Most of the biochemical reactions that sustain life happen in cellular compartments – organelles such as the Golgi and mitochondria are prime examples. Membranes that surround these organelles contain all the reactions’ necessary ingredients inside, trapping them in a delineated space for reactions to occur. Over the course of the past two decades, there’s been a growing awareness that some compartments aren’t wrapped in membranes after all. Known as biomolecular condensates, these dynamic networks of interacting proteins segregate themselves within cells through a process called phase separation, akin to how oil and water separate in a liquid mixture.

Michael Rosen, Ph.D.

Biomolecular condensates seem to be pivotal for a variety of crucial cellular tasks, including gene expression, cell signaling, and stress response. Dysfunction in these membraneless compartments has been linked to a variety of medical conditions, including neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia as well as several types of cancer.

Despite their importance in cells, many aspects of biomolecular condensate formation, regulation, and function remain mysterious. Recent research from the Rosen Lab is shedding light on these protein networks by imaging their interiors in unprecedented detail. The findings, published in Science, could eventually lead to new ways to manipulate their contents and behavior, potentially resulting in novel treatments for diseases linked with these structures.

A microscopic view in 3D

“Our hope all along was that if we could understand how molecules in a biomolecular condensate are arranged in three-dimensional space, we would understand more about how biomolecular condensates function,” said Michael Rosen, Ph.D., Chair of Biophysics. He and his colleagues were particularly interested in studying chromatin condensates, which form from DNA and associated proteins packed inside the cell nucleus and play pivotal roles in gene expression.

To reach this goal, Huabin Zhou, Ph.D., a former postdoctoral fellow in the Rosen Lab who is now an Assistant Professor in the School of Life Sciences at Westlake University in China, turned to cryo-electron tomography (cryo-ET). This variation of cryo-electron microscopy is capable of imaging biomolecules at the atomic level at cryogenic temperatures (typically below minus 170 C). However, first he had to develop a completely new protocol to overcome the challenges inherent in imaging biomolecular condensates.

Huabin Zhou, Ph.D.

Typically, he explained, scientists perform cryo-electron microscopy by spreading a dilute solution of the molecules to be imaged on a specialized grid – a setup that allows the molecules to separate sufficiently so that they aren’t touching or overlapping before the sample is subsequently frozen. However, biomolecular condensates form by transient and dynamic binding between the different proteins that make them up, causing them to touch and overlap by nature. So these standard procedures weren’t applicable.

To peer inside this crowded environment, Dr. Zhou had to freeze concentrated samples of chromatin condensates at high pressure and then use an ion beam to mill away minuscule sections. Ultimately, this produced a slab of approximately 0.1 microns thick – about a hundred times thinner than the diameter of a red blood cell – that could readily be imaged with cryo-ET.

In phase separation, size matters

Chromatin – a condensed arrangement of DNA – is made of nucleosomes (DNA wrapped around proteins called histones, like thread on a spool) connected by sections of bare DNA of varying lengths that serve as linkers. Combining Dr. Zhou’s imaging approach with computer modeling, he and his colleagues found that the size of the linkers directly impacted the shape of individual molecules and consequently how readily the chromatin phase-separated to form condensates. In turn, Dr. Zhou said, these properties might affect how easily some genes are expressed.

The research also confirmed that synthetic chromatin condensates developed in the Rosen Lab behave much like natural ones. Consequently, these synthetic, simplified models could lend significant insight on the substantially more complicated chromatin condensates found in cells.

Dr. Zhou prepares a cryo-electron microcopy sample as part of his research that elucidated the interior structures of biomolecular condensates.

In addition, he added, their investigation provides a blueprint for studying other types of biomolecular condensates through cryo-ET, which could someday shed light on how these structures operate in other cellular contexts. In his lab at Westlake University, Dr. Zhou is now studying how pathological mutations alter molecular interactions within biomolecular condensates and how these changes affect the efficiency of the molecular machinery operating inside them. He and his colleagues expect this work will clarify pathological processes underlying these mutations and provide clues for developing next-generation therapeutics.

“Structural biology has had a huge impact in understanding individual molecules and molecular machines,” Dr. Zhou said. “My hope is that the structural biology of biomolecular condensates will have a similar impact in understanding the role of these structures in health and disease.”