June 13, 2023
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Liquid-within-liquid compartments: a groundbreaking discovery in biology

by Hannah Blume
figures by Salvador Balkus

In some ways, a living cell is like a shoreline, where some creatures build their homes on rocky, solid structures while others live in shifting and dynamic sands. This ecosystem needs both rigid and fluid structures to support life, and similarly, individual cells in our bodies use both structured and fluid compartments to carry out vital functions. The existence of liquid-like cellular compartments was proven only 15 years ago, but gave rise to a new field of liquid biology, impacting life science research from cancer to aging to drug development. Liquid-like cellular compartments are essential for life and also play a role in disease, meaning a better understanding of how they arise and function may lead to groundbreaking ways of treating human illnesses.

The Classic View of Cellular Organization

The inside of a cell is a fluid environment separated from the outside world by a boundary called a membrane. Cells also use membranes to build small, functional compartments within themselves, similar to how a human body has organs that perform particular tasks. Cellular compartments like this are called “membrane-bound organelles” and include the nucleus and mitochondria (Figure 1). For the last hundred years, biologists largely thought that membrane-bound organelles were almost solely responsible for the spatial organization within the cell. In our beach analogy, this means that rocky structures were the only microenvironments known to support life. But like a sand crab’s burrow hints at life within the sand, there had been clues that another process spatially organizes cells too. 

Scientists in the 1800s saw what they described as “liquid-like” cell compartments that lacked a membrane border. One such compartment was a spherical organelle present in nearly all nucleus-containing cells including our own, which they named nucleolus for “little nucleus” (Figure 1). One scientist even suggested that liquid-like structures such as the nucleolus predated membrane-bound structures in the evolution of life. However, scientists were puzzled by how a compartment could be built and maintained in cells without a membrane border. No one had rigorous proof of a chemical process that would allow for the creation and maintenance of membrane-less compartments in cells.

Figure 1. Cells contain both membrane-bound organelles and membrane-less, liquid-like compartments. On the left, we see a cell (green) delineated from the environment by a border, the cell membrane. The cell contains mitochondria (orange) and a nucleus surrounded by the endoplasmic reticulum (dark blue), all of which are membrane-bound compartments. The nucleolus (light blue), found inside the nucleus, is a liquid-like, membrane-less compartment. On the right, we see a zoom-in on the nucleolus, where its three layers, all of which are liquid-like, are portrayed.

Proof of Liquid-Like Compartments in Cells

A watershed moment came in 2009 from a study of the molecules that instruct worm embryo cells to become sperm and eggs. Clifford Brangwynne observed that these molecules assemble in distinct structures with no membrane border, like the previously observed membrane-less nucleoli. Importantly, Brangwynne saw that these assemblies have very specific behaviors: they fuse together, split apart, flow, and drip within worm cells. In other words, these structures behave like a liquid (Figure 2). These observations suggested that membrane-less structures may indeed be compartments, though more dynamic than their membrane-bound counterparts. 

Figure 2. Worm embryo cells contain subcellular compartments called P granules (light blue). These structures exhibit behaviors characteristic of liquid droplets: they can flow, drip down a surface like the nucleus, fuse with each other, and split apart. Scientists refer to such liquid-like compartments in cells as biomolecular condensates. P granules were the first biomolecular condensate rigorously characterized. This cartoon is adapted from a data figure in Brangwynne et al., 2009.

The existence of liquid-like cellular compartments within the liquid environment of cells has an intuitive explanation from the macro world: combinations of certain liquids, such as oil and vinegar, will never fully mix. Instead, they separate into distinct liquid phases in a process known as “liquid-liquid phase separation”, or LLPS (Figure 3). LLPS occurs because the two liquids have different chemical properties that make it unfavorable for their molecules to touch. To minimize physical contact, one liquid will form droplets within the other. The formation of liquid droplets is called “condensation,” like when water condenses to form dew drops on a leaf. Scientists often call the condensation of biological liquids in cells “biomolecular condensation.” 

