Phase Separation In Systems Research Articles

Nanosecond Molecular Motion in pHP1α Liquid-Liquid Phase Separation Captured by Solid-State NMR.

The relationship among protein structure, function, and dynamics is fundamental to biological activity, particularly in more complex biomolecular systems. Solid-state and solution-state NMR techniques offer powerful means to probe these dynamics across various time scales. However, standard assumptions about molecular motion are often challenged in phase-separated systems like phosphorylated heterochromatin protein 1 alpha (pHP1α), which exhibit both solid- and solution-like characteristics. This study investigates the nanosecond molecular motions in pHP1α liquid-liquid phase separation (LLPS) using relaxation in hetNOE-filtered HSQC signals. By systematically analyzing motions captured by hetNOE-filtered HSQC and conventional HSQC, we characterize the global dynamics site-specifically in pHP1α LLPS. Our findings reveal ∼15 ns motion in the pHP1α LLPS system, suggesting the coexistence of different dynamic phases, and support previous observations on its role in chromatin organization. This work contributes to the expanding literature on phase-separated biomolecular behavior, with implications for understanding the molecular basis of chromatin compaction and genomic stability.

Trifluoroacetic Acid as a Molecular Probe for the Dense Phase in Liquid-Liquid Phase-Separating Peptide Systems.

Although trifluoroacetic acid (TFA) is not typically considered a Hofmeister reagent, it has been demonstrated to modulate biocoacervation. We show that TFA can be employed to probe specific interactions in coacervating bioinspired peptide phenylalanine (Phe) 19F-labeled at a single site, altering its liquid-liquid phase separation (LLPS) behavior. Solid-state nuclear magnetic resonance (NMR) spectroscopy revealed two dynamically distinct binding modes of TFA with Phe, resulting in a structured, dipolar-ordered complex and a more dynamic complex, highlighting the proximity between TFA and Phe. Quantum chemistry modeling of 19F chemical shift differences indicates that the structured complex is formed by the intercalation of one TFA molecule between two stacked Phe aromatic rings, possibly contributing to the stabilization of the condensed dense phase. Thus, we propose that TFA can be used as a convenient molecular probe in 19F NMR-based studies of the structure and dynamics of the dense phase in LLPS peptide systems.

Discrimination between Purine and Pyrimidine-Rich RNA in Liquid-Liquid Phase-Separated Condensates with Cationic Peptides and the Effect of Artificial Crowding Agents.

Membraneless organelles, often referred to as condensates or coacervates, are liquid-liquid phase-separated systems formed between noncoding RNAs and intrinsically disordered proteins. While the importance of different amino acid residues in short peptide-based condensates has been investigated, the role of the individual nucleobases or the type of heterocyclic structures, the purine vs pyrimidine nucleobases, is less researched. The cell's crowded environment has been mimicked in vitro to demonstrate its ability to induce the formation of condensates, but more research in this area is required, especially with respect to RNA-facilitated phase separation and the properties of the crowding agent, poly(ethylene glycol) (PEG). Herein, we have shown that the nucleotide base sequence of RNA can greatly influence its propensity to undergo phase separation with cationic peptides, with the purine-only RNA decamer (AG)5 readily doing so while the pyrimidine-only (CU)5 does not. Furthermore, we show that the presence and size of a PEG macromolecular crowder affects both the ability to phase separate and the stability of coacervates formed, possibly due to co-condensation of PEG with the RNA and peptides. This work sheds light on the presence of low-complexity long purine- or pyrimidine-rich noncomplementary repeat (AG or CU) sequences in various noncoding RNAs found in biology.

Protein-Specific Crowding Accelerates Aging in Protein Condensates.

