Using a hollow needle no wider than a speck of dust, Holly Cheng sucked up viscous blobs from within the nucleus of a frog egg cell. As the fluorescent blobs squeezed through the needle, she captured their deformations on video as she watched through a microscope.
The experiment was the culmination of more than a year of meticulous trial and error to hone the process’s many steps: extracting the nucleus from the egg cell, keeping it intact and immobile under a microscope, and preventing the needle from getting “clogged with all sorts of junk” from other parts of the egg cell.
These are just a few of the challenges Cheng worked through during her senior thesis, which is now yielding significant insights into how internal structures of the nucleolus relate to its functions, and how these functions are disrupted in disease. She is the lead author on a paper published on May 28, 2025, in the Proceedings of the National Academy of Sciences.
Cheng’s research was in the laboratory of Clifford Brangwynne, Princeton’s June K. Wu ’92 Professor of Chemical and Biological Engineering. She graduated from Princeton in 2024 with an A.B. in molecular biology and is now a Ph.D. student at the Massachusetts Institute of Technology.
Brangwynne has called the process that Cheng honed a microscopic version of bubble tea, referring to the popular creamy drink laced with tapioca pearls sucked through a wide straw. The technique, called micropipette aspiration, has long been used to study the properties of whole cells, and in recent years has been applied to cell components synthesized in test tubes. But Cheng and her colleagues were the first to successfully use it to measure parts from a living cell while they were still biologically active.
“The excitement when I had something that even partially worked” kept her motivated through some of the more frustrating times in the laboratory, said Cheng. “There’s this tiny amount of fluid under the microscope that I’m applying pressure to, and it’s interacting with the pipette. It felt kind of sci-fi, because you’re in this dark room and there’s lasers shooting at your sample and you have these joysticks controlling stuff that’s super tiny.”
Specifically, Cheng was controlling the movements of layers of the nucleolus. Inside the nucleus of plant and animal cells, the nucleolus produces ribosomes, the molecular machines that make proteins. Proteins are key to all life’s functions, from growth and metabolism to stress responses and repair.
Irregularities in nucleoli and their role in producing proteins have been linked to cancer and neurodegenerative diseases. Indeed, much remains unknown about how parts of the nucleolus work in concert to assemble ribosomes from 80 different proteins and four RNA molecules.
Cheng’s work is a major step forward. Previously, researchers tried to understand cells’ ribosome-making machines using artificial nucleoli, which are far simpler than natively produced nucleoli but don’t retain the same material properties. Cheng’s study measured the properties of nucleoli in their native environment as they actively produced ribosome components.
Direct measurements of nucleolus layers begin to link structure and function
The nucleolus is an example of a biomolecular condensate, a compartment within a cell that, rather than being bound by a biological membrane, separates from its surroundings like a drop of oil in water.
The aspiration technique, combined with microscopy-aided measurements, allowed Cheng and her co-authors to zoom in on this phenomenon. Their experiments showed how the layers of the nucleolus have different levels of viscosity and surface tension. They also revealed the crucial role of RNA molecules in maintaining these distinct properties.
“This paper shows that you can measure those properties in this living context,” said Brangwynne, who directs Princeton’s Omenn-Darling Bioengineering Institute. “I think that’s really exciting. It’s the beginning of applying of this technique in other contexts where we can embrace the full complexity of a living cell.”
Brangwynne is a pioneer in the study of biomolecular condensates, and the nucleolus has long been a key focus of his research. His team recently published a study that tracked the movements and processing of RNA molecules within the nucleolus during ribosome assembly.
“The really interesting thing about the nucleolus is that it looks different in different contexts,” said Cheng.
Nucleoli with irregular shapes can be correlated with negative cancer prognosis, she said. “And the nucleolus has this cool structure,” said Cheng. “It has three concentrically arranged sub-compartments that are each responsible for different parts of ribosome production.”
At the core of the nucleolus, the dense fibrillar component produces densely packed ribosomal RNA molecules. Within this core, smaller structures called fibrillar centers contain the genes encoding this RNA. Surrounding the core is the granular component, where the RNA is packaged together with proteins to form nascent ribosomes.
