The shape-shifting blobs that shook up cell biology

The shape-shifting blobs that shook up cell biology

For years, if you asked a scientist how they pictured the inner workings of a cell, they might have spoken of a highly organized factory, with different departments each performing specialized tasks in delineated assembly lines.

Ask now, and they might be more inclined to compare the cell to a chaotic open-plan office, with hot-desking zones where different types of cellular matter gather to complete a task and then scatter to other regions.

Everywhere scientists look in cells, throngs of proteins and RNA seem to be sticking together, coalescing into pearl-like droplets distinct from their surrounding environment. These dynamic compartments allow cells to perform essential functions, ranging from gene control and DNA repair to waste disposal and stress responses. They are often fleeting, and are unhindered by an enclosing membrane — unlike many other cellular components, such as mitochondria, which are membrane-bound. When a droplet is no longer needed, it vanishes.

These transient beads are created by a process called phase separation, which involves groups of molecules segregating themselves owing to differences in their density or the way they interact. The idea became popular among biologists a decade ago, with the number of relevant publications increasing by some 50% each year since about 2017. Biologists have invoked phase separation to explain aspects of how embryos develop, how neurons communicate, how immune systems defend against microorganisms, and much more. And when the process goes awry, diseases from cancer and diabetes to autism spectrum disorder and neurodegeneration seem to follow. The drug industry is already exploring how to target condensates as a path towards new treatments, with strategies in the works that are designed to break up troublesome aggregates or to fine-tune phase behaviours in more subtle ways.

But the field is now at a crossroads. After an initial rush to document the phenomenon in every nook and cranny of the cell, scientists are beginning to ask more detailed questions. They want to know what these globules are doing, how they form and, importantly, how to prove that these membrane-less organelles — or ‘biomolecular condensates’ as they have come to be known — are really as widespread and essential to the cell as many reports have claimed. Researchers are also responding to critics who have questioned the accuracy of some descriptions of phase separation in cells, arguing that other forces besides phase separation could have created droplets. But many biologists don’t need convincing.

“We have the observations that condensates form,” says Jonathon Ditlev, a cellular biophysicist at the Hospital for Sick Children in Toronto, Canada. “Now we need to show why they are important.”

Form and function

The design maxim that ‘form follows function’ assumes that objects are built to serve a particular purpose. Although that works for architects, it can create a puzzle for biologists, who have to reverse-engineer an entity to deduce what it is for.

Condensates come in all shapes and sizes, ranging from tiny spheres the size of a virus to more-complex structures comparable to bacteria. The main function scientists propose for all of these phase-separated droplets is as molecular crucibles. By concentrating components in one place in the cell, the droplets can speed up biochemical processes, as well as separate reactants from each other to prevent unwanted interactions. Yet this line of reasoning has been inferential at best, speculative at worst.

“There are lots of fundamental biological processes where there are papers that say phase separation plays a role,” says Tanja Mittag, a structural biologist at St. Jude Children’s Research Hospital in Memphis, Tennessee. But, she points out: “That hasn’t been shown rigorously, and so I think this needs to be worked out.”

To do this, scientists must understand not just the population of molecules that group together in a droplet, but also how they work inside it. Only then can researchers start to gather insights into why such droplets might take shape in the first place.

In Mittag’s view, the closest anyone has come to convincingly demonstrating a condensate’s purpose is an experiment by biochemist Mike Rosen at the University of Texas Southwestern Medical Center in Dallas. Last year, he and William Peeples, a former graduate student of his, showed how the kinetics of a group of enzymes could be speeded up through phase separation1. They used a system in which they could watch the droplets in 3D. Outside the condensates, enzymatic reactions progressed at a slow, steady pace; inside, the rate of activity was about 36 times faster.

Cancer cell nuclei with MYC condensates.

Cancer-linked proteins (green) gather in the nuclei of multiple myeloma cells.Credit: Jonathan Henninger

Increased local concentrations of these enzymes and their partner molecules partially explained the data, as other groups had shown. But the researchers also found that condensates gave the process extra structure: they helped to organize the enzymes spatially, providing a molecular ‘scaffold’ so that they could more easily partner with their reactants. A small amount of reactant then went further towards revving up enzymatic action, allowing greater catalytic efficiency overall. An independent study published in September demonstrated this same scaffolding effect with a broad array of enzymes2.

“You get this combined effect of increasing efficiency and increasing concentration,” says Peeples, who now works for an early-stage biotechnology company affiliated with Flagship Pioneering, a life-sciences innovation firm based in Cambridge, Massachusetts. Or, put another way, Peeples says: “You get a twofer.”

Build out

Another approach to better understanding how something works is to build it from scratch. In 2020, three independent research teams did just this with a specialized type of condensate known as a stress granule35.

