Membranes are crucial to our cells. Every cell in your body is enclosed by one. And each of those cells contains specialized compartments, or organelles, which are also enclosed by membranes.

Membranes help cells carry out tasks like breaking down food for energy, building and dismantling proteins, keeping track of environmental conditions, sending signals and deciding when to divide.

Biologists have long struggled to understand precisely how membranes accomplish these different types of jobs. The primary components of membranes — large, fat-like molecules called lipids and compact molecules like cholesterol — make great barriers. In all but a few cases, it’s unclear how those molecules help proteins within membranes do their jobs.

In a published Jan. 25 in the Proceedings of the National Academy of Sciences, a team at the 91̽�� looked at phase separation in budding yeast — the same single-celled fungus of baking and brewing fame — and reports that living yeast cells can actively regulate a process called phase separation in one of their membranes. During phase separation, the membrane remains intact but partitions into multiple, distinct zones or domains that segregate lipids and proteins. The new findings show for the first time that, in response to environmental conditions, yeast cells precisely regulate the temperature at which their membrane undergoes phase separation. The team behind this discovery suggests that phase separation is likely a “switch” mechanism that these cells use to govern the types of work that membranes do and the signals they send.

“Previous work showed that these domains can be seen in the membranes of living yeast cells,” said lead author Chantelle LeveilIe, a 91̽��doctoral student in chemistry. “We asked: If it’s important for a cell to have these domains, then if we change the cell’s environment — by growing them at different temperatures — would the cell ‘care’ and devote energy to maintaining phase separation in its membranes? The clear answer is yes, it does!”

Past research has shown that when sugar is plentiful, the yeast cell’s vacuole — an important organelle for storage and signaling — grows large and its membrane appears uniform under a microscope. But when food supplies dwindle, the vacuole undergoes phase separation, with many round zones appearing in the organelle’s membrane.

In this new study, Leveille and her co-authors — 91̽��chemistry professor , 91̽��biochemistry professor and Caitlin Cornell, previously a 91̽��doctoral student in chemistry — sought to understand whether yeast can actively regulate phase separation. Leveille grew yeast at their typical laboratory temperature of 86 F with plenty of food. After the food dwindled, the yeast cell vacuole membranes underwent phase separation, as expected. When Leveille briefly raised the temperature in the yeast’s environment by about 25 degrees Fahrenheit, the domains disappeared. Then Leveille grew yeast at a cooler temperature — 77 F instead of the normal 86 F — and discovered that the domains disappeared about 25 degrees above this new temperature. When she grew yeast in still colder conditions, at 68 F, phase separation yet again disappeared about 25 degrees higher than their growth temperature.

These experiments showed that the yeast cells always maintained phase separation in the vacuole membrane until the temperature rose about 25 degrees above their growth temperature.

“We think this is a clear sign that yeast cells are engineering the vacuole membrane in different environmental conditions to maintain this consistent state of phase separation,” said Leveille.

Phase separation in the vacuole membrane likely serves an important purpose in yeast, she added.

“This result suggests that membrane phase separation for yeast is likely a two-way door,” said Leveille. “For example, if the cells ever found food again, they would want to go back to their original state. Yeast do not want to get too far away from the transition.”

Future research could identify other membrane components that affect the vacuole membrane’s ability to phase separate, as well as the consequences of its phase separation. Biologists have known that, when the domains appear in the yeast vacuole’s membrane, the cell stops dividing. These two events may be linked because the yeast vacuole’s membrane contains two complexes of proteins that are important for cell division. When the complexes are far apart, cell division stops.

“Phase separation in the vacuole occurs right when the yeast cell needs to stop dividing because its food supply has run out,” said Merz. “One idea is that phase separation is the mechanism that the yeast cell ‘uses’ to separate these two protein complexes and stop cell division.”

In cells from yeast to humans, protein complexes embedded in membranes affect cell behavior. If additional research shows that phase separation in the yeast vacuole regulates cell division, it would likely be the first rigorous example of cell regulation through this once-overlooked property of membranes.

