Severe osmotic compression triggers a slowdown of intracellular signaling, which can be explained by molecular crowding

Abstract

Regulation of the cellular volume is fundamental for cell survival and function. Deviations from equilibrium trigger dedicated signaling and transcriptional responses that mediate water homeostasis and volume recovery. Cells are densely packed with proteins, and molecular crowding may play an important role in cellular processes. Indeed, increasing molecular crowding has been shown to modify the kinetics of biochemical reactions in vitro; however, the effects of molecular crowding in living cells are mostly unexplored. Here, we report that, in yeast, a sudden reduction in cellular volume, induced by severe osmotic stress, slows down the dynamics of several signaling cascades, including the stress-response pathways required for osmotic adaptation. We show that increasing osmotic compression decreases protein mobility and can eventually lead to a dramatic stalling of several unrelated signaling and cellular processes. The rate of these cellular processes decreased exponentially with protein density when approaching stalling osmotic compression. This suggests that, under compression, the cytoplasm behaves as a soft colloid undergoing a glass transition. Our results shed light on the physical mechanisms that force cells to cope with volume fluctuations to maintain an optimal protein density compatible with cellular functions.

Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress

“…In these latter experiments, the reduced steady-state endosome motility quickly returned to near baseline values. These data confirm previous work showing that molecular crowding contributes to deficient trafficking (14,22). However, they also indicate that molecular crowding is not the only factor involved in decreased vesicle motility after hypertonic challenge.…”

“…This observation made in various mammalian cell types is similar to the decreased vesicle trafficking observed in yeast exposed to severe osmotic stress (3 M sorbitol; i.e., 3,000 mOsmol/kg) (14). Osmotic compression per se and accompanying molecular crowding undoubtedly play a large part in the vesicle immobilization that accompanies water efflux, because the intracellular space is filled to near capacity with macromolecules and organelles under basal conditions (62).…”

“…Membrane trafficking is a major process affected by hypertonicity that probably contributes significantly to the change incurred upon challenge. Indeed, we and others have observed that endocytosis, exocytosis, and vesicular trafficking are reduced from the onset of challenge, and that the reduction can persist hours after challenge (14)(15)(16)(17)(18)(19). However, how immediate changes in the cell's biophysical parameters influence these events is unclear.…”

Analysis of osmoadaptation system in budding yeast suggests that regulated degradation of glycerol synthesis enzyme is key to near-perfect adaptation

“…5c). Van Wuytswinkel et al (2000) and recently Miermont et al (2013) have reported that at 1.4 M NaCl, the Hog1 phosphorylation and transcriptional response is delayed. Moreover, it was observed by these authors that there is persistence activation of Hog1PP even after long time (240 min in Van Wuytswinkel et al (2000)) indicating imperfect adaptation at higher stress levels.…”

“…Our model does not predict delayed activation of Hog1PP. This could be due to the fact that the effect of delay due to nuclear transport of Hog1 at higher stress (Miermont et al 2013) was not considered in our model. However, the persistent activity of Hog1PP predicted by the model is consistent with their experimental observations.…”