The uniformity and stability of cellular mass density in mammalian cell culture

Abstract

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.

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

“…5). This consistency is further supported by our recent findings that cell volume usually changes proportionally with cell mass in cultured proliferating cells, except during mitosis, resulting in a narrow distribution of cell mass density (67). However, we were able to observe more detailed discrepancies in the regulation of mass and volume growth.…”

Protein homeostasis from diffusion-dependent control of protein synthesis and degradation

“…The cytoplasm is crowded with macromolecules, with proteins being the most abundant class. In total, proteins constitute about 82% of the dry mass of a mammalian cell (Oh et al, 2022) and about 55% of an E. coli cell (Milo et al, 2009), and the cytoplasmic concentration of protein ranges from about ~75 mg/mL in mammalian cell lines (Liu et al, 2022) and Xenopus egg extracts (Green, 2009;Murray, 1991) to 200-320 mg/mL in E. coli (Elowitz et al, 1999;Zimmerman and Trach, 1991). Assuming an average protein density of 1.35 g/mL, this means that the volume fraction of a cell occupied by protein is ~6% to 25%, and the macromolecular excluded volume is higher still.…”

“…Assuming an average protein density of 1.35 g/mL, this means that the volume fraction of a cell occupied by protein is ~6% to 25%, and the macromolecular excluded volume is higher still. Although protein concentration transiently decreases during mitosis (Son et al, 2015;Zlotek-Zlotkiewicz et al, 2015), for a given cell type, the concentration of macromolecules in the cytoplasm is tightly regulated and remarkably constant (Delarue et al, 2018;Liu et al, 2022;Neurohr and Amon, 2020;Oh et al, 2022). This brings up two basic questions: why is the cytoplasmic protein concentration as high as it is, and no higher, and what mechanisms set and maintain this concentration?…”

“…In other ways, the dynamics of proteins in Xenopus extracts are typical of animal cells. Common mammalian cell lines have protein concentrations of about 75 mg/mL (Liu et al, 2022), as do Xenopus extracts (Murray, 1991), and the median protein half-lives in Xenopus extracts (Peshkin et al, 2015) and mouse NIH3T3 cells (Schwanhausser et al, 2011) are almost identical. The simplicity, manipulability, and verisimilitude of the extract system makes it an attractive choice for the present studies of how protein synthesis and degradation are affected by the cytoplasmic concentration.…”

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

“…In contrast, mammalian cells maintain a somewhat lower concentration of about 200 (90–260) mg/mL, as shown by a wide range of techniques and mammalian cell types . Normalized stimulated Raman imaging also reports on protein content. , Using this method, the densities of the mammalian cytoplasm, nucleus, and nucleolus are 75, 85, and 115 mg/mL, respectively, and vary upon perturbations such as osmotic stress, ouabain treatment (inhibition of Na + /K + ATPase), cytoskeleton disruption, cell senescence, and quiescence. Interestingly, the concentrations of protein in cell tissues vary: pancreatic islet maintains about 200 mg/mL, kidney glomerulus 100–200 mg/mL, skeletal muscle cells 200–300 mg/mL, and Zymogen granules in the pancreatic islet 300 mg/mL.…”