Above: When a cell is stressed by a salt solution, the WNK1 protein (fluorescently tagged in these images) goes from being spread diffusely throughout the cytoplasm to concentrating into droplets with liquid-like properties. The process helps cells sense crowding and regain volume.

Cells are the basic building blocks of life, but many of their inner workings remain a mystery.

For a long time, researchers mostly linked cell functions to membrane-bound organelles that handle important tasks—think mitochondria, lysosomes and the other cellular actors that many of us first saw in biology textbooks. But these conveniently cordoned-off pieces don’t account for everything that happens—or can go wrong—in a cell.

In the last decade, researchers have noticed a phenomenon that could fill in some of the gaps. Swirling around in the cytoplasm, biomolecules such as proteins can join together in liquid-like droplets, just as oils separate into droplets in water.

These membraneless “biomolecular condensates,” formed through a process called liquid-liquid phase separation, offer a new layer to scientists’ thinking about how cells are organized. And Pitt researchers are finding evidence of the protein-packed droplets’ role in a range of cell functions and dysfunctions, as in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS).

“It’s a way to compartmentalize proteins and biomolecules so that they can coordinate and do a job in a little liquid-like network,” says Chris Donnelly, a PhD assistant professor of neurobiology and scientific director of the LiveLikeLou Center for ALS Research. “What we found, and what people have been finding, is that the process can go awry.”

In about 97% of ALS cases, the patients’ cells have toxic clumps of a protein called TDP-43. Usually, TDP-43 binds with RNA as part of a number of normal, healthy processes. Issues arise when it instead binds too much with itself. Donnelly’s team has found that this starts with an aberrant liquid-liquid phase separation, with condensates of TDP-43 eventually solidifying.

Scientists don’t yet know whether TDP-43 is involved in causing ALS, but disrupting the buildup may still offer a way to treat symptoms. Donnelly and his collaborators have started a company called Confluence Therapeutics to develop synthetic nucleic acids that bind with the protein to prevent the abnormal phase separation or break up aggregates.

Enthusiasm about biomolecular condensates’ potential has been building. Pitt’s School of Medicine recently awarded its highest honor, the Dickson Prize, to Clifford Brangwynne, a Princeton University bioengineer whose work launched the field.

Thanks in part to his background in materials science, Brangwynne recognized that phase separation—a well-established process in nonliving matter—could also happen in cells. The idea didn’t catch on right away.

Carlos Camacho, a PhD associate professor of computational and systems biology, says his background in physics initially made him skeptical that liquid-liquid phase separation was relevant in biology. But after learning more and conducting experiments of his own, he became intrigued by the ways that condensates may allow disordered proteins to regulate the signals that cells send to coordinate function.

Some scientists still aren’t convinced. But in a study published in 2022 in the journal Cell, kidney researchers from Pitt and Carnegie Mellon University made one of the first clear links between condensates and cell function—and solved a longstanding mystery.

High levels of stressors, like salt or sugar, can cause a cell’s volume to decrease. Scientists long believed the cells regained their volume by somehow sensing crowding within the cell. Further research suggested an enzyme called with-no-lysine kinases, or “WNKs,” reversed cell shrinkage, but how remained unclear.

That changed after some unexpected observations made in 2016 under a microscope by Cary Boyd-Shiwarski, an MD, PhD and assistant professor of medicine in the Division of Renal-Electrolyte, and Daniel Shiwarski, a PhD assistant professor of bioengineering and medicine who was at the time a Carnegie Mellon postdoc. (The Shiwarskis are married.)

When they added a salt solution to a sample of cells, causing them to shrink, fluorescently tagged WNKs condensed into droplets, along with the molecules that activate the cells’ salt transporters. The phase separation allowed the cell to import both ions and water, quickly restoring the cell’s volume.

Arohan R. Subramanya, senior author on the study, is now eager to tie their findings back to the kidney. When potassium levels are low in the blood, WNK-dependent condensates (which the researchers call WNK bodies) form in the kidney tubule.  “All of our evidence to date indicates that WNK bodies are important for controlling salt transport, potassium balance and blood pressure,” says Subramanya, an MD associate professor of medicine.

Camacho, Donnelly and Subramanya are all part of Pitt’s Center for Protein Conformational Diseases. There, collaborations are brewing to find even more ways that condensates are involved in diseases that involve irregular protein formations.

Says Boyd-Shiwarski, “There’s a lot of potential for this to be involved in more reactions and processes within the cell than we can even fathom at this point.”

Read more from the Fall 2023 issue.