All living organisms are made of cells, which are the smallest unit of life. Plants and animals have up to trillions of cells that work together to produce ever more intricate organization and function. Within cells are organelles, or little organs, that do specific jobs. Plant and animal cells have mitochondria, for example, which generate energy, and a nucleus that contains most of the genetic information and acts as a control center. These well-known organelles are enclosed within membranes that maintain their shape and separate them from the cytoplasm, the fluid that fills the cells.
But this textbook account of cells, with its neat division of labor into tidy membrane-bound packages, is incomplete. Not all organelles have membranes, and over the past decade biologists have come to realize that membraneless organelles—such as tiny droplets of concentrated protein or other biomolecules—may be more plentiful and carry out more diverse tasks in cell function than was previously realized. Scientists call these droplets biomolecular condensates, an analogy to the droplets of water that condense on a cold glass of water on a humid day.
Their physics is somewhat of a mystery. Why don’t these little workers need walls to keep them contained, and how do they keep their elements separate from the cytoplasm around them? By understanding how condensates form and operate, we hope to finally figure out what they do.
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This research is still emerging, but scientists think the droplets play vital roles related to gene regulation, cell division and the transportation of materials within cells. There are even hints that biomolecular condensates are important in cellular processes related to human diseases, including amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. So far, however, most of the evidence about biomolecular condensates comes from test tube experiments. In the coming years, researchers aim to understand how these droplets act in the more complex environment of a living cell. As we continue to discover new types of condensates and uncover new clues about their purposes, we hope we might even find a universal theory that will describe them all.
Under a microscope, biomolecular condensates look like tiny objects adrift in a sea of cytoplasm packed with organelles and other structures. Though suspended in this liquid, they act like a liquid themselves—they’re spherical in shape and deformable when poked with a micropipette. When two droplets come into contact, they merge into one. The recent discoveries about their possible biological significance have generated interest in how biomolecular condensates form. To a biophysicist like me, this looks like a question of thermodynamics.
Thermodynamics is the branch of physics concerned with the relationship between heat and other forms of energy. Its principles apply to everything from chemical reactions to meteorology. For our purposes, thermodynamics describes liquid-liquid phase separation—the division of a fluid into two compartments (or phases) with different concentrations and compositions. A classic example is oil and water. If I pour oil into a glass of water, at first the two fluids will mix somewhat. After several minutes, however, they will separate and form two phases: one that is enriched with oil and very little water and another that contains water and very little oil. In contrast, a case where entropy wins is the combination of milk and coffee, which become well mixed.
Thermodynamics tells us that this phase separation results from a competition between entropy and energy. Entropy is the amount of disorder in a system; it favors a uniform mixture of oil and water. Energy includes the energy contained in chemical bonds within each molecule as well as the energy of interactions between molecules. In this case, the energy for oil molecules to interact with one another is lower than the energy for molecules of oil and water to interact, which drives the oil and water to split into distinct layers. The reduction of energy in the interactions between molecules outweighs the opposing contribution from entropy to stay evenly mixed.
Something similar may happen inside a cell when molecules such as proteins, DNA or RNA come together in high concentrations. These molecules may condense into tiny droplets because they have a lower energy when they stick together than when they’re dispersed throughout the cytoplasm. But in contrast to the separation of oil and water or the beads that form on the outside of a water glass, the behavior of biomolecular condensates can’t be explained by thermodynamics alone. Phase separation driven purely by thermodynamics should be stable: once oil and water separate inside a glass, they will stay that way forever. But inside the cell, many condensates exist only temporarily. For example, pyrenoids are dynamic structures that dissolve and reform between cell divisions. They’re essential for the photosynthesis process that algae use to convert sunlight into energy.
The pyrenoid is one of the earliest known biomolecular condensates. In 1782, Danish naturalist and scientific illustrator Otto Frederik Müller observed and sketched small dots in green algae, capturing these structures with the limited technology available at the time. Later, in the 1830s, German physiologists Rudolf Wagner and Gabriel Valentin independently reported their observations of another biomolecular condensate. This was during a period when scientists were beginning to realize that cells are the basic units of life. Despite the relatively crude microscopes of the era, Wagner and Valentin were able to discern tiny structures within the nuclei of neuronal cells. Thanks to later work, we now know them as the nucleolus or “the nucleus within the nucleus,” the structure that builds ribosomes, the molecular machines that turn RNA sequences into proteins. But at the time Wagner and Valentin had little idea of the nucleolus’s function. Later, in an 1899 paper published in Science, American biologist Edmund B. Wilson proposed that the cell's cytoplasm is not a uniform liquid but rather a complex mixture of liquids with suspended droplets “of different chemical nature.” Although Wilson lacked direct evidence for this idea, his model was remarkably accurate and remains a part of our modern understanding of cellular biology.
