An Introduction to Liquid-Liquid Phase Separation (LLPS)

What is Liquid-Liquid Phase Separation?

When molecules are fully dissolved in water, the most obvious phase transition they could undergo is liquid-to-solid, separating and forming solid particles. Some molecules have the ability to form droplets of a new liquid phase instead, still essentially water-based but still distinct from the surroundings. This phenomenon of liquid-liquid phase separation has been known for a long time, but in the recent decade, it has turned out to be incredibly important and surprisingly common in biology.In this text, we will take a look at this phenomenon and what it means for cellular function in health and disease, and our understanding of the origin of life.

Phase Separation in the Cell

The inside of the cell is a crowded and complicated environment, like a bustling three dimensional factory floor where tens of thousands of molecules carry out and coordinate their different functions.

One of the most important ways that cells have evolved for dealing with this complexity is the creation of separate compartments for specific functions. These cellular sub-units, called organelles, are surrounded by membranes that selectively regulate the flow of molecules. This strict division helps cells keep different reactions separate, and to maintain several very distinct chemical environments in a small space.

However, there are also ways for cells to sort molecules without locking them behind an impermeable membrane. In recent years, researchers have begun to explore how cells can create small regions where the concentration of some molecules, mainly proteins and RNA, is much higher than in the surroundings. These membrane-less organelles come together through liquid-liquid phase separation.

When cells are exposed to stress, they tend to form small granules that handle the breakdown of mRNA and therefore regulate protein expression. Other, similar structures, that handle RNA expression and DNA storage, have also been found. Since they are not protected by a lipid membrane, these are together called membrane-less organelles. A decade and a half ago, researchers were startled to discover that these structures were liquid in nature.

Liquid-Liquid Phase Separation and Disease

The central role that membrane-less organelles play in cell function and regulation also means that dysregulated phase separation can be disastrous. There is a growing body of evidence that aberrant phase separation can be a key step in the breakdown of regulation of cell division in many types of cancer.The highly increased local concentrations that define condensates create the additional risk of pushing proteins towards pathological aggregation. Several protein aggregates linked to disease, including alpha-synuclein and FUS, are now believed first to undergo liquid-liquid phase separation before they continue on the pathological pathway of solid aggregates. In many misfolding diseases, one of the biggest unresolved mysteries is the onset of aggregation, and aberrant phase separation could be one of the missing puzzle pieces.Learning more about the mechanisms of liquid-liquid phase separation could prove crucial in providing new tools to understand some of our most devastating and difficult-to-treat diseases.

A Clue to the Origin of Life?

One very exciting aspect of the discovery of phase separation of biomolecules is the fresh light it is shedding on the question about how life got started on our planet.

The formation of cell membranes has long been a perplexing step in the beginning of life. While compartmentalisation has been identified as a crucial step on the way from non-life to life, it has been difficult to imagine how the first self replicating chemical reactions managed to become enclosed in an impermeable lipid membrane. Sort of a chicken and egg problem.

Liquid-liquid phase separation provides a plausible intermediate step. One could imagine an early stage where phase separation created discrete droplets containing a locally high concentration of self replicating macromolecules, with reactants diffusing freely in and out.

Factors Influencing Liquid-Liquid Phase Separation

The dynamic nature of liquid-liquid phase separation emerges from a subtle balance of weak molecular interactions. If a molecule primarily interacts with the surrounding solvent, it will remain in a homogenous solution. If it has a very strong tendency to interact with other macromolecules, it will form solid aggregates and might fall out of the solution completely. However, in between these two extremes, macromolecules will be drawn together by weak interactions that break and form again, strong enough to cluster them together but still weak enough to remain liquid.The forces that drive liquid-liquid phase separation can be fine-tuned by solution conditions. Factors such as temperature, pH, molecular crowding, and the concentrations of salts and macromolecules can all play a crucial role. There are also several examples of molecules that are soluble on their own but will form condensate when mixed together. All this gives cells a wide selection of dials to turn in order to rapidly trigger or reverse the formation of membrane-less organelles, and it also gives researchers many avenues to explore when mapping out the determinants of phase behaviour.

Outlook

Now is a fascinating time for this emerging field of research. Researchers of diverse disciplines, from cell biology, medicine, and biochemistry to physical chemistry, polymer science, and biophysics, are joining forces to share their insights and explore this complex and dynamic phenomenon.

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