“Phase separation could be a common, reversible mechanism to modulate many, many types of cellular properties,” said Keller.

Cornell is now a postdoctoral researcher at the University of California, Berkeley. The research was funded by the National Institutes of Health and the National Science Foundation.

For more information, contact Keller at slkeller@uw.edu and Merz at merza@uw.edu.

Cells — the building blocks of our bodies — are encapsulated by membranes. The same goes for the specialized compartments within our cells.

These membranes are extremely thin, oily films, containing proteins and fatty molecules called lipids. For decades, scientists have argued about how cell membranes organize and maintain distinct regions enriched in particular protein and lipid types. These regions are thought to influence cellular activities, such as the signaling that controls both normal cellular growth and the growth of cancerous cells.

In a paper in the , scientists at the 91̽�� show for the first time that the complex distribution of molecules within a membrane of a living yeast cell arises through demixing. Also known as phase separation, demixing is a simple physical process that is similar to the action that causes droplets of oil to separate from vinegar in a salad dressing.

“Cells have a toolbox with a variety of resources to help them complete a variety of tasks,” said senior author , a 91̽��professor of chemistry. “By teaming up with , a 91̽��professor of biochemistry and a yeast expert, we’ve shown that phase separation is one of those tools to shape membranes and their functions within a living system.”

The 91̽��researchers were inspired by pictures of a genetically engineered strain of yeast in which membrane proteins fluorescently glowed. The proteins lit up intracellular, membrane-bound compartments called vacuoles. The vacuoles looked like miniature green balls patterned with dark polka dots. Those polka dots, the researchers realized, looked nearly identical to membrane regions that arise from phase separation in two types of non-living systems: simple, artificial membranes created in a lab and membranes shed from cells under severe stress.

“The membranes of living systems contain many different types of fats, proteins and other molecules,” said co-lead author , a lecturer at 91̽��Tacoma who conducted this research when he was a 91̽��doctoral student in chemistry. “Each of these types of molecules harbors different physical and chemical properties with the potential to affect the properties of the membrane as a whole. We and other groups have hypothesized that this variety of molecules would allow membranes to phase separate by composition into discrete regions.”

First, the team discovered that the polka dots that appear on vacuole membranes can merge quickly. This behavior is consistent with fluid phases, just as droplets in a recently-shaken oil and vinegar salad dressing quickly coalesce when they collide. Next, the team found that phase separation in the membranes of yeast vacuoles depends on temperature. When the researchers warmed the yeast above 90 degrees Fahrenheit, the two liquid phases merged into one — the polka dots vanished. As the yeast cells were cooled back to room temperature, the phase-separated regions reappeared.

“Scientists had never previously shown that phase-separated liquids can co-exist in the membranes of living cells,” said co-lead author , a 91̽��doctoral student in chemistry. “To show that phase separation occurs, we had to reliably track the distribution of proteins within membranes, show that they formed regions like in artificial systems and that these regions would merge in response to changing environmental conditions.”

Now that the researchers have shown that living membranes can undergo phase separation, future work is needed to show how cells regulate phase separation. This could be through the action of genes, environmental conditions or a combination of factors.

“Our finding that phase separation can drive membrane organization in yeast suggests that similar processes may operate in other cells, including human cells,” said Merz. “Again, we see the power of model systems such as yeast, fruit flies and worms in our exploration of fundamental physiology. 91̽��has been at the forefront of yeast genetics and cell biology for over 60 years.”

“There is incredible potential here to unlock how different types of cells form and maintain unique structures — and how different structures are formed even within the same cell,” said Keller.

Caitlin Cornell, a 91̽��doctoral student in chemistry, is a co-author. The research was funded by the National Science Foundation and the National Institutes of Health.

###

For more information, contact Keller at skeller@chem.washington.edu or 206-543-9613 and Merz at merza@uw.edu or 206-616-8308.

Grant numbers: DGE-1256082, MCB-1402059, T32GM008268, GM077349.