By Jerimiah Oetting
Cliff Brangwynne was seeing cells in the sidewalk again.
It was another long day in the lab at Harvard Medical School, where Brangwynne would often work late nights, staring at cells. Sometimes he spent so much time staring at cells through the microscope that the cells would follow him home, their shapes imprinted on his vision. Walking late at night, he’d see them dancing over the buildings and the empty streets and sidewalks.
Though Brangwynne was in his college years, he wasn’t a student — in fact, some would call him a drop-out. He’d been enrolled at Carnegie Mellon University, a ﬁrst-generation college student, when a mixture of burnout and wanderlust prompted him to take a year off midway through his degree. At ﬁrst he thought he would take a yearlong trip to Latin America. But he was interested in materials science — he liked how it described the world in terms of math and physics. He also loved biology: he loved that innumerable cells could self-assemble into organisms that eventually walk around and talk about philosophy.
Brangwynne suspected that his two interests, biology and materials science, were more connected than his coursework suggested.
“I knew the two ﬁelds were related, because cells are doing all these crazy things that reﬂect weird material properties and states — ﬂowing and oozing and moving around,” said Brangwynne, now a professor of chemical and biological engineering at Princeton. “But the biologists I would talk to knew nothing about the materials, and the materials people I talked to knew nothing about biology.”
A Scientiﬁc American article would ﬁnally connect the dots. Written by Harvard scientist Donald Ingber, “The Architecture of Life” described the structure of a cell in the same way that an engineer might explain the architecture of a building, down to the biological materials of its construction. Ingber brought together materials science and biology in a way that resonated with Brangwynne’s interests. He was so excited about Ingber’s ideas that he wrote him a letter. When Brangwynne took the year off from college, instead of heading south on his yearlong trip, he headed to Harvard to take a job in Ingber’s lab. As Brangwynne recalls, the experience was nothing short of magical.
Maybe it was all the late nights staring through a microscope, pondering the materials of cells. Or perhaps it was the work-induced hallucinations that followed —the soft, squishy building blocks of life, stuck in his vision, superimposed on the cold and rigid bricks of Boston. Whatever caused it, Brangwynne saw cells and their structures a little differently than other scientists, and it would lead him to a major discovery that had been hiding in plain sight.
All shook up
Oil and vinegar don’t mix. Even a vigorously shaken bottle of vinaigrette dressing will eventually separate into two distinct layers. The two layers are both liquids, so this separation is known as a phase separation — two liquids, segregating from each other because of their characteristic chemistries.
Phase separation, so well-known in salad dressings, was unheard of in cellular biology. But Brangwynne, with his background in materials science, knew that everything, including cells and salads, adheres to the same laws of physics. If phase separation could happen in a bottle at dinner, why not within a cell?
That’s the question Brangwynne asked himself when, once again, cells appeared to be playing tricks on his vision. He had returned to Carnegie Mellon to ﬁnish his undergraduate degree in 2001, and then went back to Harvard where he earned his Ph.D. working with David Weitz, a pioneer in the ﬁeld of soft-matter physics — the physics of “squishy materials.” Then he headed to Max Planck Institute of Molecular Cell Biology and Genetics to work with Tony Hyman, a leader in the study of cellular structures.
After squashing some cells from a roundworm and looking at them under a microscope, Brangwynne observed a structure, which scientists had long assumed to be a solid, instead blobbing apart and coalescing in lava lamp fashion. The supposedly solid structures were behaving much like droplets of oil in vinaigrette dressing. Despite the liquid environment of the cell, the droplets remained distinct from their surroundings. They came together to form bigger blobs and broke apart into smaller ones.
With Hyman and colleagues from the lab, Brangwynne published this observation in the journal Science in a 2009 paper, which described the liquid-like behavior of the structures. He correctly suspected that the droplets he saw weren’t just a curiosity speciﬁc to roundworms — they were an entirely overlooked form of cellular organization.
Today, these structures are known as “biomolecular condensates,” because, although made from proteins and other molecules, they form in a manner similar to the condensation of water on a windowpane on a rainy day, or a dewdrop on a blade of grass. When concentrations of certain proteins disperse in the cell’s liquid, they begin to stick together, forming larger and larger droplets. But unlike normal condensation, they don’t glob together randomly. The proteins bind together in speciﬁc ways that create a functional structure.