“We were studying nucleoli inside frog oocytes because these nucleoli are biochemically and structurally very similar to the nucleoli that we [humans] have, but they’re much, much larger,” said Cheng. The diameter of a single frog cell nucleolus is comparable to that of an entire average human cell — big enough to manipulate the structure’s different layers with a needle.
Cheng used micropipette aspiration to suck up successive layers of fluorescently labeled nucleoli from frog egg cells (oocytes), recording the process on video under the microscope.
She worked with James Roggeveen, then a graduate student in Howard Stone’s lab in the Department of Mechanical and Aerospace Engineering, to convert the video data into measurements of the compartments’ physical properties. Roggeveen is now a postdoctoral fellow at Harvard University.
These measurements showed that the outer layer of the nucleolus, the granular component, is liquid-like, and the inner core is more solid-like, much like a tapioca bubble floating in a glass of tea. In one set of experiments, Cheng treated nucleoli with an enzyme to degrade RNA molecules. After this treatment, the core was more liquid-like — showing that its material properties are dependent on the RNA itself.
Frustration, persistence, and the excitement of discovery
Cheng began working on the project during her sophomore year at Princeton. She had just joined Brangwynne’s lab and learned about the recent work of Zheng Shi, an assistant professor at Rutgers University. Shi’s lab was using micropipette aspiration to study droplets made from purified proteins. Brangwynne and Cheng wondered if they could use the technique to look at nucleoli in their native context, and because of their size, nucleoli from frog egg cells seemed like ideal candidates.
Cheng spent the following summer in Shi’s lab at Rutgers, bringing frog eggs from Princeton to test as she learned the minutiae of micropipette aspiration. She worked closely with graduate student Huan Wang, who completed a Ph.D. this spring and is also a co-author on the paper.
“Holly just ran with the idea and made it all work — learning the technique and bringing it to Princeton and combining it with the frog system,” said Brangwynne. It was a “really incredible” example of the transformative potential of undergraduate thesis research, he said.
During that summer in Shi’s lab, Cheng said, she only got the technique to work on frog cell nucleoli a couple of times.
In the fall, she waited for new equipment to arrive at Brangwynne’s lab before beginning the experiments again in December. By February, she succeeded in aspirating the nucleolus layers carefully enough to produce a video, “but it wasn’t very usable,” she said. “The resolution was way too low. The whole setup was pretty shaky and difficult to analyze.”
It took another year to perfect the technique to the point where she consistently got usable data, she said. She experimented with different ways of steadying the needle and different coatings to keep the nucleus in place and prevent the needle from getting clogged.
Timing was also a challenge, since the nuclei only remain biologically active for about two hours after being put in a petri dish. Even before that time, because the nucleus is separated from the cell, “the quality of the preparation is going to degrade,” said Cheng.
“I would prepare [the nucleus] at my bench and then run it over to the microscope room, stick it under the microscope and try to put everything together pretty quickly,” she said.
In the end, most of the data analyzed for the publication was produced during just a few weeks — and those weeks were exhilarating for Cheng.
“The first few times I got really high-resolution videos, I was super excited,” she said. “I sprinted up the stairs and grabbed my friend”— another undergraduate doing research in the Brangwynne lab. “Sometimes she would be in the middle of something and then I would just FaceTime and show her the video from the microscopy room.”
Working with researchers from different departments to refine her experiments and measurements was a highlight of her Princeton experience, said Cheng. Her senior thesis research was honored with a Sigma Xi Book Award from the Department of Molecular Biology and a Calvin Dodd MacCracken Senior Thesis/Project Award from the School of Engineering and Applied Science.
“I learned so much from getting to work with all these incredible scientists who have different expertise. It’s very cool to see how you can combine biology with materials science and fluid dynamics to answer these questions,” she said. The experience “made me really excited to continue doing research and want to pursue a Ph.D.”
The paper, “Micropipette aspiration reveals differential RNA-dependent viscoelasticity of nucleolar subcompartments,” was published May 28 in the Proceedings of the National Academy of Sciences. Co-authors included Cheng, Roggeveen, Wang, Stone, Shi and Brangwynne. Support for this project was provided in part by the Princeton Center for Complex Materials, the St. Jude Research Collaborative on the Biology and Biophysics of RNP granules, and the Howard Hughes Medical Institute. Cheng received support from the Elkins Family Senior Thesis Fund.