Crowded rooms: how researchers at Carnegie Mellon University and the University of Pittsburgh solved a cellular mystery.
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 volume?
Their research, recently published in the journal Cellshows how the researchers connected the dots on a riddle that was originally presented hopefully three decades ago.
“We were doing live fluorescence imaging experiments that were unrelated to this study, and when we added saline to the cells, the internal cytoplasmic material quickly turned into a fluorescent lava lamp,” said Daniel Shiwarski, Ph.D., postdoctoral researcher. researcher at Carnegie Mellon University, describing how he and his wife, co-lead author Cary Boyd-Shiwarski, MD, Ph.D., turned a chance experiment into an unexpected discovery.
In this video, WNK kinases (a type of enzyme) are fluorescent and diffuse throughout the cell. When exposed to saline solution, they coalesce into larger droplets, resembling the bright green paste of a lava lamp. This process, called “phase separation”, is the cell’s way of knowing to bring both water and ions back, 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, like I was 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 outside stressor, such as high levels of salt or sugar, their volume can shrink. In the early 1990s, scientists believed that cells regained their volume by somehow tracking their protein concentration, or how “crowded” the cell was. However, they did not know how the cell felt about the clutter.
Then, in the early 2000s, With-No-Lysine Kinases, or “WNKs”, were identified as a new type of enzyme. For years, scientists have hypothesized that WNK kinases reverse cell shrinkage, but how they did it was unexplained.
The new study solves both puzzles by revealing how WNK kinases activate the “switch” that restores cell volume balance through a process known as phase separation.
“The inside of a cell contains cytosol, and generally people think of this cytosol as diffuse, with all sorts of molecules floating around in a perfectly mixed solution,” said lead author Arohan R. Subramanya, MD, professor aggregated with Renal-Electrolyte. Division of Pitt’s School of Medicine and staff physician of 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 clumps of protein and tiny droplets, and then when stress like overcrowding happens, they come together into big droplets that you can often see under a microscope.
These liquid-like droplets were the “lava lamp” that Shiwarski and Boyd-Shiwarski saw on that fateful day when they experimented with adding saline solution to the cells. They had fluorescently labeled the WNKs, which diffused throughout the cytosol, causing the whole cell to glow. When salt was added, the WNKs came together, forming large neon-green globules that oozed around the cell like goo in a lava lamp.
The team characterized what they believed to be phase separation, which is when WNKs condense into droplets with the molecules that activate the cell’s salt transporters. This step allows the cell to import both ions and water, returning the cell volume to its original state in 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 who don’t believe that phase separation is physiologically relevant,” explained Boyd-Shiwarski, assistant professor in the Renal-Electrolyte Division at Pitt’s School of Medicine. “They think it’s something that happens in a test tube when you overexpress proteins, or it happens as a disease process, but it doesn’t really happen in normal healthy cells.”
But over the past six years, the team has conducted several studies using stressors similar to the fluctuations that occur in the human body to show that WNK phase separation is a functional response to crowding.
Recovering 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 this back into the kidney.”
Other WNKs activate salt transport into renal tubule cells when potassium levels are low by forming specialized condensates by 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 does not have immediate clinical applications, the team is excited to take what they have 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 sense molecular crowding and rescue cell volume via 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, October 31, 2022, Cell.
The study was funded by the National Institutes of Health and the US Department of Veterans Affairs.
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