Stability of Intracellular Protein Concentration under Extreme Osmotic Challenge

Cell volume (CV) regulation is typically studied in short-term experiments to avoid complications resulting from cell growth and division. By combining quantitative phase imaging (by transport-of-intensity equation) with CV measurements (by the exclusion of an external absorbing dye), we were able to monitor the intracellular protein concentration (PC) in HeLa and 3T3 cells for up to 48 h. Long-term PC remained stable in solutions with osmolarities ranging from one-third to almost twice the normal. When cells were subjected to extreme hypoosmolarity (one-quarter of normal), their PC did not decrease as one might expect, but increased; a similar dehydration response was observed at high concentrations of ionophore gramicidin. Highly dilute media, or even moderately dilute in the presence of cytochalasin, caused segregation of water into large protein-free vacuoles, while the surrounding cytoplasm remained at normal density. These results suggest that: (1) dehydration is a standard cellular response to severe stress; (2) the cytoplasm resists prolonged dilution. In an attempt to investigate the mechanism behind the homeostasis of PC, we tested the inhibitors of the protein kinase complex mTOR and the volume-regulated anion channels (VRAC). The initial results did not fully elucidate whether these elements are directly involved in PC maintenance.