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FET (FUS-EWSR1-TAF15) family proteins form mesoscale clusters under physiological conditions at concentrations well below the threshold for phase separation. However, how ATP, an amphiphilic molecule and essential cellular metabolite, affects this clustering remains unclear. Here, I show that ATP modulates the size of subsaturation mesoscale clusters in a concentration-dependent manner. At low concentrations (1–2 mM), ATP promotes clustering by acting as a molecular crosslinker, leading to larger assemblies. At a moderate concentration (5 mM), clusters become smaller but remain stable, whereas at a higher concentration (10 mM), the cluster size is reduced. Other amphiphilic molecules, including sodium xylene sulfonate, sodium toluene sulfonate, and hexanediol, display comparable concentration-dependent effects. These observations cannot be explained solely by hydrotropic or kosmotropic mechanisms; instead, they arise from non-specific interactions between amphiphilic molecules and protein. Thus, the intrinsic chemical features of small molecules and FET proteins collectively govern mesoscale clustering at subsaturation concentrations. ATP and other amphiphilic molecules are known to maintain protein solubility at saturation concentrations by dissolving or preventing phase-separated condensates, but whether similar effects occur in subsaturation mesoscale clusters has remained underexplored. Focusing on the FET family of proteins, the author shows that ATP and related amphiphilic molecules such as sodium xylene sulfonate, sodium toluene sulfonate, and hexanediol modulate cluster size in a concentration-dependent manner, acting as mild crosslinkers that promote clustering at low concentrations and at higher concentrations saturate protein interaction sites leading to reduced cluster size.

It has long been proposed that phase-separated compartments can provide a basis for the formation of cellular precursors in prebiotic environments. However, we know very little about the properties of coacervates formed from simple peptides, their compatibility with ribozymes or their functional significance. Here we assess the conditions under which functional ribozymes form coacervates with simple peptides. We find coacervation to be most robust when transitioning from long homopeptides to shorter, more pre-biologically plausible heteropeptides. We mechanistically show that these RNA–peptide coacervates display peptide-dependent material properties and cofactor concentrations. We find that the interspacing of cationic and neutral amino acids increases RNA mobility, and we use isothermal calorimetry to reveal sequence-dependent Mg 2+ partitioning, two critical factors that together enable ribozyme activity. Our results establish how peptides of limited length, homogeneity and charge density facilitate the compartmentalization of active ribozymes into non-gelating, magnesium-rich coacervates, a scenario that could be applicable to cellular precursors with peptide-dependent functional phenotypes. Phase-separated compartments have long been proposed as precursors to cellular life. Now, it has been shown that RNA–peptide protocells are more robust when formed using shorter (rather than longer) peptides, and that peptide sequence determines the functional materials properties of these compartments.

Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via complex coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides. Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via coacervation. Here authors demonstrate that a coacervate microenvironment supports RNA catalysis whilst selectively sequestering RNA based on length.

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.

Phase separation and percolation contribute to phase transitions of multivalent macromolecules. Contributions of percolation are evident through the viscoelasticity of condensates and through the formation of heterogeneous distributions of nano- and mesoscale pre-percolation clusters in sub-saturated solutions. Here, we show that clusters formed in sub-saturated solutions of FET (FUS-EWSR1-TAF15) proteins are affected differently by glutamate versus chloride. These differences on the nanoscale, gleaned using a suite of methods deployed across a wide range of protein concentrations, are prevalent and can be unmasked even though the driving forces for phase separation remain unchanged in glutamate versus chloride. Strikingly, differences in anion-mediated interactions that drive clustering saturate on the micron-scale. Beyond this length scale the system separates into coexisting phases. Overall, we find that sequence-encoded interactions, mediated by solution components, make synergistic and distinct contributions to the formation of pre-percolation clusters in sub-saturated solutions, and to the driving forces for phase separation. Biomolecular condensates form via phase separation of multivalent macromolecules. Phase separation is governed by solubility whereas multivalence drives percolation, also known as gelation. The authors in this work identify the distinct energy and length scales that influence phase separation versus percolation.

Biomolecular phase separation is an emerging theme for protein assembly and cellular organisation. The collective forces driving such condensation, however, remain challenging to characterise. Here we show that tracking the dilute phase concentration of only one component suffices to quantify composition and energetics of multicomponent condensates. Applying this assay to several disease- and stress-related proteins, we find that monovalent ions can either deplete from or enrich within the dense phase in a context-dependent manner. By analysing the effect of the widely used modulator 1,6-hexanediol, we find that the compound inhibits phase separation by acting as a solvation agent that expands polypeptide chains. Extending the strategy to in cellulo data, we even quantify the relative energetic contributions of individual proteins within complex condensates. Together, our approach provides a generic and broadly applicable tool for dissecting the forces governing biomolecular condensation and guiding the rational modulation of condensate behaviour. Biomolecular phase separation arises from collective molecular interactions and is emerging as a key theme for biological function. Here the authors propose a broadly applicable method to quantify these interactions based on compositional and energetic parameters.

