Material Properties Of Condensates Research Articles

Optogenetic control of mRNA condensation reveals an intimate link between condensate material properties and functions

Biomolecular condensates, often assembled through phase transition mechanisms, play key roles in organizing diverse cellular activities. The material properties of condensates, ranging from liquid droplets to solid-like glasses or gels, are key features impacting the way resident components associate with one another. However, it remains unclear whether and how different material properties would influence specific cellular functions of condensates. Here, we combine optogenetic control of phase separation with single-molecule mRNA imaging to study relations between phase behaviors and functional performance of condensates. Using light-activated condensation, we show that sequestering target mRNAs into condensates causes translation inhibition. Orthogonal mRNA imaging reveals highly transient nature of interactions between individual mRNAs and condensates. Tuning condensate composition and material property towards more solid-like states leads to stronger translational repression, concomitant with a decrease in molecular mobility. We further demonstrate that β-actin mRNA sequestration in neurons suppresses spine enlargement during chemically induced long-term potentiation. Our work highlights how the material properties of condensates can modulate functions, a mechanism that may play a role in fine-tuning the output of condensate-driven cellular activities.

Small molecules in regulating protein phase separation.

Biomolecular condensates formed by phase separation are involved in many cellular processes. Dysfunctional or abnormal condensates are closely associated with neurodegenerative diseases, cancer and other diseases. Small molecules can effectively regulate protein phase separation by modulating the formation, dissociation, size and material properties of condensates. Discovery of small molecules to regulate protein phase separation provides chemical probes for deciphering the underlying mechanism and potential novel treatments for condensate-related diseases. Here we review the advances of small molecule regulation of phase separation. The discovery, chemical structures of recently found small molecule phase separation regulators and how they modulate biological condensates are summarized and discussed. Possible strategies to accelerate the discovery of more liquid-liquid phase separation (LLPS)-regulating small molecules are proposed.

Time-Dependent Material Properties of Aging Biomolecular Condensates from Different Viscoelasticity Measurements in Molecular Dynamics Simulations.

Biomolecular condensates are important contributors to the internal organization of the cell material. While initially described as liquid-like droplets, the term biomolecular condensates is now used to describe a diversity of condensed phase assemblies with material properties extending from low to high viscous liquids, gels, and even glasses. Because the material properties of condensates are determined by the intrinsic behavior of their molecules, characterizing such properties is integral to rationalizing the molecular mechanisms that dictate their functions and roles in health and disease. Here, we apply and compare three distinct computational methods to measure the viscoelasticity of biomolecular condensates in molecular simulations. These methods are the Green-Kubo (GK) relation, the oscillatory shear (OS) technique, and the bead tracking (BT) method. We find that, although all of these methods provide consistent results for the viscosity of the condensates, the GK and OS techniques outperform the BT method in terms of computational efficiency and statistical uncertainty. We thus apply the GK and OS techniques for a set of 12 different protein/RNA systems using a sequence-dependent coarse-grained model. Our results reveal a strong correlation between condensate viscosity and density, as well as with protein/RNA length and the number of stickers vs spacers in the amino acid protein sequence. Moreover, we couple the GK and the OS technique to nonequilibrium molecular dynamics simulations that mimic the progressive liquid-to-gel transition of protein condensates due to the accumulation of interprotein β-sheets. We compare the behavior of three different protein condensates, i.e., those formed by either hnRNPA1, FUS, or TDP-43 proteins, whose liquid-to-gel transitions are associated with the onset of amyotrophic lateral sclerosis and frontotemporal dementia. We find that both the GK and OS techniques successfully predict the transition from functional liquid-like behavior to kinetically arrested states once the network of interprotein β-sheets has percolated through the condensates. Overall, our work provides a comparison of different modeling rheological techniques to assess the viscosity of biomolecular condensates, a critical magnitude that provides information on the behavior of biomolecules inside condensates.

Determining Thermodynamic and Material Properties of Biomolecular Condensates by Confocal Microscopy and Optical Tweezers.

While the roles of biomolecular condensates in health and disease are being intensely studied, it is equally important that their physical properties are characterized in order to achieve mechanistic understanding. Here we share some of the protocols developed in our lab for measuring thermodynamic and materials properties of condensates. These include a simple method for determining the droplet-phase concentrations of condensate components on a confocal microscope, and a method for determining the viscoelasticity of condensates by optical tweezers. These protocols are either generally applicable to biomolecular condensates or are unique for their characterization.

RNA at the surface of phase-separated condensates impacts their size and number

Although it is now recognized that specific RNAs and protein families are critical for the biogenesis of ribonucleoprotein (RNP) condensates, how these molecular constituents determine condensate size and morphology is unknown. To circumvent the biochemical complexity of endogenous RNP condensates, the use of programmable tools to reconstitute condensate formation with minimal constituents can be instrumental. Here we report a methodology to form RNA-containing condensates in living cells programmed to specifically recruit a single RNA species. Our bioengineered condensates are made of ArtiGranule scaffolds composed of an orthogonal protein that can bind to a specific heterologously expressed RNA. These scaffolds undergo liquid-liquid phase separation in cells and can be chemically controlled to prevent condensation or to trigger condensate dissolution. We found that the targeted RNAs localize at the condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. The recruitment of RNA changes the material properties of condensates by hardening the condensate body. Moreover, the condensate size scales with RNA surface density; the higher the RNA density is, the smaller and more frequent the condensates are. These results suggest a mechanism based on physical constraints, provided by RNAs at the condensate surface, that limit condensate growth and coalescence.

Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate

SignificanceA large subclass of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.

Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases.

Cells are compartmentalized by numerous membrane-enclosed organelles and membraneless compartments to ensure that a wide variety of cellular activities occur in a spatially and temporally controlled manner. The molecular mechanisms underlying the dynamics of membrane-bound organelles, such as their fusion and fission, vesicle-mediated trafficking and membrane contactmediated inter-organelle interactions, have been extensively characterized. However, the molecular details of the assembly and functions of membraneless compartments remain elusive. Mounting evidence has emerged recently that a large number of membraneless compartments, collectively called biomacromolecular condensates, are assembled via liquid-liquid phase separation (LLPS). Phase-separated condensates participate in various biological activities, including higher-order chromatin organization, gene expression, triage of misfolded or unwanted proteins for autophagic degradation, assembly of signaling clusters and actin- and microtubule-based cytoskeletal networks, asymmetric segregations of cell fate determinants and formation of pre- and post-synaptic density signaling assemblies. Biomacromolecular condensates can transition into different material states such as gel-like structures and solid aggregates. The material properties of condensates are crucial for fulfilment of their distinct functions, such as biochemical reaction centers, signaling hubs and supporting architectures. Cells have evolved multiple mechanisms to ensure that biomacromolecular condensates are assembled and disassembled in a tightly controlled manner. Aberrant phase separation and transition are causatively associated with a variety of human diseases such as neurodegenerative diseases and cancers. This review summarizes recent major progress in elucidating the roles of LLPS in various biological pathways and diseases.