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A team of NYU chemists and physicists are using cutting-edge tools—holographic microscopy and super-resolution imaging—to unlock how cells build and grow tiny, dynamic droplets known as biomolecular condensates.

For the first time, scientists measured the protein content and growth dynamics of individual biomolecular condensates without disturbing them, gaining insights that may shape future drug development and disease modeling.

Biomolecular condensates manage vital cellular functions, from regulating genes to responding to stress. Until now, studying them has involved distorting them.

"It's been the elephant in the room for scientists," said Saumya Saurabh, assistant professor of chemical biology at NYU and the senior author of the new study, in the Journal of the American Chemical Society. "Our research provides a precise and noninvasive way to study biomolecular condensates."

"Being able to see 'under the hood' for the first time has revealed some big surprises about this important class of systems," said study author David Grier, professor of physics and director of the Center for Soft Matter Research at NYU.

Peering into the unknown

Biomolecular condensates are microscopic structures that concentrate specific molecules, like proteins and nucleic acids, without being enclosed by a membrane. This process, known as phase separation, is crucial for organizing cellular biochemistry. While the NYU study focuses on these dynamic droplets in vitro—in a controlled laboratory setting—the fundamental principles they uncover are directly applicable to understanding their behavior within living cells.

"Often compared to oil-and-water droplets, the intricate reality of biomolecular condensates, as revealed by our findings, goes far beyond simple liquid-liquid phase separation," noted Saurabh.

To study biomolecular condensates under the microscope, researchers have traditionally been limited to using fluorescent tags or two-dimensional surfaces, both of which can significantly disturb the droplets' behavior. This is a critical challenge, as these condensates are remarkably sensitive to their environment.

"I was surprised by their complex and incredibly sensitive response to different ionic species. Even a small change in ionic valency drastically altered both condensate concentration and dynamics," said Julian von Hofe, a Ph.D. candidate in Saurabh's group, who is the first author on the study.

To overcome these issues, the researchers sought a way to examine condensates in real time to gather information without damaging them. Their solution: a system that slowly flows thousands of droplets through a holographic microscope.

Holographic precision meets single-molecule resolution

Grier's lab has pioneered the use of holographic microscopy, which uses lasers and lenses to create three-dimensional images, or holograms, of particles that are captured on video for analysis. This technique allows scientists to flow particles in a solution so that they can be clearly seen and individually characterized—without the need for fluorescent labels or attachment to a surface.

Applying this novel, label-free method to condensates formed by PopZ, a bacterial protein crucial for cell growth, the researchers first aimed to precisely measure the concentration of proteins within condensates. Inspired by Benjamin Franklin's eighteenth-century experiment, which used an oil slick to infer a single molecule's length, the team measured the volume of a single protein to determine the protein concentration inside condensates.

Using this idea, they found that relevant biomolecules could concentrate proteins more than tenfold inside condensates. However, the way that the observed condensates grew was unexpected and defied classical models of growth, leading them to pursue single-molecule imaging.

To unravel the complex internal architecture and dynamics, the team utilized super-resolution imaging—a Nobel Prize-winning technology and a main forte for Saurabh's research. These data revealed that condensates were not simple uniform droplets but exhibited intricate nanoscale organization, a realm 1,000 to 100,000 times smaller than the width of a human hair. The findings were strongly supported by molecular dynamics simulations, which provided atomic-level insights into these enigmatic assemblies.

"Our collaboration has introduced fast, precise, and effective methods for measuring the composition and dynamics of macromolecular condensates," said Grier.

From droplets to diseases and drug delivery

Understanding how biomolecular condensates are organized and grow may hold clues for treating a range of illnesses, from cancer and infectious diseases to neurological disorders.

"In a disease like ALS, the proteins that form plaques in disease are fluid condensates in good health. Understanding how a spherical condensate forms into a deadly plaque is an opportunity to better understand ALS," said Saurabh.

In addition, scientists recently discovered that many drug molecules end up inside biomolecular condensates in the cells. This sequestering of drugs within condensates may help explain why drugs that are made to target a specific protein still cause side effects.

With this new approach to analyzing condensates, scientists can now measure small differences in condensate composition and architecture as new molecules partition inside them.

"For example, we can now explore the chemical space of drug modifications to precisely control their partitioning, achieving the specificity needed to prevent them from entering condensates," said Saurabh. "This opens new avenues for how we think about designing drugs and their potential side effects."

Other study authors include Jatin Abacousnac, Mechi Chen, Moeka Sasazawa, and Ida Javér Kristiansen of NYU, as well as Soren Westrey of Carnegie Mellon University.