MS01 - CDEV-05

Protein Condensates in the Cell Nucleus

Monday, July 14 at 10:20am

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Tharana Yosprakob (University of Alberta)

Description:

This mini-symposium highlights the collaboration between experimentalists and mathematicians to understand the complex nuclear processes that control genome organization, protein interactions, and function. In the nucleus, protein condensates are membraneless partitions of molecules that are formed by the interaction of proteins and DNA. These condensates are involved in regulating gene expression and DNA structure. On the experimental side, we investigate how these condensates affect gene activity and DNA organization by exploring their material states and functions. In addition, single molecule tracking is used to track nuclear particles and observe how proteins move within the nucleus and interact to form these condensates. On the theoretical side, we showcase mathematical models to explain condensate formation: one based on liquid-liquid phase separation (LLPS), which describes how proteins and DNA spontaneously cluster together, and another based on reaction-diffusion, which explains how molecules interact and move to create and maintain condensates.

Michael Hendzel

University of Alberta

"Nuclear Microenvironments and Intranuclear Transport"

There is a poorly-defined transition in the size-dependent transport properties of molecules in the nucleoplasm. The most studied molecules, proteins and protein complexes, are small enough to diffuse freely through the nucleoplasm. That is not true of larger molecules but the transition between these two states and the underlying reason is poorly understood. Of particular interest is pre-mRNA and mRNA, which are significantly larger than most protein/protein complexes and must be trafficked to the nuclear pore for export. We have been studying size-dependent transport of small molecules, RNA, and particles of defined diameters to define the transport properties of the nucleoplasm and nuclear compartments. We confirm that mRNA transport is discontinuous and that mRNAs frequently become transiently trapped within the nucleoplasm. These transport properties are very similar to what is observed with 40 nm fluorescent particles microinjected into nuclei suggesting that this reflects a sharp size- dependent transition to obstructed diffusion characterized by transient caging. In comparing two cell lines, one cancer (U2OS) and a normal cell line from mouse (C2C12). These differ in their spatial organization and local densities of chromatin and, remarkably, show an order of magnitude difference in both the confinement volumes and the diffusion coefficients observed between the two cell lines. The cancer cell line showed much more rapid transport properties. Since most transport studies have been performed in cancer cell lines, this raises the possibility that find dramatic differences in the transport of both mRNAs and fluorescent beads. In this presentation, I will review the transport properties of molecules through the nucleoplasm and its compartments and discuss our new results that suggest a surprising range of biophysical properties of the nucleoplasm across cell types.

Kelsey Gasior

University of Notre Dame

"Molecular Interactions and Intracellular Phase Separation"

Found in both the nucleus and the cytoplasm, intracellular phase separation allows for the formation of liquidlike droplets that localize molecules, such as proteins and RNAs. Many RNA- binding proteins interact with different RNA species to create compartments necessary for cellular function, such as polarity and nuclear division. Additionally, the proteins that promote phase separation are frequently coupled to multiple RNA binding domains and several RNAs can interact with a single protein, leading to a large number of potential multivalent interactions. This work focuses on a multiphase, Cahn-Hilliard (CH) diffuse interface model to examine the RNA- protein interactions and competition driving intracellular phase separation. By combining the CH approach with a Flory-Huggins free energy scheme, biologically-relevant mass action kinetics, and phase-dependent diffusion, this model explores how molecular dynamics control droplet- scale phenomena. In-depth analysis using numerical simulations and combined sensitivity techniques, such as Morris Method Screening and Sobol’, shows the depth of complications underlying even the simplest droplet field properties, such as the time of separation and composition of the droplet field. These results show that while specific mathematical parameters can be set to push a system to phase separate, it shares control of the droplet field with the rates at which the protein and RNA can interact. Ultimately, this targeted and thorough approach to intracellular condensates begins to peel back the layers of complex molecular dynamics governing the formation and evolution of these droplets that contribute to cellular function.

Justin Knechtel

Cross Cancer Institute, University of Alberta

"Single Molecule Tracking of KMT5C in Chromatin Compartments"

Our genome is packaged into chromatin, a dynamic DNA-protein complex organized into distinct functional states defined by epigenetic modifications. These states give rise to spatially segregated chromatin compartments, such as euchromatin and heterochromatin, which differ in molecular composition and biophysical properties. KMT5C, a histone lysine methyltransferase that specifically targets histone H4 lysine 20 (H4K20), shows a striking pattern of chromatin compartmentalization: while it is both highly enriched and mobile within heterochromatin, it exhibits minimal exchange with the neighboring euchromatin. To investigate this behavior, we performed single particle tracking of KMT5C in living cells and quantitatively characterized its kinetic properties within and between chromatin compartments. We applied Hidden Markov Modeling to resolve discrete states of motion and leveraged our tracking data as a microrheological tool to assess the physical state of chromatin. These findings provide new insight into how the material properties and molecular organization of chromatin regulate protein dynamics within the nucleus.

Tharana Yosprakob

University of Alberta

"Spatial Organization and Dynamics of Nuclear Proteins"

The protein KMT5C regulates gene transcription and maintains genome integrity. It interacts with CBX5 proteins and is enriched within chromocenters, which are distinct regions in the nucleus where chromatin serves as an organizing scaffold. Although both proteins are similar in size and co-localize in chromocenters, fluorescence recovery after photobleaching (FRAP) reveals different mobility characteristics: CBX5 moves freely within and between chromocenters, whereas KMT5C is limited to movement within chromocenters. To understand these differences, we developed a reaction-diffusion model that incorporates diffusion and binding/unbinding dynamics between KMT5C, CBX5, and chromatin. Using multiple-timescale analysis, we show that the correct diffusion equation for this situation differs from Fick’s Law and predicts a non-uniform steady state concentration, which results in regions of condensation rather than a uniform distribution. Simulated bleaching experiment using this model is consistent with experimental FRAP result, indicating that differential enrichment of KMT5C and CBX5 arises primarily from their binding and unbinding interactions.

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Annual Meeting for the Society for Mathematical Biology, 2025.