More than a century passed before we gained significant further insight. Advances in microscopy played an important role by allowing biologists to zoom in and watch events inside a cell unfold in real time instead of seeing them only in static images. In a landmark 2009 study, researchers examined membraneless organelles called P granules in embryonic cells of the roundworm Caenorhabditis elegans, a favorite species for lab studies. P granules contain proteins and RNA that aid in determining which cells eventually become sperm and eggs in early embryonic development. Using fluorescent dyes, the scientists showed that proteins move rapidly inside the P granules, suggesting that they are well mixed and constitute a distinct phase that is separate from the cytoplasm. Additionally, the researchers found that the P granules are round and deform just like a liquid would. And when two droplets come close to each other, they fuse together. This was some of the first direct evidence that biomolecular condensates can form within a living cell.
Since this discovery, other teams have found that biomolecular condensates arise in various types of cells. Although the biological function of these condensates is not yet known in many cases, the emerging picture suggests that cells use phase separation as a way to control multiple important biological processes at once. Understanding more of the physics of how condensates form could help us gain a clearer picture of what they do.
I came to physics in a roundabout way. I entered college as a biology major, taking the introductory classes on cell biology, genetics, ecology and evolution. I was fascinated by how something as small as a cell could be so complicated. But I also had to take introductory physics courses, and I was drawn to the quantitative nature of physics. Some calculations would take pages and pages to arrive at a solution. I found the process attractive, but the car collisions and swinging pendulums of introductory physics problems didn’t inspire me. I dreamed of investigating biology quantitatively using the laws of physics.
I went on to do my graduate studies at the University of California, Berkeley. Toward the end of my Ph.D., I began what is now one of my main research projects, studying phase separation inside the cell. One area of my work involved a condensate that forms in early development inside T cells, crucial components of our immune system. The protein that makes up the condensate is called linker for activation of T cells (LAT), and it regulates a key signaling pathway during development and during infection. LAT is attached to the inside of the cell membrane, and it forms a condensate within the membrane, which is essentially a two-dimensional liquid. My collaborators at U.C. Berkeley have been studying how LAT condensates form in test tube experiments with molecules of LAT adhered to an artificial membrane. This process, they’ve found, requires adding “glue” proteins to the solution on the opposite side of the membrane. These glue proteins bind to individual molecules of LAT and connect them to other LAT molecules to mimic the interactions in live cells.
I had the privilege of collaborating with these researchers on a recent study where they measured the time it takes for a condensate to form after adding glue proteins to a uniform solution of LAT. They found that this phase transition time is a function of temperature. As a theorist, I set out to understand what determines that time scale. Together with my adviser at U.C. Berkeley, chemist David Limmer, I developed a numerical model that included relevant biological information, such as the rate at which LAT molecules link up with one another and spread out on the surface of the membrane and the maximum number of connections that each LAT molecule can form with other LAT molecules. In our simulations, LAT builds up tiny clusters of 10 to 20 proteins at the beginning of the condensation process. At intermediate time scales, these clusters begin to fuse together to form a fiberlike network. Eventually this network consolidates into roundish blobs, and two distinct phases arise—one enriched with LAT and the other with very little LAT but lots of other proteins. During this last step of the process, which takes the longest, there are very few available binding sites in the fiberlike structures, which have to slowly move and bend to make the remaining binding sites available. Our simulation results for both the growth of the LAT clusters and the dynamics of molecules within the clusters agree well with the experiments done by my collaborators. Together our work shows how a particular type of condensate forms in cells with a life-sustaining function.
Another area I’m working on now is phase separation within green algae that live worldwide in soil and freshwater and gather energy from the sun. Just as in plants on land, this process—photosynthesis—relies on green pigment molecules inside an organelle called the chloroplast. But in contrast to land plants, these green algae also have a biomolecular condensate called a pyrenoid within each chloroplast that has a high concentration of enzymes needed to convert carbon dioxide from the air into sugars for energy. I want to understand the biophysics of how these condensates form. In collaboration with biologists who are investigating pyrenoids in the lab, I’m studying how the composition and size of these biomolecular condensates can be tuned for specific biological functions. We hope this work may one day allow us to genetically engineer land plants that produce pyrenoids, which may allow them to grow in areas with little sun.
