Extended Data Fig. 11. Timescales of osmotic potential buffering.
Gradual changes in temperature, external osmolarity, and the intracellular concentration of soluble cytosolic macromolecules are countered by the active transport of small solutes, which maintain Ψπ without appreciable volume change2. Over shorter timescales, severe external osmotic stress stimulates net movement of water over the plasma membrane and accompanying volume adjustments, with cell volume eventually restored by the concerted activity of various solute transport systems (so called Regulated Volume Increase or Decrease, RVI/D)21 in the longer term. We propose that rapid changes in biomolecular condensation of many different IDR proteins is the mechanism by which cells accommodate acute physiological fluctuations in temperature, hydrostatic and osmotic pressure, that normally help to maintain cell volume homeostasis. The following addresses the likely mechanisms that operate over longer timescales, where an acute challenge to intracellular water potential is sustained, supported by some relevant observations. (a) Timescale of relaxation of condensation upon sustained challenge to intracellular Ψπ by a mild external hyperosmotic shock. Human U2OS cells transiently expressing FusLC-GFP were subjected to a sustained +75 mOsm l−1 hyperosmotic treatment and the degree of FusLC-GFP was assessed qualitatively by live SDCM over long timescales (see methods). The behaviour of four different cells is shown. Notice the heterogeneity of the long-term adaptation of cells: condensates in some cells completely dissolved within one hour (cell #4), while it can take >5 h for other cells (cell #1). This is expected as the level of protein condensation is a continuous variable, not a binary process, that results from changes in the equilibrium between fully hydrated and co-localized, partially hydrated monomers, and thus variations are expected in the cell population over long timescales according to differences in cell cycle or metabolic activity for instance. Also note that some condensates remain stable even when all the other condensates in the cell have disappeared (blue arrows). Nevertheless, the timescale of relaxation of phase separation is consistent with established mechanisms of osmoregulation and the behaviour of other IDR proteins43,50,78,79. Images correspond to maximum intensity z-projections (20 planes; Δ0.75 µm). Scale bar: 10 µm. (b) In vitro assays of kinase activity for purified WNK1 and General Control Nonderepressible 2 (GCN2) kinases upon varying osmotic potential via addition of indicated concentration of PEG-20kDa. In contrast with the GCN2 control, WNK1 activity increases with osmotic potential, as the free:structured water ratio decreases (see refs. 2,49,101 for more details). (c) Proposed model of short- and long-term buffering of water potential in cells using a rapid but sustained temperature decrease as an example. At short time scales (sub-second), protein condensation quickly buffers the free:structured water ratio in the cytosol whilst maintaining cell volume. At this point, the cell has reduced capacity to buffer Ψπ and volume against further hypothermal or external hyperosmotic challenges, where cessation of the challenge would allow a rapid (passive) return to the starting condition. If the challenge is maintained (>minutes), it will elicit a change in the activity of Ψπ-sensitive proteins, such as WNK1 kinase whose auto-phosphorylation is stimulated by a fall in free:structured water ratio, leading to increased phosphorylation and activity of substrate effectors such as OXSR1/SPAK kinases2,21. The resultant change in phosphoproteome composition acts to restore Ψπ-buffering capacity around a new set point by: 1) phosphorylation of Ψπ-buffering proteins observed by mass spectrometry (Fig. 2, Extended Data Fig. 5); 2) compensatory electroneutral transport of small solutes via changes in transporter activity e.g. SLC12A family members are regulated by WNK-OXRS/SPAK signalling2,21; 3) longer term changes in gene expression and proteome composition. Together these processes function to restore the condensation state of the Ψπ-buffering proteins, and therefore the ability of the cell to buffer around the new osmotic setpoint that has been established. Challenges that exceed the cell’s innate Ψπ-buffering capacity are expected to additionally result in a cell volume change that is resolved by the same established mechanisms2,29. This framework of thinking is fully consistent with the recent results from Boyd-Shiwarski and colleagues50.