The most well-studied cellular structures are bound by membranes, which separate their inner machinery against the milieu inside the cell. But these droplets don’t need a membrane; they exclude their surroundings the way that oil droplets exclude water.
By foregoing the complications of a membrane, the cellular structures can form and dissolve depending on changing conditions, revealing the cell as a much more dynamic and malleable environment than previously thought — more like a lava lamp with its undulating interior than a table lamp with ﬁxed parts.
Since 2009, the droplets have been discovered as key parts of dozens of processes, including cellular division and gene expression. They’ve been implicated in degenerative diseases, like Alzheimer’s and ALS. Some scientists even speculate that these blobs of molecules, simpler than any cell or structure that requires a membrane, may have been the precursors to the earliest forms of life on Earth.
Much like the blobs themselves, the research spawned by their discovery deﬁes boundaries. A growing number of scientists, at Princeton and beyond, are coalescing around Brangwynne and his discovery, ﬁnding condensates within their own study systems by collaborating over methods that cross disciplines.
“I don’t think this has been oversold in any way,” said Ned Wingreen, a professor of molecular biology at Princeton who also trained in physics. “This is a real revolution. It is truly, literally rewriting the textbooks.”
Glomming onto the next big thing
Crack open a biology textbook, and you invariably will ﬁnd an illustrated version of a cell: a central nucleus, surrounded by colorful squiggles and bean-shaped capsules. In this picture-perfect universe, globular proteins ﬂoat dutifully through the cell’s liquid interior to ﬁnd their perfectly shaped counterparts. They connect, perform their function, then separate, drifting off into the cartoon sunset.
“We’re absolutely kidding ourselves with those diagrams,” said Wingreen, the Howard A. Prior Professor in the Life Sciences, and professor of molecular biology and the Lewis-Sigler Institute for Integrative Genomics.
Wingreen studies the physics underlying biological systems. In contrast to those neat diagrams in textbooks, everything inside the cell is constantly bouncing around randomly. In this complicated soup of moving and shaking proteins and molecules, it’s no wonder that biologists assumed a membrane was critical to exclude all the cellular riffraff from the work happening inside the cell’s most important structures.
But many cellular processes occur only if the right proteins are in the right place at the right time. How this happens inside a cell’s busy interior remains a mystery.
“Here comes phase separation to the rescue,” Wingreen said. When the cell produces enough of a speciﬁc protein, they begin to glom together. As they do, the droplet that forms contains a high concentration of those proteins, making it much more likely that they will accomplish their task.
Wingreen uses the example of DNA repair — a process that occurs within the cell’s nucleus. Sometimes, DNA suffers a double-stranded break. The repairing proteins could form a droplet around the break, repair it, and then dissolve.
For a theoretical biophysicist like Wingreen, studying this process is “a theorist’s dream.” But he was only mildly curious about Brangwynne’s blobs until he attended a 2015 meeting held by the Princeton Center for Theoretical Science that was focused on condensate research. Many consider it the ﬂedgling ﬁeld’s ﬁrst important workshop.
“It was deﬁnitely a who’s who of people in the ﬁeld,” Wingreen said of the meeting, which he co-organized with Brangwynne and Mikko Haataja, professor of mechanical and aerospace engineering. The list of attendees and speakers included many scientists who have since made big discoveries involving the cellular droplets, including many currently working at Princeton.
“It was like when a band plays a small venue before they got cool,” Brangwynne said.
The number of researchers was still small and “cultish” back then. But after the workshop, the idea started to gain traction across cellular biology. Citations for Brangwynne’s 2009 paper skyrocketed. Brangwynne says he knew his discovery was important, but “it became a really big deal. And it’s hard to claim I knew that was going to happen — of course I was passionate about the science we were doing, but I didn’t realize the whole world would become similarly obsessed with it.”
For Wingreen, the meeting proclaimed in no uncertain terms that these liquid droplets were going to be transformative for cellular biology. “I’ve got to be working in this ﬁeld,” he recalled thinking.
Wingreen wasn’t alone. Countless other scientists would soon ﬁnd blobs of proteins in their study systems, offering new and exciting ways to understand mysterious cellular processes.
“Brangwynne brainwashed me!” said Mike Levine, the director of Princeton’s Lewis-Sigler Institute for Integrative Genomics. Levine has studied gene regulation, the “most interesting biological process on Earth,” for more than 40 years. Now, he thinks condensates play a pivotal role in that process.