The observation of Liquid-Liquid Phase Separation (LLPS) in biological cells has dramatically shifted the paradigm that soluble proteins are uniformly dispersed in the cytoplasm or nucleoplasm. The LLPS region is preceded by a one-phase solution, where recent experiments have identified clusters in an aqueous solution with 10 2 -10 3 proteins. Here, we theoretically consider a core-shell model with mesoscale core, surface, and bending properties of the clusters’ shell and contrast two experimental paradigms for the measured cluster size distributions of the Cytoplasmic Polyadenylation Element Binding-4 (CPEB4) and Fused in Sarcoma (FUS) proteins. The fits to the theoretical model and earlier electron paramagnetic resonance (EPR) experiments suggest that the same protein may exhibit hydrophilic, hydrophobic, and amphiphilic conformations, which act to stabilize the clusters. We find that CPEB4 clusters are much more stable compared to FUS clusters, which are less energetically favorable. This suggests that in CPEB4, LLPS consists of large-scale aggregates of clusters, while for FUS, clusters coalesce to form micron-scale LLPS domains. This paper models how proteins form clusters before undergoing phase separation. It finds that some proteins, like Cytoplasmic Polyadenylation Element Binding-4 (CPEB4), form stable clusters that later aggregate, while others, like Fused in Sarcoma (FUS), form transient clusters that grow and coalesce into larger condensates.

Recent advances in cell biology enable precise molecular perturbations. The spatiotemporal organization of cells and organisms, however, also depends on physical processes such as diffusion or cytoplasmic flows, and strategies to perturb physical transport inside cells are not yet available. Here, we demonstrate focused-light-induced cytoplasmic streaming (FLUCS). FLUCS is local, directional, dynamic, probe-free, physiological, and is even applicable through rigid egg shells or cell walls. We explain FLUCS via time-dependent modelling of thermoviscous flows. Using FLUCS, we demonstrate that cytoplasmic flows drive partitioning-defective protein (PAR) polarization in Caenorhabditis elegans zygotes, and that cortical flows are sufficient to transport PAR domains and invert PAR polarity. In addition, we find that asymmetric cell division is a binary decision based on gradually varying PAR polarization states. Furthermore, the use of FLUCS for active microrheology revealed a metabolically induced fluid-to-solid transition of the yeast cytoplasm. Our findings establish how a wide range of transport-dependent models of cellular organization become testable by FLUCS. Mittasch et al. show that controlling cytoplasmic flow via focused-light-induced cytoplasmic streaming (FLUCS), a non-invasive technique, can be used to invert asymmetric cell division in Caenorhabditis elegans zygotes.

Lecture given at an American Physical Society meeting at Caltech on 29 December 1959 - Physicist Richard Feynman The past decade has witnessed an exponential growth in nanomaterial science and research for a variety of biomedical applications, such as drug and gene delivery, anticancer therapeutics, diagnostics and imaging, tissue engineering and stem cell based therapies. In particular, nanosized hydrogels, so-called nanogels, have gained attention for biomedical applications due to their high water content, 3D structure and high biocompatibility as well as their relatively large size that enables them to encapsulate biomacromolecules like proteins or genes (3,4). [...]nanogels have been shown to be excellent scaffolds for preparing composites as a novel class of advanced materials, which comprise both nanogels and other constituents such as polymers or inorganic nanoparticles. According to IUPAC, a composite is defined as a “multicomponent material comprising multiple, different (non-gaseous) phase domains in which at least one type of phase domain is a continuous phase” (5). The same nanogel composites loaded with growth factors show continuous release for up to 28 days, which stimulate the regeneration of the urethral muscle tissue surrounding the urethral wall and promote the recovery of its biological function (24). [...]nanogels of lithium-neutralized polyacrylic acid are being combined with a biodegradable polyhydroxybutyrate matrix for bone regeneration and drug delivery (6).

We analyzed the protein corona of thermoresponsive, poly( -isopropylacrylamide)- or poly( -isopropylmethacrylamide)-based nanogels. Traces of protein corona detected after incubation in human serum were characterized by proteomics and dynamic light scattering in undiluted serum. Apolipoprotein B-100 and albumin were the main components of the protein coronae. For dendritic polyglycerol-poly( -isopropylacrylamide) nanogels at 37°C, an increase in adsorbed immunoglobulin light chains was detected, followed by partially reversible nanogel aggregation. All nanogels in their hydrophilic state are colloidally stable in serum and bear a dysopsonin-rich protein corona. We observed strong changes in NG stability upon slight alterations in the composition of the protein coronae according to nanogel solvation state. Nanogels in their hydrophilic state possess safe protein coronae.