Macromolecular crowding agents, such as poly(ethylene glycol) (PEG), are often used to mimic cellular cytoplasm in protein assembly studies. Despite the perception that crowding agents have an inert nature, we demonstrate and quantitatively explore the diverse effects of PEG on the phase separation and maturation of protein condensates. We use two model proteins, the FG domain of Nup98 and bovine serum albumin (BSA), which represent an intrinsically disordered protein and a protein with a well-established secondary structure, respectively. PEG expedites the maturation of Nup98, enhancing denser protein packing and fortifying interactions, which hasten beta-sheet formation and subsequent droplet gelation. In contrast to BSA, PEG enhances droplet stability and limits the available solvent for protein solubilization, inducing only minimal changes in the secondary structure, pointing toward a significantly different role of the crowding agent. Strikingly, we detect almost no presence of PEG in Nup droplets, whereas PEG is moderately detectable within BSA droplets. Our findings demonstrate a nuanced interplay between crowding agents and proteins; PEG can accelerate protein maturation in liquid-liquid phase separation systems, but its partitioning and effect on protein structure in droplets is protein specific. This suggests that crowding phenomena are specific to each protein-crowding agent pair.

Hybrid Polymer-Liquid Electrolytes and Their Interfaces Towards Electrode Materials for Lithium Ion Batteries

The escalating demand for efficient, safe, and sustainable energy storage solutions has highlighted lithium-ion batteries (LIBs) as pivotal in the transition towards renewable energy and electrification of society. Despite their widespread adoption, driven by superior energy density, longevity, and low self-discharge rates, LIBs present challenges concerning safety, cost, and environmental impact. The liquid electrolytes traditionally used in LIBs show safety hazards due to their flammability and potential for leakage which impede the utilization of high-voltage cathode materials, thus limiting overall battery performance and reliability.This study focuses on the exploration and implementation of hybrid polymer-liquid electrolytes (HEs) as a novel approach to circumvent these challenges. HEs, characterized by their unique combination of solid and liquid phases, aim to leverage the benefits of both electrolyte types to enhance safety while maintaining good thermal stability and ionic conductivity. Particularly, the research is focused on the process known as polymerization-induced phase separation (PIPS) consisting in the synthesis of HEs with distinct phase-separated systems, where a liquid ion-conducting phase percolates the macropores and mesopores of the formed thermoset encapsulating phase. The electrochemical performance of such a system depends on the chemical interaction between the liquid and the polymer phase and on the nm-scale morphology of the structure [1]. In this context, the pivotal role of interfaces within LIBs is analyzed, with an emphasis on the electrochemical processes at electrode-electrolyte interfaces that are crucial for the operational efficiency and lifespan of batteries. The study investigates commercial cathode materials highlighting their respective merits and challenges in relation to HEs. The primary objective of this investigation is to evaluate the feasibility of infusing cathode materials and to show that PIPS is possible within both micron-sized and nano-sized confined spaces. By incorporating these HE-infused electrodes into half-cell configurations, the study aims to prove their compatibility with high voltage electrode materials exhibiting energy density and battery performance comparable with traditional systems. A significant focus is placed on assessing the morphological and electrochemical stability of HEs after cycling, validating their practicality and effectiveness in enhancing battery safety while preserving efficiency.This research endeavors to push the boundaries of current battery technology, proposing innovative solutions to longstanding challenges. By exploring the synergistic effects of HEs with a broader selection of cathode materials, the study aims to contribute valuable insights towards the development of safer, more efficient, and environmentally friendly LIBs. Acknowledgements The Swedish Energy Agency (grant #48488) is gratefully acknowledged for financial support.

Spatial Control over Reactions via Localized Transcription within Membraneless DNA Nanostar Droplets.

Biomolecular condensates control where and how fast many chemical reactions occur in cells by partitioning reactants and catalysts, enabling simultaneous reactions in different spatial locations of a cell. Even without a membrane or physical barrier, the partitioning of the reactants can affect the rates of downstream reaction cascades in ways that depend on reaction location. Such effects can enable systems of biomolecular condensates to spatiotemporally orchestrate chemical reaction networks in cells to facilitate complex behaviors such as ribosome assembly. Here, we develop a system for developing such control in synthetic systems. We localize different transcription templates within different phase-separated, membraneless DNA nanostar (NS) droplets─programmable, in vitro liquid-liquid phase separation systems for partitioning of substrates and localization of reactions to membraneless droplets. When RNA produced within such droplets is also degraded in the bulk, droplet-localized transcription creates RNA concentration gradients. Consistent with the formation of these gradients, toehold-mediated strand displacement reactions involving transcripts are 2-fold slower far from the site of transcription than when nearby. We then demonstrate how multiple such gradients can form and be maintained independently by simultaneous transcription reactions occurring in tandem, each localized to different NS droplet types. Our results provide a means for constructing reaction systems in which different reactions are spatially localized and controlled without the need for physical membranes. This system also provides a means for generally studying how localized reactions and the exchange of reaction products might occur between protocells.

