Iris B. A. Smokers

Raster image correlation spectroscopy (RICS) is a nondestructive, fluorescence-based method to probe molecular diffusion and effective viscosity in biomolecular condensates. By measuring fluorescence intensity autocorrelations of fluorescent probes in a scanning confocal microscope image, RICS enables the calculation of effective, local condensate viscosities both in reconstituted condensates and in living cells. Here we demonstrate the use of RICS as a powerful tool to probe local diffusion and length-scale-dependent viscosities in multiphase coacervates and stress granules without disrupting them. We quantify the local viscosity in core and shell regions of multiphase coacervates, and inside stress granules and the surrounding cytoplasm in HEK293T cells without relying on labeled proteins. By comparing different probe types, we further show that globular probes such as folded proteins and quantum dots experience a length-scale-dependent viscosity in condensates, whereas dextran-based polymeric probes exhibit an apparent size-independent diffusivity over three orders of magnitude in molecular weight. We attribute these findings to a progressive collapse with increasing size of the polymer chains inside condensates, combined with a weakening of sticky probe-condensate interactions. These findings emphasize the need for due care when using probes to characterize condensate properties. Altogether, our results show that RICS is a useful tool for studying condensate properties that can be accessed on conventional confocal microscopes.

Phosphorus is essential for extant and nascent life. However, phosphate’s low terrestrial abundance and the poor aqueous solubility of phosphorus-bearing minerals pose major hurdles for prebiotic chemistry. Here, we show that silicate-phosphate immiscibility in volcanic melts could have provided a widely available mechanism for the local enrichment of phosphate. Starting with a phosphorus-enriched Archean melt, we observe that rapid cooling results in the formation of immiscible glassy droplets with up to 21 weight % phosphorus pentoxide, which can be delivered to the surface by volcanic processes. In aqueous leaching, we detect millimolar phosphate concentrations, which enable the synthesis of phosphorylating agents such as polyphosphates and imidazole phosphate with up to 34% yield. These results demonstrate how volcanic processes can enrich phosphate to fuel prebiotic chemistry.

Biomolecular condensates (BCs) are ubiquitous compartments that regulate key functions in cells. BCs are surrounded by a complex intracellular environment, of which amino acids (AAs) are prominent components. However, it is unclear how AAs interact with condensate components and influence the material properties of condensates. Here, we demonstrate that phase separation is suppressed with glycine by using model heterotypic condensates composed of nucleophosmin 1 and ribosomal ribonucleic acid. The condensate density decreases, and the dynamics within the condensate increase. We find that glycine weakly binds to amide groups in the protein backbone and aromatic groups in the side chains, weakening the backbone-backbone interactions between neutral and charged disordered proteins while strengthening the interactions between aromatic stickers. This leads to different modulations of the phase behaviour in condensates formed by π/cation-π interactions and charge complexation. We further show that a modulation effect on BCs is observed for other proteinogenic AAs and can be transferred to short homopeptides. These insights offer strategies to modulate the dynamic properties of BCs in vivo.

Biomolecular condensates modulate various ion-dependent cellular processes and can regulate subcellular ion distributions by selective uptake of ions. To understand these processes, it is essential to uncover the molecular grammar governing condensate-ion interactions. Here, we use nuclear magnetic resonance (NMR) spectroscopy of ions and model condensate components to quantify and spatially resolve selective ion binding to condensates and show that these interactions follow the "law of matching water affinities", resulting in strong binding between proteins and chaotropic anions and between nucleic acids and kosmotropic cations. Ion uptake into condensates directly follows binding affinities, resulting in selective uptake of strong-binding ions but exclusion of weak-binding ions. Ion binding further shapes the condensate microenvironment by altering the composition, viscosity, and interface potential. Such changes can have profound effects on biochemical processes taking place inside condensates, as we show for RNA duplex formation. Our findings provide a new perspective on the role of condensate-ion interactions in cellular bio- and electrochemistry and may aid the design of condensate-targeting therapeutics.

Phosphorus is an essential element of life, as observable in modern life forms that have perfected phosphorus accumulation and recycling. Prebiotic chemistry, on the other hand, was likely challenged by the low abundance and poor solubility of phosphorus minerals on early Earth.Current experimental approaches to solve this so-called phosphate problem mainly focus on the solubilization of phosphate minerals like apatite or reduced phosphorus species in extraterrestrial schreibersite. However, the possibilities for prebiotic chemistry through magmatic evolution and phosphate enrichment in basaltic melts have not yet been considered.Here, we show the formation of phosphate-rich glass droplets, driven by liquid immiscibility in basaltic melts, and explore its downstream utilization for prebiotic chemistry. We synthesized two glasses mimicking an average composition of 2.2 – 3.8 Ga old basalts containing more than 0.5 wt.% phosphorus, and doped them with 1 and 10 wt.% P2O5 respectively. In the latter, unmixing lead to the formation of Fe-P-rich droplets in a Si-rich matrix with a partition coefficient of DP ~3.9. Subsequent leaching behavior of the synthesized glass was investigated over a range in pH and organic solvents delivering up to 5 mM dissolved phosphate. The concentration in the experimental leachate was sufficient to form di- and triphosphate afterwards at elevated temperatures and phosphorylation of adenosine was possible.We further found that our initially produced leachates were able to trigger the synthesis of imidazole phosphate with up to 34 % yield and the downstream phosphorylation of glycerol in wet/dry cycles with up to 17 % yield. Our results thus show an alternative source of phosphate for prebiotic chemistry through its early enrichment in the magma and present an alternative approach to solving the phosphate problem for the origin of life.