These storage bubbles contain protein and RNA, and are formed in response to cellular or environmental hardships, helping to sequester and protect crucial cellular tools until conditions improve. But just as a cluttered wardrobe can create dangerous amounts of dust or become a fire hazard in the home, so, too, can stress granules cause harm in the cell if they are not cleared up in a timely fashion.

Scientists had previously studied how phase-separated droplets work by fabricating simple versions of them, and by tweaking natural condensates in cells using drug inhibitors and genetic tools, to examine what happens if they are disturbed. But the three groups were the first to faithfully stitch together condensate replicas from the bottom up. Using a combination of experimental techniques, theory and detailed atomic simulations, they deciphered many of the biophysical rules governing condensate formation.

For instance, they showed how one particular scaffolding protein seems to be the epicentre of stress-granule assembly. When the cell encounters adversity, this protein, called G3BP1, changes shape, prompting nearby RNA molecules to link up with it and promote clustering. Empowered by this key mechanistic insight, researchers are now beginning to probe how these compartments dynamically form and fragment, and which molecules drive each part of their life cycle.

“That’s the power of in vitro reconstitution,” says Peiguo Yang, a cell biologist at Westlake University in Hangzhou, China, who worked on one of the studies3.

Another of the teams has since explored, in unpublished work, how disease-linked proteins affect condensates. Condensates usually have a squishy consistency. But in the presence of these proteins, the structures become more rigid, leading to the types of protein clumps in cells that underpin many neurodegenerative disorders. “We can actually see aggregation happening inside the granules we’ve built,” says Simon Alberti, a biochemist at the Technical University of Dresden in Germany, who constructed the granules.

In transition

Efforts such as these should go a long way towards resolving one of the biggest controversies in the condensate field — how exactly they form.

Much of the evidence that these blobs are created by phase separation comes from test-tube experiments that might not reflect conditions in living cells, notes Amy Gladfelter, a cell biologist at the University of North Carolina, Chapel Hill — especially because these condensates are orders of magnitude bigger than their natural counterparts. “We’ve been lured to study these large, very luscious droplets that are macroscopic and charismatic,” she said at an online meeting. Held to discuss open questions and challenges in condensate biology, it was convened by the German research foundation (DFG) and the newly launched Center for Biomolecular Condensates at Washington University in St. Louis, Missouri, in late October.

But many crucial functions might be happening at scales that scientists can’t see. Researchers also disagree over the precise mechanisms by which molecules might become concentrated into membrane-less compartments, and these processes are hard to see with even the best technology. So, although biologists have spent the past decade seeing condensates all over the place — in test-tube experiments, in cells and in animal models — some critics fear that many of those observations might turn out to be mirages.

Part of the challenge of working out whether a blob is a product of phase separation is the wide variation in how they look and what they are made of. In their landmark 2009 paper6, the first to identify fluid-like, phase-separated blobs, cell biologist Tony Hyman at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, and biophysicist Cliff Brangwynne, now at Princeton University in New Jersey, described corpuses of RNA and protein coming together and breaking apart like beads of water on a pane of glass.

“They appeared similar to liquid drops wetting a surface,” the authors wrote. (Hyman and Brangwynne won the prestigious 2023 Breakthrough Prize in Life Sciences for this work.)

The researchers ascribed the phenomenon to ‘liquid–liquid phase separation’ (LLPS), a demixing process analogous to the coarsening of oil droplets suspended in vinegar. LLPS seemed to be everywhere in the cell — in small bodies in the nucleus, at sites of gene activity and in structures involved in cell division (see ‘A crowd of condensates’).

A crowd of condensates: an infographic that shows the function of a selection of condensates that form in a neuron

Nik Spencer/Nature

But some of these blobs behaved more like solids than liquids, or they took on a gooey, gel-like consistency. Realizing that more-complex biophysics was at play than just liquids pulling apart, in 2017 Hyman and Rosen coined7 a catch-all name for these compartments: biomolecular condensates. The name left open how these assemblages of proteins and nucleic acids took shape or became undone. “It was deliberately supposed to be mechanism-free,” Rosen explains.

In addition to the oil-and-vinegar demixing process, physical and chemical interactions between specific parts of these networked structures matter, too. For instance, one hotspot of condensate assembly turns out to be the wobbly bits of protein that lack stable 3D structures, and which interact with other molecules and solvents to guide phase separation. Further experiments and theory showed that a huge number of forces work together to create condensates.

Some in the community have sought to inject precision into the field and guide researchers in finding out whether a blob forms through phase separation or in some other way.

Mittag and computational biophysicist Rohit Pappu, director of the Washington University condensates centre, put together a framework that stipulates how to check that a condensate is really present, including the difference in density inside and outside a blob, and physical crosslinking between the molecules inside8. And they suggested ways to test for phase separation — such as experiments designed to show the concentration thresholds above which droplets form, because of either transitions in density or physical interactions, or both.