Lately scientists are beginning to get a better handle on how droplets within cells form. We know that condensates can arise when the energy required for proteins to cluster together is less than the energy they would need to disperse evenly through the cytoplasm. It seems to happen through two main mechanisms. One involves having the right conditions to facilitate chemical bonds so that individual protein molecules can bond with one another.
A second mechanism to form condensates thermodynamically involves proteins that contain so-called intrinsically disordered regions (IDRs), or regions where the sequence of amino acids that make up the protein is highly repetitive. These IDRs may contain amino acids with positive or negative charges that attract other amino acids with the opposite charge and repel ones with the same charge. Because of the distribution of charges along the molecule, proteins with IDRs may also have complex interactions with water that contribute to the way they fold and transition to a separate phase.
In contrast to these passive, thermodynamically driven condensates, other condensates form through “active” processes that require burning molecular fuel for energy. Sharon Glotzer, now a professor at the University of Michigan, developed a theory using statistical physics to show that energy-consuming chemical reactions can alter how condensates form, leading to multiple small droplets instead of a single condensate. For instance, imagine a system of two types of particles, A and B, where A sticks to A and B sticks to B but A and B repel each other. According to passive thermodynamics, we’d expect this system to separate into one phase containing many A particles and another phase containing many B particles. If the system also includes a chemical reaction that burns energy to turn A to B and B to A, however, a single condensate can become unstable and break up. As a result, instead of two phases—mostly A and mostly B—many small circular droplets can form, full of either A or B particles. Other scientists have since found that the size and total number of these droplets is related to how much energy is used in the chemical reactions.
Research by Glotzer’s team and others suggests this mechanism helps stabilize centrosomes, liquidlike cellular structures that help coordinate cell division. During the cell cycle, centrosomes double in size, split into two, then move to opposite sides of the cell. The two centrosomes form a mitotic spindle—a bundle of ropelike components that align the chromosomes and pull them apart during cell division. According to theory, the energy consumed by the assembly and disassembly of centrosomes controls the growth and size of these biomolecular condensates and also allows two centrosomes to coexist without merging into one droplet. This suggests that the cell has control knobs to tune the spatial and temporal organization needed to orchestrate cell division.
Another active area of interest for researchers is the question of how condensates slowly change over time. One example is a protein known as FUS, which binds to DNA and RNA inside the cell nucleus, repairs DNA and regulates genes and their products. Mutations in the gene that encodes FUS cause an inherited form of ALS, also known as Lou Gehrig’s disease. In test tube experiments, this mutated version of FUS forms condensates that resemble the clumps of FUS protein found in brain tissue from people who have died of ALS. Recent studies suggest the properties of these in vitro FUS condensates change over time. In one study, Louise Jawerth, now an assistant professor at Leiden University in the Netherlands, and her colleagues bound droplets of FUS to plastic beads that could be manipulated with a laser tweezer, allowing them to investigate how the droplets deform when pulled. Interestingly, the droplets became more dense as they aged, requiring more force to deform.
These studies mark the beginning of our understanding of the complex dynamics of condensates. Although the biophysics of this phenomenon are not well understood, an increase in local concentrations of FUS proteins seems to trigger the phase transition into an aggregate state similar to the clumps of protein implicated in ALS. Computer simulation studies on similar proteins show that local concentrations can increase by the strengthening of bonds between proteins over time or by increasing the number of those bonds, a process called gelation. Similar processes might underlie the development of protein aggregates tied to other neurodegenerative disorders, such as the amyloid fibrils found in the brains of people with Alzheimer’s disease or the synuclein protein deposits implicated in Parkinson’s disease. One interesting hypothesis is that normal physiological conditions support only a liquid state of these proteins, and disease is associated with a transition from a liquid to a solid aggregate state.
The realization that biomolecular condensates are ubiquitous and diverse is an exciting development, but we have still a long way to go to understand their true nature and function. Progress is speeding up, and we hope to see exciting discoveries to come about how biomolecular condensates form, how they age, and how they affect cells and larger organisms.