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

University of Pittsburgh and Carnegie Mellon University scientists have solved a decades-old mystery about how cells control their size.

Crowded Rooms: How Carnegie Mellon University and University of Pittsburgh Researchers Solved the Mystery of the Cell.

A surreal video of stressed cells under a microscope has inspired a group of kidney physiologists and biologists from the University of Pittsburgh and Carnegie Mellon University to investigate a mystery: How do cells control their size?

Their research recently published in the journal cellshowing how researchers connected the dots in a puzzle that was initially introduced three decades ago with a little bit of luck.

“We were doing live fluorescence imaging experiments unrelated to this study, and when we added the salt solution to the cells, the inner cytoplasm quickly turned into a fluorescent lava lamp,” said Daniel Schwarsky, PhD, postdoctoral researcher. A research fellow at Carnegie Mellon University, he describes how he and his wife, co-lead author Carrie Boyd-Schwarsky, MD, turned part of the experiment into an unexpected discovery.

In this video, WNK kinases (a type of enzyme) fluoresce and circulate throughout the cell. When exposed to a salt solution, it clumps into larger droplets, looking like the bright green goo in a lava lamp. This process, called “phase separation,” is how the cell knows it needs to return both water and ions, returning to their original state within seconds. Credit: Boyd-Shiwarski, et al., The Cell (2022)

“I looked at her,” he said, “and she asked me what was going on, as I was supposed to know.” And I said, “I have no idea, but I think it might be something important!” “

When cells are suddenly exposed to an external stressor, such as high levels of salt or sugar, they can decrease in volume. In the early 1990s, scientists believed that cells somehow restore their volume by tracking their protein concentration, or how “crowded” the cell is. However, they weren’t aware of how crowded the dungeon could feel.

Daniel Schwarsky, Arohan Subramanya, and Carrie Boyd Schwarsky

From left to right: D. Daniel Schwarsky, Dr. Arohan Subramanya, and Dr. Carrie Boyd Schwarsky. Credit: Jake Carlson/UPMC

Then, in the early 2000s, With-No-Lysine Kinases, or “WNKs,” were identified as a new type of enzyme. For years, scientists assumed that WNK kinases reversed cell shrinkage, but how they did this was unexplained.

The new study solves both mysteries by revealing how WNK kinases activate a “switch” that returns cell volume to homeostasis through a process known as phase separation.

said senior author Arohan R. Subramanya, MD, assistant professor in the Renal-Electrolyte Department at Pitt College of Medicine and a physician at the VA Pittsburgh Healthcare System. “But there was this paradigm shift in our thinking about how the cytosol works. It’s really like an emulsion with a bunch of little protein clumps and droplets, and then when stress like overcrowding happens, they clump together into big droplets that you can often see with a microscope.”

Those liquid-like droplets were the “lava lamp” that Schwarsky and Boyd Schwarsky were seeing on that fateful day when they experimented with adding a salt solution to cells. They fluorescently labeled the WNKs, which spread throughout the cytosol, causing the entire cell to glow. When salt was added, the WNKs clumped together, forming large neon-green globules that ooze around the cell like the gunk in a lava lamp.

The team described what they were seeing as phasic separation, which is when WNK condenses into droplets with molecules that activate salt transporters in the cell. This step allows the cell to import both ions and water, returning the cell volume to its original state within seconds.

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

“There are a lot of people who don’t think phase separation is physiologically relevant,” explained Boyd Schwarsky, MD, assistant professor in the electrolyte department at Pitt College of Medicine. “They think it’s something that happens in a test tube when you overexpress proteins or that it happens as a disease process but doesn’t actually happen in normal healthy cells.”

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

Restoring cell volume has implications for human health, Subramanya explained, too: “One of the reasons we’re so excited is that the next step for us is to put this back into the kidneys.”

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

Although the new discovery will not have immediate clinical applications, the team is excited to take what they learned and explore the links 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 potassium balance disorders.

Reference: “WNK Kinases Sensing Molecular Crowding and Rescue Cell Size Across Phase Separation” By Carrie R Boyd-Schwarsky, Daniel J Schwarsky, Sean E. Griffiths, Rebecca T Beecham, Logan Norrell, Daryl E Morrison, John Wang, Jacob Mann, William Tennant, Eric N. Anderson, Jonathan Franks Michael Calderon, Kelly A. Connolly, Muhammed Omar Cheema, Claire J. Weaver, Lupika J. Nkashama, Claire C. Wickerly, Kathryn E. Keery, Uday Pandey, Christopher J. Donnelly, Dandan Sun, Aileen R. Rodan and Arohan R. Subramanya, Oct. 31, 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.

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