According to Mittag, this more-formal definition of the process is a “really important step forward in terms of our conceptual understanding of phase separation”. But, she acknowledges, it has also raised the scientific bar in a way that is creating more questions. “And so, in the end,” Mittag says, “I actually think we’re not really past the controversies.”

A critical step

Much of that pushback has come from Robert Tjian, a biochemist at the University of California, Berkeley. In 2019, he and his colleagues published a widely read commentary9 that cast doubt on the field’s scientific rigour — a critique made more resonant by a news article in the journal Science.

Tjian says he appreciates what scientists such as Mittag and Pappu are doing to address his concerns. And he welcomes the move beyond simplistic explanations. “This is obviously still a rather complex and ill-defined field,” Tjian says, and he looks forward to proponents of phase separation performing “actual discerning experiments”. Many in the field acknowledge that his caution has pushed them to be more exacting in their science.

A minority of researchers still hold fast to their scepticism, however. Andrea Musacchio, a mechanistic cell biologist at the Max Planck Institute of Molecular Physiology in Dortmund, Germany, published a scathing appraisal of the field earlier this year10. The framework put forward by Pappu and Mittag “essentially wipes out the entire literature on phase separation so far”, he says. Many condensate researchers say his critique is based on flawed arguments and an incomplete reading of the literature.

Few others take Musacchio’s harsh view. And as biophysicist Josh Riback at Baylor College of Medicine in Houston, Texas, points out, it’s only natural for scientific understanding to mature with time. When it comes to such a new concept, he says, “you want to start simple and then build up complexity”.

Drugging droplets

Despite the debates in academia, drug hunters are embracing the concept. Condensate-focused biotech companies such as Dewpoint Therapeutics in Boston, Massachusetts, have collectively raised more than US$500 million since 2019, and established companies have signed partnership deals with condensate start-ups.

Most companies interested in phase separation are prioritizing drug development for cancer and neurological disorders, two disease classes frequently linked to condensates that have gone awry. Sometimes, these condensates contain toxic proteins, and the simplest therapeutic manoeuvre is to dissolve them with drugs or prevent them from forming in the first place (see ‘Druggable droplets’).

Druggable droplets: a graphic that shows how drugs can be used to remove, alter or target condensates and their contents.

Nik Spencer/Nature

In motor neuron disease (amyotrophic lateral sclerosis), for example, many disease mutations can make condensates more viscous than usual, leading to dense aggregates that are a hallmark of the degenerative neuromuscular condition. In cancer, proteins that encourage or suppress tumours can end up in the wrong compartments or at the wrong levels, leading to tumour growth.

Etern Therapeutics, based in Shanghai, China, has a drug candidate for cancer in early clinical trials. The experimental medicine, named ETS-001, targets a tumour-associated enzyme. As company co-founder and chief executive Jidong Zhu and his colleagues have shown11, mutated forms of this enzyme accumulate in condensates, leading to a signalling cascade that can spur runaway cell growth. ETS-001 binds to the enzyme, blocking condensate formation and stifling the tumour. Last month, Zhu and his collaborators described another drug candidate for prostate cancer that disrupts condensates that are thought to make such cancers resistant to certain standard therapies12.

Other diseases might require more careful handling of condensates. The world’s largest drug maker, Pfizer, headquartered in New York City, is working with Dewpoint to develop condensate-targeted treatments for a form of myotonic dystrophy, a rare genetic disorder that affects muscles and other body systems. In this disease, the condensates, which tend to accumulate in the wrong locations in affected cells, need to be stabilized rather than destroyed.

Dewpoint is focused on other diseases that also require this nuance. Company biochemist Phi Luong and his team have been working on an undisclosed neurodegenerative disease, and have found that the affected cell’s nucleolus, a dense spherical condensate in the cell’s nucleus where protein-making ribosomes come together, takes on an abnormal shape in diseased cells. Breaking up the nucleolus entirely would kill the cell. So the goal is to find drug candidates with subtler, more restorative capabilities — “not ones that are just kind of a sledgehammer,” Luong says.

And because many drugs tend to accumulate in condensates, the blobs themselves could represent a new delivery strategy, allowing drugs to concentrate at desired sites of action.

In effect, condensates provide a middle ground between targeting molecules and whole cells. They are a way of understanding cells “that you don’t really get at by looking at the individual building blocks”, says Tuomas Knowles, a biophysicist at the University of Cambridge, UK, who co-founded and is chief technology officer of Transition Bio, headquartered in Cambridge, Massachusetts.

By one count, hundreds, if not thousands, of disease states might be fuelled by condensate-linked mechanisms13. “You can’t look at any organ system or any related disease without considering the possibility that the pathogenic mutation is causing a dysregulated condensate,” says Rick Young, a biologist at the Whitehead Institute in Cambridge, Massachusetts, and Dewpoint co-founder, who, with his co-authors, published the analysis in July.

“There isn’t a cellular process that’s been studied that is not now known to involve condensates,” Young says. “It involves damn near everything.”

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