Biomolecular condensates (BCs) are versatile intracellular compartments that help organize intracellular space and perform diverse biological functions. In living cells, BCs are surrounded by a complex environment, of which amino acids (AAs) are prominent components. However, it is unclear how AAs interact with condensate components and influence the formation and material properties of condensates. Here, we show that phase separation is suppressed with increasing glycine concentration by using model heterotypic condensates made of nucleophosmin 1 (NPM1) and ribosomal ribonucleic acid (RNA): the condensate density is decreased and the dynamics inside the condensate are increased. We find that glycine weakly binds to amide groups in the protein backbone and aromatic groups in the side chains with a 1~2 M affinity, weakening the backbone-backbone interactions between neutral and charged disordered proteins while strengthening the interactions between aromatic stickers. This leads to different modulations of the phase behaviour in condensates formed by cation/π-π interactions and condensates formed by charge complexation. We further show that a similar modulation effect on BCs is observed for other proteinogenic AAs and can be transferred to short peptides. These insights offer a better understanding of BC stability, with implications for new strategies to modulate the dynamic properties of BCs in vivo.

Biomolecular condensates modulate various ion-dependent cellular processes and can regulate subcellular ion distributions by selective uptake of ions. However, the molecular grammar governing condensate-ion interactions is poorly understood. Here, we use NMR spectroscopy of ions and model condensate components to quantify and spatially resolve selective ion binding to condensates and show that these interactions follow the law of matching water affinities, resulting in strong binding between proteins and chaotropic anions, and between nucleic acids and kosmotropic cations. Ion uptake into condensates directly follows binding affinities, resulting in selective uptake of strong-binding ions, but exclusion of weak-binding ions. Ion binding further shapes the condensate microenvironment by altering the composition, viscosity and interface potential. Such changes can have profound effects on biochemical processes taking place inside condensates, as we show for RNA duplex formation. Our findings provide a new perspective on the role of condensate-ion interactions in cellular bio- and electrochemistry and may aid design of condensate-targeting therapeutics.

Accurate determination of biomolecular condensate volume reveals that destabilization of condensates can lead to either swelling or shrinking of condensates, giving fundamental insights into the regulation of the volume of cellular condensates. Determination of the volume of biomolecular condensates and coacervate protocells is essential to investigate their precise composition and impact on (bio)chemical reactions that are localized inside the condensates. It is not a straightforward task, as condensates have tiny volumes, are highly viscous, and are prone to wetting. Here, we examine different strategies to determine condensate volume and introduce two new methods, with which condensate volumes of 1 μL or less (volume fraction 0.4%) can be determined with a standard deviation of 0.03 μL. Using these methods, we show that the swelling or shrinking of condensates depends on the degree of physical cross-linking. These observations are supported by Flory-Huggins theory and can have profound effects on condensates in cell biology.

Phase‐separated compartments can localize (bio)chemical reactions and influence their kinetics. They are believed to play an important role both in extant life in the form of biomolecular condensates and at the origins of life as coacervate protocells. However, experimentally testing the influence of coacervates on different reactions is challenging and time‐consuming. We therefore use a numerical model to explore the effect of phase‐separated droplets on the kinetics and outcome of different chemical reaction systems, where we vary the coacervate volume and partitioning of reactants. We find that the rate of bimolecular reactions has an optimal dilute/coacervate phase volume ratio for a given reactant partitioning. Furthermore, coacervates can accelerate polymerization and self‐replication reactions and lead to formation of longer polymers. Lastly, we find that coacervates can ‘rescue’ oscillating reaction networks in concentration regimes where sustained oscillations do not occur in a single‐phase system. Our results indicate that coacervates can direct the outcome of a wide range of reactions and impact fundamental aspects such as yield, reaction pathway selection, product length and emergent functions. This may have far‐reaching implications for origins of life, synthetic cells and the fate and function of biological condensates.

Accurate determination of biomolecular condensate volume reveals that destabilization of condensates can lead to either swelling or shrinking of condensates, giving fundamental insights into regulation of the volume of cellular condensates. Determination of the volume of biomolecular condensates and coacervate protocells is essential to investigate their precise composition and impact on (bio)chemical reactions that are localized inside the condensates. It is not a straightforward task, as condensates have tiny volumes, are highly viscous and prone to wetting. Here we examine different strategies to determine condensate volume, and introduce two new methods, with which condensate volumes of 1 μL or less (volume fraction 0.4%) can be determined with a standard deviation of 0.03 μL. Using these methods we show that the swelling or shrinking of condensates depends on the degree of physical crosslinking. These observations are supported by Flory-Huggins theory and can have profound effects on condensates in cell biology.