell mass homeostasis requires a balance between stochastic noise and control mechanisms. In unperturbed cells with an intact G1/S circuitry, the weights of control mechanisms from the heaviest to the lightest are the growth rate modulation, G1 length regulation, and nonG1 length regulation. When G1/S control is perturbed, the impact of the G1 length regulation becomes smaller, and the nonG1 length regulation and growth rate modulation become larger to compensate. When the growth rate modulation is suppressed, the G1 length regulation plays a more prominent role in compensating for the reduced impact of growth rate modulation.</p

Cell mass variation is regulated throughout the cell cycle.

(A, B) Cell mass CV change with cell cycle progression measured in live RPE-1 (n = 89) (A) and HeLa cells (n = 223) (B). The red solid lines denote the cell mass CV of the population; the pink shadows show the 95% confidence interval; the dashed line indicates the G1/S transition. (C–H) The profiles of how cell mass CV changes with cell cycle progression at cell mass homeostasis measured in fixed RPE-1 (C), HeLa (D), U2OS (E), and HT1080 (F) cells, as well as RPE-1 cells that had reached the new cell mass homeostasis with 50 nM palbociclib (G) or 100 nM rapamycin (H). The cell cycle stages were identified by DNA content and log(mAG-hGeminin) as illustrated in S4B–S4F, S4H, and S4J Fig; the late G1 and S phases are indicated by areas shaded in purple and orange, respectively; error bars are the standard error of CV, (), where n is the cell number at the corresponding cell cycle stage (n > 135 for all conditions). The data underlying this figure and the scripts used to generate the plots are available on the Open Science Framework at osf.io/3kyvw. CV, coefficient of variation.</p

Image1_The uniformity and stability of cellular mass density in mammalian cell culture.pdf

Cell dry mass is principally determined by the sum of biosynthesis and degradation. Measurable change in dry mass occurs on a time scale of hours. By contrast, cell volume can change in minutes by altering the osmotic conditions. How changes in dry mass and volume are coupled is a fundamental question in cell size control. If cell volume were proportional to cell dry mass during growth, the cell would always maintain the same cellular mass density, defined as cell dry mass dividing by cell volume. The accuracy and stability against perturbation of this proportionality has never been stringently tested. Normalized Raman Imaging (NoRI), can measure both protein and lipid dry mass density directly. Using this new technique, we have been able to investigate the stability of mass density in response to pharmaceutical and physiological perturbations in three cultured mammalian cell lines. We find a remarkably narrow mass density distribution within cells, that is, significantly tighter than the variability of mass or volume distribution. The measured mass density is independent of the cell cycle. We find that mass density can be modulated directly by extracellular osmolytes or by disruptions of the cytoskeleton. Yet, mass density is surprisingly resistant to pharmacological perturbations of protein synthesis or protein degradation, suggesting there must be some form of feedback control to maintain the homeostasis of mass density when mass is altered. By contrast, physiological perturbations such as starvation or senescence induce significant shifts in mass density. We have begun to shed light on how and why cell mass density remains fixed against some perturbations and yet is sensitive during transitions in physiological state.</p