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For years, if you asked a scientist how they imagined the inner workings of cells, they might say a well-organized factory with different departments performing specialized tasks
on a demarcated assembly line.
Now asking this question, they may be more inclined to compare the cell to a chaotic open office, with hot desk areas where different types of cellular material are clustered together to complete a task and then dispersed to other areas
.
Everywhere scientists look at cells, they can see clusters of proteins and RNA that seem to stick together, combining into pearly droplets
that are very different from their surroundings.
These dynamic compartments enable cells to perform essential functions, from gene control and DNA repair to waste disposal and stress response
.
They are usually transient and not surrounded by membranes — unlike other cellular components, such as mitochondria, which are membrane-bound
.
When a drop is no longer needed, it disappears
.
These ephemeral beads are produced through a process called phase separation, which involves isolating
groups of molecules from each other due to differences in molecular density or interaction patterns.
The idea became popular among biologists 10 years ago, and the number of related publications has grown by about 50%
per year since 2017.
Biologists invoke phase separation to explain how embryos develop, how neurons communicate, how the immune system fends off microbes, and more
.
When this process goes wrong, diseases like cancer, diabetes, autism spectrum disorder, and neurodegeneration seem to follow
.
The pharmaceutical industry is already exploring how to target condensates as a pathway to develop new therapies, and strategies currently being studied aim to break up troublesome aggregates or fine-tune phase behavior
in more subtle ways.
But the field is now at a crossroads
.
After initially hastily recording the phenomenon of every nook and cranny of the cell, the scientists began to ask more detailed questions
.
They want to know what these globules are doing, how they form, and more importantly, how to prove that these membraneless organelles — or "biomolecular condensates" — are really as widespread as many reports claim and are vital
to cells.
The researchers also responded to some critics' skepticism about the accuracy of the description of phase separation in cells, arguing that forces other than phase separation may also produce droplets
.
But many biologists don't need to convince them
.
Jonathon Ditlev, a cellular biophysicist at Children's Hospital Toronto in Canada, said: "We observed the formation
of condensate.
Now we need to prove why they matter
.
”
The design principle of "form follows function" assumes that objects are built for a specific purpose
.
While this is feasible for architects, it presents a conundrum for biologists, who must reverse engineer an entity to deduce its purpose
.
Condensates come in all shapes and sizes, from tiny spheres the size of viruses to more complex structures
comparable to bacteria.
Scientists propose that the main function of all these phase separation droplets is to act as molecular
crucibles.
By concentrating components in one place within the cell, droplets can accelerate biochemical processes and separate reactants from each other to prevent unwanted interactions
.
However, this reasoning is inference at best and speculation
at worst.
Tanja Mittag, a structural biologist at St.
Jude's Children's Research Hospital in Memphis, Tennessee, said, "There are many fundamental biological processes that have papers showing that phase separation plays a role
.
" But, she noted, "This hasn't been rigorously proven, so I think it needs to be looked out
.
"
To do this, scientists need to understand not only the number of molecules clumped together in the droplet, but also how they work
in the droplet.
Only then will researchers begin to gain insight into why
these droplets form in the first place.
In Mittag's view, the most convincing demonstration of the role of condensate was an experiment
by Mike Rosen, a biochemist at the University of Texas Southwestern Medical Center in Dallas.
Last year, he and his former graduate student William Peeples showed how phase separation can accelerate the kinetics of a group of enzymes
.
They used a system
that could observe droplets in 3D.
Outside of the condensate, the enzyme's reaction proceeds at a slow and steady pace; And inside, the speed of activity is about 36 times
.
Cancer-associated proteins (green) accumulate in
the nucleus of multiple myeloma cells.
Source: Jonathan Henninger
As other research groups have shown, the increase in local concentrations of these enzymes and their partner molecules partly explains these data
.
But the researchers also found that the condensate gives the process additional structure: They help enzymes organize spatially, providing a molecular "scaffold" so they can more easily work
with reactants.
A small number of reactants further accelerates the action of the enzyme, making the overall catalytic efficiency higher
.
An independent study published in September showed the same scaffolding effect
on a large number of enzymes.
"You get this combination of increased efficiency and increased focus," said Peeples, who now works at an early-stage biotech company
owned by Flagship Pioneer, a Cambridge, Massachusetts-based life sciences innovator.
Or, to put it another way, Peeples says, "You get a double.
"
Another way to better understand how things work is to build it
from scratch.
In 2020, three separate research teams completed this work
using a specialized type of condensate known as stress particles 3-5.
These storage bubbles contain proteins and RNA that are formed when cells or environments are difficult and help isolate and protect critical cellular tools until conditions improve
.
