Andre, A. A. M. | bioRxiv Science

bioRxiv2022

Crowding-induced phase separation and solidification by co-condensation of PEG in NPM1-rRNA condensates

The crowdedness of the cell calls for adequate intracellular organization. Biomolecular condensates, formed by liquid-liquid phase separation of intrinsically disordered proteins and nucleic acids, are important organizers of cellular fluids. To underpin the molecular mechanisms of protein condensation, cell-free studies are often used where the role of crowding is not investigated in detail. Here, we investigate the effects of macromolecular crowding on the formation and material properties of a model heterotypic biomolecular condensate, consisting of nucleophosmin (NPM1) and ribosomal RNA (rRNA). We studied the effect of the macromolecular crowding agent PEG, which is often considered an inert crowding agent. We observed that PEG could induce both homotypic and heterotypic phase separation of NPM1 and NPM1-rRNA, respectively. Crowding increases the condensed concentration of NPM1 and decreases its equilibrium dilute phase concentration, while no significant change in the concentration of rRNA in the dilute phase was observed. Interestingly, the crowder itself is concentrated in the condensates, suggesting that co-condensation rather than excluded volume interactions underlie the enhanced phase separation by PEG. Fluorescence recovery after photobleaching (FRAP) measurements indicated that both NPM1 and rRNA become immobile at high PEG concentrations, indicative of a liquid-to-gel transition. Together, these results shed new light onto the role of synthetic crowding agents in phase separation, and demonstrate that condensate properties determined in vitro depend strongly on the addition of crowding agents. STATEMENT OF SIGNIFICANCELiquid-liquid phase separation of proteins and nucleic acids leads to the formation of biomolecular condensates. To mimic biomolecular condensates in vitro, polymeric crowding agents, such as PEG, are often added. Such crowding agents are considered to make in vitro solutions more physiologically relevant, by mimicking the high cellular macromolecule concentrations. However, these crowding agents are commonly selected for their commercial availability and solubility in water, and their influence on phase separation and the physicochemical properties of condensates are seldom studied. Here we use biophysical methods to show that PEG induces phase separation of a model condensate through co-condensation rather than volume exclusion. As a consequence, crowding changes the partitioning, concentrations and viscoelastic properties of the condensates significantly, which sheds new light onto studies aimed at quantifying the material properties of biomolecular condensates.

biophysics↗

bioRxiv2021

ATP:Mg2+ shapes condensate properties of rRNA-NPM1 in vitro nucleolus model and its partitioning of ribosomes

Nucleoli have viscoelastic gel-like condensate dynamics that are not well represented in vitro. Nucleoli models, such as those formed by nucleophosmin 1 (NPM1) and ribosomal RNA (rRNA), exhibit condensate dynamics orders of magnitude faster than in vivo nucleoli. Here we show that an interplay between magnesium ions (Mg2+) and ATP governs rRNA dynamics, and this ultimately shapes the physical state of these condensates. Using quantitative fluorescence microscopy, we demonstrate that increased RNA compaction occurs in the condensates at high Mg2+ concentrations, contributing to the slowed RNA dynamics. At Mg2+ concentrations above 7 mM, rRNA is fully arrested and the condensates are gels. Below the critical gel point, NPM1-rRNA droplets age in a temperature-dependent manner, suggesting that condensates are viscoelastic materials, undergoing maturation driven by weak multivalent interactions. ATP addition reverses the dynamic arrest of rRNA, resulting in liquefaction of these gel-like structures. Surprisingly, ATP and Mg2+ both act to increase partitioning of NPM1-proteins as well as rRNA, which influences the partitioning of small client molecules. By contrast, larger ribosomes form a halo around NPM1-rRNA coacervates when Mg2+ concentrations are higher than ATP concentrations. Within cells, ATP levels fluctuate due to biomolecular reactions, and we demonstrate that a dissipative enzymatic reaction can control the biophysical properties of in vitro condensates through depletion of ATP. This enzymatic ATP depletion also reverses the formation of the ribosome halos. Our results illustrate how cells, by changing local ATP concentrations, may regulate the state and client partitioning of RNA-containing condensates such as the nucleolus. Significance StatementO_LIThere is a significant discrepancy between the dynamics of in vitro nucleolus models and in vivo nucleoli - with the latter more gel-like. C_LIO_LIThe interplay between Mg2+ ions, ATP and the nucleolus components - specifically RNA - governs the dynamics, and ultimately the physical state, of nucleolus-like condensates. C_LIO_LIWe show that the nucleolus are dynamically adapting condensates, responding to local ATP concentrations through Mg2+-induced compaction of the RNA, and reversible relaxation when ATP binds Mg2+ again. Other condensates containing RNA probably respond in similar ways to Mg2+ and ATP. C_LI

biophysics↗