Exploring biomolecular condensate networks’ internal structure

Biomolecular condensates are a newly appreciated way for our body to organize biomolecules such as proteins, DNA and RNA. These dynamic compartments, which often take the form of droplets, organize biomolecules into a dense, tightly packed and regulated hub exactly where they are needed to perform a job. They then disperse when they are no longer needed. Our understanding of the breadth and function of biomolecular condensates is still rapidly expanding, as is how much we know about the molecular networks underlying their internal organization. Richard Kriwacki, PhD, Department of Structural Biology, has taken a particular interest in unearthing the complex networks that feature within biomolecular condensates.

Recent discussions on this topic have extended beyond the biological world and entered the physical world, exploring the nature of biomolecular condensates from the perspective of materials. These materials can appear to be more liquid-like, glass-like, gel-like, or perhaps behave like a “viscoelastic” putty, endowing the behavior of a liquid and a solid. How biomolecules behave within condensates and how this defines their material properties is vital to truly understanding their roles in biology and disease.

Neutron scattering illuminates condensates’ internal network

Kriwacki, in a recent Nature Communications publication in collaboration with Washington University at St. Louis, Pusan National University, and Oak Ridge National Laboratory, leveraged the power of small-angle neutron scanning and molecular dynamics simulations to explore the inhomogeneous nature of condensates. The work, according to Kriwacki, has been a long time coming. “Around 2014, I presented at a workshop in Telluride and was talking about our limited understanding of how molecules are organized within condensates,” Kriwacki said. “[Co-author] Christopher Stanley from Oak Ridge National Labs was there and educated me about the power of neutron scattering to access that information.”

Small-angle neutron scattering (SANS) is a technique researchers use when they want to observe the shape and size of biomolecules as they exist in solution. With this technique, no freezing or crystallization is necessary the way it is with other complementary techniques, such as cryo-electron microscopy or X-ray crystallography. Neutrons are also less harmful to the sample than electrons or X-rays and can penetrate better through dense samples. The neutrons are deflected or “scattered” at very small angles as they interact with the sample. This scattering produces a finely detailed fingerprint of the sample enabling researchers to extract information such as sizes and shapes.

For studying the internal network of condensates, the use of neutron scattering was the breakthrough idea that Kriwacki needed. “According to Chris, the experiments were, at the time, the most interesting SANS results that he had recorded for a biomolecular system,” Kriwacki expressed. “That was where this project was born. It led to many of the experiments with mutants described in the paper where we sought to manipulate the dimensions of the network within these model condensates.”

These findings formed the basis of a 2016 eLife publication describing the nucleolus’ internal organization network. The nucleolus is the cell’s largest biomolecular condensate, which is a manufacturing hub in the nucleus responsible for ribosome production. The researchers discovered the critical role of the protein nucleophosmin (NPM1) in forming the intricate network within the condensate.

Inhomogeneous nature of condensates revealed

There was more to discover, and the SANS data offered the perfect opportunity to collaborate with Rohit Pappu, PhD, Gene K. Beare Distinguished Professor of Biomedical Engineering at the McKelvey School of Engineering at Washington University in St. Louis. As a computational biophysicist, Pappu has made significant contribution to developing molecular dynamics tools which leverage SANS data to replicate the internal environment of condensates. These models used particles with defined interaction rules to represent whole proteins. Such “coarse-grained” simulations have been vital to studying the complex nature of biomolecular condensates.

“Through the simulations Rohit and his team performed, the NPM1 molecules appeared to have two diffusive behaviors.” said Kriwacki. “Some of them appear to be diffusing very rapidly like in a gas, and then others much more slowly, forming a network structure. And, of course, they’re constantly exchanging with each other.”

These findings imply that the underlying molecular structure of condensates is not random, and that there are, in fact, network connections between biomolecules that have an order based on the size and shape of the molecules that make up the network.

“Our prior understanding of how biomolecular condensates form had called for evidence of inhomogeneous network-like organization of molecules within the fluid,” said Pappu. “This collaboration provided new evidence leading to the description of condensates as spatially inhomogeneous, viscoelastic, network fluids.”

Kriwacki hopes to leverage the insights gained from studying NPM1 condensates to further explore the complexities of the nucleolus, with its numerous components and layers.

“It's early days yet in really understanding how this type of organization contributes to function,” he said, “And while this is a simple model system, I think it provides key insights into the organization of molecules within condensates and how they can exhibit different diffusive properties. The ability to have different diffusive properties may be tied in with the functions that biomolecules and condensates perform.”