Surreal video of stressed cells helps biologists solve decades-old mystery

Crowded rooms: How Carnegie Mellon University and University of Pittsburgh researchers solved a cellular mystery.

A surreal video of stressed cells under a microscope inspired a group of kidney physiologists and biologists at the University of Pittsburgh i Carnegie Mellon University to investigate a mystery: how do cells control their volume?

Their research, which was recently published in the journal cellshows how researchers connected the dots in a puzzle that was initially presented three decades ago with a bit of luck.

“We were doing live fluorescence imaging experiments unrelated to this study, and when we added a salt solution to the cells, the internal cytoplasmic material quickly turned into a fluorescent lava lamp,” he said. said Daniel Shiwarski, Ph.D., a postdoctoral fellow. researcher at Carnegie Mellon University, describing how he and his wife, co-senior author Cary Boyd-Shiwarski, MD, Ph.D., turned some serendipitous experimentation into an unexpected discovery.

In this video, WNK kinases (a type of enzyme) are fluorescent and diffuse throughout the cell. When exposed to a salt solution, they coalesce into larger droplets, similar to the glowing green mass of a lava lamp. This process, called “phase separation,” is how the cell knows it needs to get water and ions back into it, returning to its original state within seconds. Credit: Boyd-Shiwarski, et al., Cell (2022)

“I looked at her and she asked me what was going on, how was she supposed to know,” he said. “And I said, ‘I have no idea, but I think it’s probably something big!’

When cells are suddenly exposed to an external stress, such as high levels of salt or sugar, their volume can decrease. In the early 1990s, scientists believed that cells regained their volume by somehow keeping track of their protein concentration, or how “crowded” the cell was. However, they were not aware of how the cell senses crowding.

Then, in the early 2000s, lysine-less kinases, or “WNKs,” were identified as a new type of enzyme. For years, scientists theorized that WNK kinases reversed cell shrinkage, but how they did it was not clear.

The new study solves both puzzles by revealing how WNK kinases turn on the “switch” that restores cell volume to equilibrium through a process known as phase separation.

“The inside of a cell contains cytosol, and people generally think of this cytosol as diffuse, with all kinds of molecules floating around in a perfectly mixed solution,” said lead author Arohan R. Subramanya, MD, Associate Professor of Renal-Electrolyte. Division of Pitt College of Medicine and VA Pittsburgh Health System Staff Physician. “But there’s been this paradigm shift in our thinking about how the cytosol works. It’s really like an emulsion with lots of little protein clusters and droplets, and then when a stress like crowding occurs, it they aggregate into large droplets that can often be seen under a microscope.”

Those liquid-like droplets were the “lava lamp” that Shiwarski and Boyd-Shiwarski were seeing that fateful day when they experimented with adding a salt solution to the cells. They had fluorescently labeled WNKs, which diffused throughout the cytosol, causing the entire cell to glow. When salt was added, the WNKs clumped together, forming large neon green globules that flowed through the cell like the pinch of a lava lamp.

The team characterized what they were seeing as phase separation, which is when WNKs condense into droplets along with molecules that activate the cell’s salt transporters. This step allows the cell to import both ions and water, returning the cell’s volume to its original state within seconds.

Phase separation is an emerging area of ​​interest, but whether or not this process was an important part of cell function has been controversial.

“There are a lot of people out there who don’t think that phase separation is physiologically relevant,” explained Boyd-Shiwarski, an assistant professor in the Division of Renal Electrolytes at the Pitt School of Medicine. “They think it’s something that happens in a test tube when proteins are overexpressed or occurs as a pathological process, but it doesn’t really happen in normal healthy cells.”

But over the past six years, the team conducted multiple studies using stressors similar to the fluctuations that occur within the human body to show that phase separation of WNKs is a functional response to crowding.

The recovery of cell volume also has implications for human health, Subramanya explained: “One of the reasons we’re so excited is that the next step for us is to get it back into the kidney.”

Other WNKs activate salt transport within renal tubule cells when potassium levels are low by forming specialized condensates through phase separation, called WNK bodies. Modern Western diets tend to be low in potassium, so while attempting to regulate cell volume, WNK bodies may contribute to salt-sensitive hypertension.

While the new discovery won’t have immediate clinical applications, the team is excited to take what they’ve learned and explore the connections between WNKs, phase separation, and human health. Ultimately, their work may lead to a better understanding of how to prevent strokes, high blood pressure, and disorders of potassium balance.

Reference: “WNK Kinases Detect Molecular Crowding and Rescue Cell Volume Through Phase Separation” by Cary R. Boyd-Shiwarski, Daniel J. Shiwarski, Shawn E. Griffiths, Rebecca T. Beacham, Logan Norrell, Daryl E. Morrison, Jun Wang, Jacob Mann, William Tennant, Eric N. Anderson, Jonathan Franks, Michael Calderon, Kelly A. Connolly, Muhammad Umar Cheema, Claire J. Weaver, Lubika J. Nkashama, Claire C. Weckerly, Katherine E. Querry, Udai Bhan Pandey, Christopher J. Donnelly, Dandan Sun, Aylin R. Rodan, and Arohan R. Subramanya, 31 Oct 2022, cell.
DOI: 10.1016/j.cell.2022.09.042

The study was funded by the National Institutes of Health and the US Department of Veterans Affairs.