"Cell" was first proposed 30 years ago, and the answer is still available

A team of kidney physiologists and biologists from the University of Pittsburgh and Carnegie Mellon shot a psychedelic film of stressed cells under a microscope, which set them on a journey to solve a mystery: How do cells control their own size?

Their study, published today in the journal Cell, explains how scientists used a little serendipitous discovery to connect the dots in a puzzle first proposed 30 years ago
.

Dr.
Daniel Shiwarski, a postdoctoral researcher at Carnegie Mellon University, described how he and his wife, Dr.
Cary Boyd-Shiwarski, Ph.
D.
, turned a serendipitous experiment into an unexpected discovery, saying, "We were doing a real-time fluorescence imaging experiment unrelated to this study, and when we added a saline solution to the cells, the cytoplasmic material inside quickly turned into a fluorescent lava lamp
.
" "I looked at her and she asked me what had happened, as if I should have known," he said
.
"But I said, 'I don't know, but I think it might be something important!'"

When cells are exposed to sudden external stressors, such as high levels of salt or sugar, their volume decreases
.
In the early '90s, scientists proposed that cells could somehow monitor protein concentrations, or "crowding" within cells, to restore volume
.
But they don't know how the cells feel overcrowded
.

Then, at the beginning of the 21st century, an enzyme called "lysine kinases" (WNKs) was discovered
.
For years, scientists suspected that WNK kinases reversed cell contraction, but how they did so is also a mystery
.

The new study solves both puzzles, revealing how WNK kinase activates a "switch" that restores cell volume to equilibrium
through a process called phase separation.

"Inside the cell contains a cytosol, which is generally thought to be diffuse, with various molecules floating in a perfectly mixed solution," said
senior author Arohan R.
Subramanya, MD.
"But our thinking about how cytosols work has undergone a paradigm shift
.
It's like an emulsion with a bunch of tiny, tiny clusters of proteins and droplets, and then when pressure happens, like crowding, they clump together to become large droplets that you can often see
with a microscope.
”

On the day Shiwarski and Boyd-Shiwarski experimented with adding a saline solution to cells, the liquid-like droplets they saw were "lava lamps.
"
They labeled WNKs with fluorescence, and these WNKs diffused in the cytoplasm, causing the whole cell to glow.

When the salt is added, the WNKs clump together to form giant neon-green balls that ooze around the cells, like a sticky substance
in a lava lamp.

The team described what they saw as phase separation, where WNKs condense into droplets
along with molecules that activate the cell salt transporter.
This step allows the cell to introduce ions and water, returning the volume of the cell to its original state
in a matter of seconds.

Phase separation is an emerging area of research, but whether this process is an important part of cell function has been debated
.

"There are a lot of people who don't believe phase separation is physiologically related," explains
Boyd-Shiwarski, an assistant professor in the Department of Renal Electrolytes at Pitt School of Medicine.
"They think it's something that happens when you overexpress a protein in a test tube, or something that happens as a pathological process, but it doesn't really happen
in normal, healthy cells.
"

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

Cell volume recovery also has implications for human health, Subramanya explains: "One of the reasons we're so excited is that the next step is that we're going to put it back in the kidneys
.
"

When potassium content is low, other WNKs form specialized condensates, known as WNK bodies, by phase separation, which activate salt transport
within tubular cells.
The modern Western diet is generally low in potassium, so when trying to regulate cell volume, WNK bodies may cause salt-sensitive hypertension
.

While the new findings won't be immediately available to the clinic, the research team is excited to use their findings to explore the link between
WNKs, phase separation and human health.
Ultimately, their work may lead to a better understanding of how to prevent stroke, high blood pressure and potassium imbalance
.

WNK kinases sense molecular crowding and rescue cell volume via phase separation