Protein droplets help cells respond to osmotic stress

Liquid-liquid phase separation can occur when drops or layers of one liquid, like oil, form alongside another liquid, like water. It has been widely observed within cells, where proteins can condense out of the cytoplasm despite being evenly mixed at first. Whether these protein globules have a function has been hotly debated.

“At this point, there’s no aspect of biology, I think it’s fair to say, that has not been suggested to be impacted by or controlled by or facilitated by phase separation,” says Clifford Brangwynne, a Princeton University biophysicist who recently received the 2023 Breakthrough Prize in Life Sciences for work on phase separation in biology. But it has been hard to demonstrate conclusively that phase separation drives cell processes.

Now, Arohan Subramanya of the University of Pittsburgh and colleagues have shown that phase separation is key for cells to sense an excess of salt or other solutes in their surroundings and start damage-control measures (Cell 2022, DOI: 10.1016/j.cell.2022.09.042).

When a cell’s environment has a higher solute concentration than the cytoplasm, water rushes out of the cell. That’s bad news for the cell, which shrivels and begins to malfunction as molecules crowd together. Healthy cells, however, will rebound using ion transporters that pump in solutes until balance is restored.

Kinases known as with-no-lysines, or WNKs, coordinate these ion transporters by sensing cell shrinkage and molecular crowding. Researchers in Subramanya’s lab found that WNKs handle these tasks by forming phase-separated droplets moments after a salt or molecular crowding stress is applied and that those droplets orchestrate the ion transporter signal. They observe phase separation in WNKs from many species.

The phase separation, and not just the WNKs’ enzymatic activity, is key. The authors generated several truncated versions of a WNK that still had enzymatic activity but could not phase separate. Cells with these versions “have a very sluggish response and volume recovery,” Subramanya says. However, they do eventually begin to recover, apparently using other mechanisms that are slower to kick in.

Researchers have observed these phase-separated droplets, which they call condensates, in response to many stimuli, but this study is “a direct illustration of how a condensate that forms via phase separation contributes to cellular physiology,” says Rohit Pappu, a biophysicist at Washington University in St. Louis, who was not involved with the work.

Brangwynne says that the paper reflects a general transition in the field from describing the properties of phase-separated condensates to beginning to explore their physiology. “We’re only beginning to understand all the ways in which condensates facilitate biological function,” he says.