Molecular transport of vitamin B6 from whey protein and agarose composite gels using diffusion blending law modelling

In this investigation, whey protein and agarose were employed as the phase-separated biopolymer system, with vitamin B6 acting as the diffusant. Fourier-transform infrared (FTIR) spectroscopy affirmed the absence of chemical interactions among all constituents within the experimental parameters. X-ray diffraction (XRD) analysis corroborated the uniform dissolution of vitamin B6 within the composite low-solid mixtures. Confocal scanning laser microscopy elucidated the topology of the matrix, providing tangible evidence of the phase-separated whey protein-agarose networks. Small-deformation dynamic oscillation in-shear was employed to establish a rheological blending law model, predicting the phase volume and effective concentration of the individual components (whey protein and agarose) within their respective domains. Subsequently, a diffusion study was conducted, advocating a novel blending law for molecular transport to estimate the theoretical diffusion coefficient of vitamin B6 in the composite gel by leveraging the effective concentration of each polymer within its phase. The outcomes were positively compared to the observed diffusion coefficient of the vitamin from the composite gel using UV–vis spectroscopy. These results underscore the viability of the blending-law diffusion theory in elucidating the molecular transport of hydrophilic vitamins released from aqueous biopolymer composite gels.

Investigating Different Dynamic pHP1α States in Their KCl-Mediated Liquid-Liquid Phase Separation (LLPS) Using Solid-State NMR (SSNMR) and Molecular Dynamic (MD) Simulations.

Chromatin phase separation is dynamically regulated by many factors, such as post-translational modifications and effector proteins, and plays a critical role in genomic activities. The liquid-liquid phase separation (LLPS) of chromatin and/or effector proteins has been observed both in vitro and in vivo. However, the underlying mechanisms are largely unknown, and elucidating the physicochemical properties of the phase-separated complexes remains technically challenging. In this study, we detected dynamic, viscous, and intermediate components within the phosphorylated heterochromatin protein 1α (pHP1α) phase-separated system by using modified solid-state NMR (SSNMR) pulse sequences. The basis of these sequences relies on the different time scale of motion detected by heteronuclear Overhauser effect (hetNOE), scalar coupling-based, and dipolar coupling-based transfer schemes in NMR. In comparison to commonly utilized scalar coupling-based methods for studying the dynamic components in phase-separated systems, hetNOE offers more direct insight into molecular dynamics. NMR signals from the three different states in the protein gel were selectively excited and individually studied. Combined with molecular dynamics (MD) simulations, our findings indicate that at low KCl concentration (30 mM), the protein gel displays reduced molecular motion. Conversely, an increase in molecular motion was observed at a high KCl concentration (150 mM), which we attribute to the resultant intermolecular electrostatic interactions regulated by KCl.

Study of Phase Separation and Reversibility in CO2-Responsive Superamphiphile Microemulsions.

Phase separation of microemulsions occupies a key position in many applications, such as oil recovery, nanomaterial synthesis, and chemical reactions. Achieving an intelligent response is crucial to microemulsion development and application. For this reason, in this study, CO2-responsive superamphiphilic molecules were developed as rapidly switchable oil-in-water microemulsions. These superhydrophilic molecules with linear structures were produced by electrostatic interaction of stearic acid and aminotrimethyltriazine COSM-1 in a 1:1 molar ratio. The introduction of n-butanol as a cosurfactant in the CO2-responsive microemulsion system resulted in the spontaneous formation of stable microemulsions. The effect of the addition of n-butanol on the carbon dioxide-responsive microemulsion system was investigated, and the optimum amount of n-butanol was determined by optimization. After exposure to CO2 for 30 s, the superhydrophilic molecules decomposed into inactive components at the interface, leading to a complete phase separation of the microemulsion into oil and water phases. The system was purged with N2 at 60 °C for 10 min to remove the CO2, and the phase separation system was transformed into a clear microemulsion, which was then evaluated for its properties. The rapid response and complete demulsification of these surface CNFS superamphiphiles to CO2 suggest that they have promising applications in product separation, microemulsion recovery, and enhanced crude oil recovery.