Brangwynne’s identification of stable and functional liquid-like compartments in the worm embryo established that cells can indeed spatially organize their contents through this chemical process in addition to using membrane-bound organelles. His 2009 paper also established methods for documenting and investigating biomolecular condensates and their chemical properties. These methods are now classic techniques that researchers around the world use to understand the functional importance of biomolecular condensation. 

Figure 3. A chemical process like mixing oil and vinegar also occurs in living cells. Oil and vinegar undergo liquid-liquid phase separation, a chemical process where two liquid substances cannot mix because of their different chemical properties. Instead of mixing, one liquid will form droplets within the other. We now know that liquid-liquid phase separation occurs in living cells too.

Fluid Compartments in Health and Disease

Since LLPS was established as a bona fide mode of cellular organization, scientists have shown that biomolecular condensates are involved in functions as wide-ranging as temperature sensing in plant cells and organizing neuronal synapses in mammals. But how do we know that the liquid nature of these compartments is important for cellular functions rather than just a quirk of biology? To answer this question, several labs have looked to the nucleolus, which in healthy cells acts like an assembly line to build a piece of cellular machinery called the ribosome. Brangwynne’s lab wanted to check whether the apparent fluidity of the nucleolus was important for its function. To do so, the scientists made nucleoli act more like jello than a liquid and found that the assembly line accumulated too many of some parts and too few of others. This suggests that the liquidity of the nucleolus is key to its function–similar to how a sand crab depends on the physical properties of sand to survive and cannot burrow into rock instead.

There is increasing evidence that biomolecular condensation is also involved in human illness. A 2023 paper from Denes Hnisz’s lab investigated the effect of disease-causing mutations on the liquid-like properties of the nucleolus. The scientists studied cells containing mutations seen in human patients to investigate how these mutations lead to illness at a molecular level (Figure 4). The different mutations they investigated were all within a single gene and produced a similar change to the electrical state, or overall charge, of the protein encoded by that gene. Electrical state is one of the chemical properties that can affect LLPS, prompting the scientists to test whether the mutated proteins phase separate differently than the regular proteins. The researchers found that the mutated proteins are “stickier,” clumping together and acting less like a liquid. They also found that the mutant proteins gathered in the cells’ nucleoli and made these organelles less liquid-like, too (Figure 4). Since the nucleolus must behave like a liquid to function normally, it is possible that the health conditions of patients with these mutations arise in part from more “solid” nucleoli. This study provides an example of disease-causing mutations altering the liquidity of biomolecular condensates; additionally, cancer and aging research is also revealing links between biomolecular condensate properties and human illness. 

Figure 4. There is evidence that biomolecular condensates may be involved in human diseases. The Hnisz lab studied patient-derived mutations in a protein that normally phase separates and acts like a liquid in cells (left). The mutations lead to altered phase separation of the protein, resulting in less liquid-like nucleoli (right). The altered chemical properties of nucleoli may contribute to the anatomical and health issues experienced by these patients.

Findings like those from the Hnisz lab raise the possibility that some diseases may one day be treated by targeting biomolecular condensates and their chemical properties. Fifteen years ago, when the first proof of liquid compartments was published, we did not understand enough about condensates to consider LLPS a viable avenue for therapeutics. Now, the combination of fundamental and disease-relevant condensate research is creating a new way of understanding—and possibly treating—human disease.

Hannah Blume is a second-year PhD student studying circadian rhythms in the Program in Neuroscience at Harvard.
Salvador Balkus is a first-year PhD student in Biostatistics at the Harvard T.H. Chan School of Public Health. You can connect with him on LinkedIn.

Cover image by Konyvesotto from pixabay.

For more information:

  • For a more detailed overview of biomolecular condensation, read this review article by the Brangwynne lab.
  • Listen to this podcast about the possible links between biomolecular condensation and neurodegeneration.
  • Read this perspective article to learn more about the relationship between biomolecular condensation, disease, and therapeutics.

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