Date Published: February 4, 2019
Publisher: Public Library of Science
Author(s): Katarina Trajkovic, Clarissa Valdez, Daniel Ysselstein, Dimitri Krainc, Vladimir Trajkovic.
The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies.
In vitro cell maintenance entails permanent cycling of cell populations between sparse and confluent states which occurs between passages. This cycling leads to continuous readjustment of cells to fluctuating conditions at individual and cell population levels. For instance, growing cell populations gradually exhaust available nutrients from the media, which in turn leads to altered cellular proteostasis and metabolism [1–4]. Progressive loss of available space between cells leads to a decrease in cell surface area adhering to extracellular matrix, which affects cell size  and the number of focal adhesions, as well as the associated focal adhesion kinase (FAK) signaling . A concomitant increase in intercellular contacts and E-cadherin homophilic binding at the cell surface stimulate Hippo signaling pathway leading to contact inhibition of proliferation which prevents unlimited population growth [7–9]. On a single-cell level, local crowding-dependent changes in FAK signaling create cell-to-cell variability in lipid composition [10, 11]. Moreover, seemingly unrelated processes such as endocytosis and viral infection depend on the local crowding and population context of each individual cell .
In this study, we show that markers of multiple cellular compartments adapt to variations in cell density. Autophagic/lysosomal proteins are regulated by mTOR and FAK signaling pathways that sense changes in nutrient availability and cell crowding in growing cell populations. The accompanying changes in the expression levels of plasma membrane and nuclear markers are consistent with allometric scaling of the respective compartments. We also demonstrate how selective adaptation of protein expression levels to cell density may confound experimental outcomes, and suggest a rational experimental design that minimizes cell density as a source of variability.