Just like oil and water are both liquids but they don't coalesce into one, it's thought that cells use phase separation to keep certain molecules and pieces of cell machinery condensed together in a kind of membrane-less compartment. This can ensure that some reactions take place. There are also some proteins that can act like liquids or condensates, and some of these proteins don't seem to mix well. But we still have a lot to learn about the function and behavior of these liquid-like proteins in cells.
"The separation between two liquids that won't mix, like oil and water, is known as liquid-liquid phase separation and it's central to the function of many proteins," said Sagar Setru, a 2021 Princeton University Ph.D. graduate.
These liquid-like proteins don't dissolve; they form condensates with other copies of themselves or a few other proteins. Since scientists discovered these membrane-less organelles only a few years ago, researchers are just beginning to study what they do.
"In molecular biology, the study of proteins that form condensed phases with liquid-like properties is a rapidly growing field," said Bernardo Gouveia, a Princeton University graduate candidate.
Setru and Gouveia are co-first authors of a new study reported in Nature Physics investigating one protein in particular.
"We were curious about the behavior of the liquid-like protein TPX2. What makes this protein special is that it does not form liquid droplets in the cytoplasm as had been observed before, but instead seems to undergo phase separation on biological polymers called microtubules," explained Setru. "TPX2 is necessary for making branched networks of microtubules, which is crucial for cell division. TPX2 is also overexpressed in some cancers, so understanding its behavior may have medical relevance."
Microtubules have a tubular structure that can shrink, grow, and form branched networks from ones that already exist. TPX2 condenses in places where new microtubules will emerge; the TPX2 globules recruit other proteins that are required for microtubule growth.
In this work, the researchers investigated how globules of TPX2 form on a microtubule. The team engineered microtubules and TPX2 proteins that were marked by fluorescent tags so the scientists could see the action. The microtubules were exposed to TPX2, and the investigators captured the process using atomic force microscopy.
"We found that TPX2 first coats the entire microtubule and then breaks up into droplets that are evenly spaced apart, similar to how morning dew coats a spider web and breaks up into droplets," said Gouveia.
Setru, Gouveia, and colleagues determined that a physical phenomenon known as the Rayleigh-Plateau instability is causing this; it explains why water falling from faucets breaks down into droplets. "It is surprising to find such everyday physics in the nanoscale world of molecular biology," noted Gouveia.
The researchers found that TPX2 levels determine how big the TPX2 globules are and how far apart they're spaced on microtubules. This can explain why microtubule branching is disrupted in cancer cells, which have abnormally high levels of TPX2.
"We used simulations to show that these droplets are a more efficient way to make branches than just having a uniform coating or binding of the protein all along the microtubule," said Setru.