But just as a messy wardrobe can produce a lot of dangerous dust or become a fire hazard at home, stress particles can also cause damage
to cells if not cleaned up in time.
Scientists have previously studied how phase separation droplets work by making simple phase separation droplets and using drug inhibitors and genetic tools to tune the natural coagulates in the cells to examine what happens if
they are disturbed.
But the three groups were the first to faithfully stitch together condensed replicas from bottom to top
.
By combining experimental techniques, theory, and detailed atomic simulations, they deciphered many of the biophysical rules
that govern condensate formation.
For example, they showed that a particular scaffold protein appears to be the center of
stress particle assembly.
When a cell encounters adversity, the protein, called G3BP1, changes shape, prompting nearby RNA molecules to bind to it, promoting aggregation
.
Supported by this critical mechanistic insight, the researchers now set out to explore how these compartments dynamically form and divide, and which molecules drive each part
of their life cycle.
"That's the power of in vitro reconstruction," said
Peiguo Yang, a cell biologist at Westlake University in Hangzhou, China, who participated in one of the studies.
Another team explored how disease-related proteins affect condensate
in unpublished work.
The condensate usually has a slimy consistency
.
However, in the presence of these proteins, the structure becomes more rigid, leading to the type of protein clumps in the cell, which underlie
many neurodegenerative diseases.
Simon Alberti, a biochemist at the Technical University of Dresden in Germany, said: "We can actually see aggregation
happening inside the particles we build.
" He constructed these particles
.
These efforts should go a long way
toward resolving one of the biggest controversies in the condensate space: how exactly condensate forms.
Amy Gladfelter, a cell biologist at the University of North Carolina at Chapel Hill, points out that much of the evidence that these spots are produced by phase separation comes from test-tube experiments that may not reflect conditions in living cells — especially since the condensates are several counts
larger than natural condensates.
"We've always been drawn to study these huge, very sweet droplets, which are macroscopic and charismatic
," she said at an online conference.
The conference, convened in late October by the German Research Foundation (DFG) and Washington University's newly established Center for Biomolecular Condensation in St.
Louis, Missouri, was convened in late October to discuss open issues and challenges
in cohesion biology.
But many important functions may occur
beyond the reach of scientists.
Researchers also disagree on the precise mechanisms by which molecules are concentrated into membraneless chambers, processes that are difficult to observe
even with the best techniques.
So while biologists have seen condensed matter everywhere in test-tube experiments, cells and animal models over the past decade, some critics worry that these observations could be a mirage
.
Part of the challenge in figuring out whether a spot is a product of phase separation is that they vary greatly in appearance and composition
.
In a landmark 2009 paper, Tony Hyman, a cell biologist at the Max Planck Institute for Molecular Cell Biology and Genetics in Dresden, and Cliff Brangwynne, a biophysicist at Princeton University in New Jersey, described in a landmark 2009 paper that clumps of RNA and protein clump together and then divide, like droplets of water on glass
.
This is the first paper
to identify fluid-like phase separation spots.
"They look like droplets
on wetted surfaces," the authors write.
(Hyman and Brangwynne received the prestigious 2023 Life Sciences Breakthrough Award
for this.
) )
The researchers attribute this phenomenon to "liquid-liquid phase separation" (LLPS), a decomposition process
similar to the coarsening of oil droplets suspended in vinegar.
LLPS seems to be ubiquitous in cells – in small bodies in the nucleus, at sites of gene activity, in structures involved in cell division (see "Swarm of condensates"
).
But some of these spots behave more like solids than liquids, or they exhibit a viscous, gel-like consistency
.
Hyman and Rosen realized that there was more complex biophysics at play than just the separation of liquids, and in 2017 they created an all-encompassing name for these compartments: biomolecular condenses
.
The name does not explain how combinations of these proteins and nucleic acids
form or disappear.
Rosen explains: "It was intentionally designed to be mechanical
.
In addition to the decomposition process of oil and vinegar, the physical and chemical interactions between specific parts of these network structures are also important
.
For example, a hot spot in condensate assembly is the wobbling sites of proteins that lack a stable 3D structure, and they interact with other molecules and solvents to guide phase separation
.
Further experiments and theories show that a large number of forces work together to produce condensed matter
.
Some in the community have tried to inject precision into the field and guide researchers to find out whether spots form
through phase separation or otherwise.
Mittag and Rohit Pappu, a computational biophysicist and director of the Condensate Center at the University of Washington, built a framework for how to check whether a condensate is actually present, including differences in density inside and outside the condensate, and physical cross-links
between molecules inside the condensate.
They also propose ways to test phase separation — such as designing experiments to show droplet formation above the concentration threshold because of a shift in density or physical interaction, or both
.
According to Mittag, this more formal definition of process is "a very important step forward in our conceptual understanding of separation.