System size effects on the free energy landscapes from molecular dynamics of phase-separating bilayers.

The "lipid raft" hypothesis proposes that cell membranes contain distinct domains of varying lipid compositions, where "rafts" of ordered lipids and cholesterol coexist with disordered lipid regions. Experimental and theoretical phase diagrams of model membranes have revealed multiple coexisting phases. Molecular dynamics (MD) simulations can also capture spontaneous phase separation of bilayers. However, these methods merely determine the signof the free energy change upon phase separation-whether or not it is favorable-but not the amplitude. Recently, we developed a workflow to compute the free energy of phase separation from MD simulations using the weighted ensemble method. However, while theoretical treatments generally focus on infinite systems and experimental measurements on mesoscopic to macroscopic systems, MD simulations are comparatively small. Therefore, if we are to put the results of these calculations into the appropriate context, we need to understand the effects the finite size of the simulation has on the computed free energy landscapes. In this study, we investigate this phenomenon by computing free energy profiles for a model phase-separating system as a function of system size, ranging from 324 to 10 110 lipids. The results suggest that, within the limits of statistical uncertainty, bulk-like behavior emerges once the systems contain roughly 4000 lipids.

Raft-like lipid mixtures in the highly coarse-grained Cooke membrane model.

Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins. They are believed to underlie the experimentally observed lateral heterogeneity of eukaryotic plasma membranes and implicated in many cellular processes, such as signaling and trafficking. Ternary model membranes consisting of saturated lipids, unsaturated lipids, and cholesterol are common proxies because they exhibit phase coexistence between a liquid-ordered (lo) and liquid-disordered (ld) phase and an associated critical point. However, plasma membranes are also asymmetric in terms of lipid type, lipid abundance, leaflet tension, and corresponding cholesterol distribution, suggesting that rafts cannot be examined separately from questions about elasticity, curvature torques, and internal mechanical stresses. Unfortunately, it is challenging to capture this wide range of physical phenomenology in a single model that can access sufficiently long length- and time scales. Here we extend the highly coarse-grained Cooke model for lipids, which has been extensively characterized on the curvature-elastic front, to also represent raft-like lo/ld mixing thermodynamics. In particular, we capture the shape and tie lines of a coexistence region that narrows upon cholesterol addition, terminates at a critical point, and has coexisting phases that reflect key differences in membrane order and lipid packing. We furthermore examine elasticity and lipid diffusion for both phase separated and pure systems and how they change upon the addition of cholesterol. We anticipate that this model will enable significant insight into lo/ld phase separation and the associated question of lipid rafts for membranes that have compositionally distinct leaflets that are likely under differential stress-like the plasma membrane.

Constructing a Janus Catholyte/Cathode Structure: A New Strategy for Stable Zn-Organic Batteries.

Organic materials are promising candidates for the electrodes of aqueous zinc-ion batteries due to their nonmetallic nature, environmental friendliness, and cost-effectiveness. However, they often suffer from significant dissolution during the charge-discharge process, which poses a major hurdle to their practical applications. Inspired by membrane-less organelles in cells, a simple and versatile strategy is proposed-constructing a Janus catholyte/cathode structured electrode based on liquid-liquid phase separation, in which redox-active organic molecules are confined in the liquid state within the activated carbon, thereby eliminating the volume effect and preventing their diffusion into the electrolyte. The customization of phase separation systems by leveraging the hydrophobicity/hydrophilicity differences of various anions is successfully demonstrated. This approach allows for precise regulation of ion cluster/coordination structures, enabling the confinement of active substances while ensuring efficient ion transport. Consequently, the as-constructed Zn||Janus catholyte/cathode cells exhibit superior reversible rate capacity (186mAhg-1 at 5.0Ag-1) and remarkable cycling performance (retention of 72.5% after 12000 cycles). The strategy in building Janus catholyte/cathode structured electrodes breaks free from the limitations imposed by traditional solid-state electrodes, offering tremendous opportunities for exploring diverse advanced battery systems.