Turning genes into proteins requires a series of steps involving enzymes, which ﬁrst transcribe double-stranded DNA into single-stranded RNA, and later translate RNA into proteins. But how do these enzymes know which genes to transcribe and which to ignore? How does the whole process operate with such precision?
The old idea was that one by one, RNA polymerase, the enzyme responsible for transcription, would swoop in, transcribe a gene, and swoop away. But recent innovations in imaging techniques, pioneered by Princeton’s Thomas Gregor, professor of physics and the Lewis-Sigler Institute for Integrative Genomics, showed that there were actually great distances between all of these pieces of cellular machinery. Somehow, genes were being activated from afar, and the enzymes were still ﬁnding the right places to go.
“I believe Brangwynne-style liquid condensates will be the explanation to these observations,” said Levine, Princeton’s Anthony B. Evnin ’62 Professor in Genomics and a professor of molecular biology.
Instead of a single RNA polymerase ﬁnding its DNA partner, Levine thinks a large droplet full of the enzymes likely forms around areas of the genome that are activated for transcription. In other areas, droplets exclude those RNA polymerase enzymes, like oil excludes water, repressing transcription and effectively shutting off those genes.
The implications of this discovery, according to Levine, would be enormous. “The key to how genomes are organized in functional units may all be driven by these dynamic condensates,” he said. “This is going to have a lot of legs, and a lot of impact on the future of genomics.”
Levine is careful to say these ideas are still “wildly speculative” at this point. But recent experiments in his lab have resulted in videos of “clear-cut condensates,” formed during transcription — blobs that can fuse together and break apart in a dynamic, liquid fashion.
To study these processes requires leveraging the expertise of many Princeton faculty members, combining classical genomics, computational biology and biophysics.
“Phase separation is really in the sweet spot of what Princeton is all about, in terms of encouraging interdisciplinary research,” he said. “Princeton is a powerhouse in the ﬁeld.”
The explosion of work in the area also underscores Princeton’s strength in building from fundamental discoveries in basic science toward innovations in bioengineering that could have wide beneﬁts for human health and the environment.
Brangwynne, for example, is collaborating with Assistant Professor José Avalos, who is jointly appointed in the Department of Chemical and Biological Engineering and the Andlinger Center for Energy and the Environment. Their work seeks to engineer synthetic condensates that help turn yeast cells into chemical factories, including potentially the production of advanced biofuels.
Across several departments in the School of Engineering and Applied Science, faculty members are bringing computational approaches to studying condensates, including Athanassios Panagiotopoulos, the Susan Dod Brown Professor of Chemical and Biological Engineering, and Professor Mikko Haataja and Assistant Professor Andrej Košmrlj in the Department of Mechanical and Aerospace Engineering. In the Department of Chemistry, Assistant Professor William Jacobs adds to the deep bench of Princeton theorists interested in this problem.
A future phase
With much attention focused on this new way of looking at the biological world, it is not surprising that Brangwynne, who in addition to his appointment at Princeton is a Howard Hughes Medical Institute investigator, has received some attention. He has won numerous awards, including a John D. and Catherine T. MacArthur Foundation fellowship, and in 2020 was named a Laureate for the Blavatnik National Awards for Young Scientists.
One of Brangwynne’s favorite sayings is that biological systems are the most richly textured forms of matter in the universe.
“Black holes, quasars, supercomputers —nothing can compete with the complexity of even the most basic bacterial cell,” he said.
Cellular droplets have revealed the strange and dynamic ways that cells organize themselves. They defy the notion that important cellular processes depend on the trappings of a membrane. How these delicate processes evolved is an open question.
“The cell is like a universe,” Brangwynne said. “Any place you point to with a very ﬁne needle, you could spend a lifetime studying it. And people do.”
But despite centuries of scientists looking at cells under microscopes, Brangwynne’s blobs had evaded discovery until a decade ago. Seeing them required a more ﬂuid view of science, and demanded a vision unfettered by the boundaries of scientiﬁc disciplines. It required thinkers, like Brangwynne, who see things a little differently.
How many mysteries still remain in the blind spots?
“Phase separation is one foundational physical principle,” Brangwynne said. “I think it’s probably one of many that have yet to be discovered.”
In recent papers in the journal Cell, Princeton research teams reported the development of new tools that harness light to accurately probe intracellular phase separation — the process by which the chaotic liquid matter inside cells transforms into functioning cellular compartments called membraneless organelles.