"
But, she acknowledges, it also raises the scientific bar to some extent, creating more problems
.
"So, in the end," Mittag said, "I actually don't think we're really out of the controversy
yet.
" ”
Most of the objection came from Robert Tjian
, a biochemist at the University of California, Berkeley.
In 2019, he and his colleagues published a widely read review questioning the scientific rigor of the field — a criticism that resonated
even more with a news article in the journal Science.
Tjian said he appreciates the efforts
of scientists like Mittag and Pappu to address his concerns.
He welcomed
that approach that went beyond simplistic interpretation.
"This is clearly still quite complex and poorly defined territory," said Tjian, who expects "actual recognition experiments"
from separated proponents.
Many in the field acknowledge that his caution has prompted them to be more rigorous
in scientific research.
However, a small number of researchers still hold on to their
skepticism.
Earlier this year, Andrea Musacchio, a mechanistic cell biologist at the Max Planck Institute for Molecular Physiology in Dortmund, Germany, published a scathing review
of the field.
The framework proposed by Pappu and Mittag "essentially erases the entire literature on phase separation to date," he said
.
Many condensed matter researchers say his criticism is based on flawed arguments and incomplete readings
of the literature.
Few people hold Moussacchio's harsh views
.
Josh Riback, a biophysicist at Baylor College of Medicine in Houston, Texas, notes that it's
natural for scientific understanding to mature over time.
When it comes to such a new concept, he says, "You start simple and then add complexity.
"
Despite the debate in academia, drug seekers are embracing the concept
.
Since 2019, condensate-focused biotech companies, such as Dew Point Therapy in Boston, Massachusetts, have collectively raised more than $500 million, and established companies have signed partnership agreements
with condensate startups.
Most companies interested in phase separation are prioritizing the development of drugs for cancer and neurological disorders, two groups often associated
with malfunctioning condensation.
Sometimes, these condensates contain toxic proteins, and the simplest treatment is to dissolve them with drugs or stop their formation
in the first place.
For example, in motor neuron disease (amyotrophic lateral sclerosis), many disease mutations can make the condensate more viscous than usual, leading to dense aggregation, which is a hallmark
of degenerative neuromuscular disease.
In cancer, proteins that promote or inhibit tumors may end up in the wrong compartment or level, causing tumor growth
.
Shanghai, China-based Etern Therapeutics has a cancer drug candidate in early-stage clinical trials
.
The experimental drug, called ETS-001, targets an enzyme
associated with tumors.
The company's co-founder and CEO and his colleagues have demonstrated that mutated forms of this enzyme accumulate in the condensate, leading to a signaling cascade that stimulates uncontrolled cell growth
.
ETS-001 binds to enzymes, blocks the formation of coagulants and inhibits tumors
.
Last month, Zhu and his collaborators described another drug candidate to treat prostate cancer that destroys condensate that is thought to make prostate cancer resistant to
certain standard therapies.
Other diseases may require more careful handling of the coagulation
.
Pfizer, the world's largest drugmaker based in New York, is working with Dew Point to develop a condensation-targeted therapy
for myotonic dystrophy, a rare genetic disorder that affects muscles and other body systems.
In this disease, condensate tends to accumulate in the wrong place of infected cells and needs to be stabilized rather than destroyed
.
Dew point studies focus on other diseases
that also require this nuance.
Phi Luong, a biochemist at the company, and his team have been studying an undisclosed neurodegenerative disease and found that nucleoli (dense spherical coagulants in the nucleus of the nucleus that make proteins clump together) take on an abnormal shape
in the diseased cells.
Completely breaking up the nucleoli kills the cells
.
So our goal is to find drug candidates with more subtlety, more resilience — "not just a sledgehammer," Luong said
.
Since many drugs tend to accumulate in the condensate, the spots themselves may represent a new dosing strategy that allows the drug to be concentrated at the desired site of
action.
In effect, the agglomerate provides a middle ground
between the targeted molecule and the entire cell.
Tuomas Knowles, a biophysicist at the University of Cambridge in the United Kingdom, says they are a way to understand cells that "you can't really understand by looking at individual building blocks," said Tuomas Knowles, a Cambridge, Massachusetts-based company of Transition Biosciences
.
According to statistics, there are hundreds, if not thousands, of disease states that may be triggered by condensate-related mechanisms
.
"You can't look at any organ system or any related disease without considering the possibility of pathogenic mutations causing coagulate dysregulation," said
Rick Young, a biologist at the Whitehead Research Institute in Cambridge, Massachusetts.
He is the co-founder of Dew Point Labs, and together with his co-authors, published this analysis in July
.
Young said: "The cellular processes that have been studied so far are all related to
condensation.
This involves almost everything
.
”