Revealing nanoscale structure and interfaces of protein and polymer condensates via cryo-electron microscopy.

Liquid-liquid phase separation (LLPS) is a ubiquitous demixing phenomenon observed in various molecular solutions, including in polymer and protein solutions. Demixing of solutions results in condensed, phase separated droplets which exhibit a range of liquid-like properties driven by transient intermolecular interactions. Understanding the organization within these condensates is crucial for deciphering their material properties and functions. This study explores the distinct nanoscale networks and interfaces in the condensate samples using a modified cryo-electron microscopy (cryo-EM) method. The method involves initiating condensate formation on electron microscopy grids to limit droplet growth as large droplet sizes are not ideal for cryo-EM imaging. The versatility of this method is demonstrated by imaging three different classes of condensates. We further investigate the condensate structures using cryo-electron tomography which provides 3D reconstructions, uncovering porous internal structures, unique core-shell morphologies, and inhomogeneities within the nanoscale organization of protein condensates. Comparison with dry-state transmission electron microscopy emphasizes the importance of preserving the hydrated structure of condensates for accurate structural analysis. We correlate the internal structure of protein condensates with their amino acid sequences and material properties by performing viscosity measurements that support that more viscous condensates exhibit denser internal assemblies. Our findings contribute to a comprehensive understanding of nanoscale condensate structure and its material properties. Our approach here provides a versatile tool for exploring various phase-separated systems and their nanoscale structures for future studies.

Elastin-like polypeptide coacervates as reversibly triggerable compartments for synthetic cells

Compartmentalization is a vital aspect of living cells to orchestrate intracellular processes. In a similar vein, constructing dynamic and responsive sub-compartments is key to synthetic cell engineering. In recent years, liquid-liquid phase separation via coacervation has offered an innovative avenue for creating membraneless organelles (MOs) within artificial cells. Here, we present a lab-on-a-chip system to reversibly trigger peptide-based coacervates within cell-mimicking confinements. We use double emulsion droplets (DEs) as our synthetic cell containers while pH-responsive elastin-like polypeptides (ELPs) act as the coacervate system. We first present a high-throughput microfluidic DE production enabling efficient encapsulation of the ELPs. The DEs are then harvested to perform multiple MO formation-dissolution cycles using pH as well as temperature variation. For controlled long-term visualization and modulation of the external environment, we developed an integrated microfluidic device for trapping and environmental stimulation of DEs, with negligible mechanical force, and demonstrated a proof-of-principle osmolyte-based triggering to induce multiple MO formation-dissolution cycles. In conclusion, our work showcases the use of DEs and ELPs in designing membraneless reversible compartmentalization within synthetic cells via physicochemical triggers. Additionally, presented on-chip platform can be applied over a wide range of phase separation and vesicle systems for applications in synthetic cells and beyond.

Affinity-Controlled Partitioning of Biomolecules at Aqueous Interfaces and Their Bioanalytic Applications.

All-aqueous phase separation systems play essential roles in bioanalytical and biochemical applications. Compared to conventional oil and organic solvent-based systems, these systems are characterized by their rich bulk and interfacial properties, offering superior biocompatibility. In particular, phase separation in all-aqueous systems facilitates the creation of compartments with specific physicochemical properties, and therefore largely enhances the accessibility of the systems. In addition, the all-aqueous compartments have diverse affinities, with an important property known as partitioning, which can concentrate (bio)molecules toward distinct immiscible phases. This partitioning affinity imparts all-aqueous interfaces with selective permeability, enabling the controlled enrichment of target (bio)molecules. This review introduces the basic principles and applications of partitioning-induced interfacial phenomena in a typical all-aqueous system, namely aqueous two-phase systems (ATPSs); these applications include interfacial chemical reactions, bioprinting, and assembly, as well as bio-sensing and detection. The primary challenges associated with designing all-aqueous phase separation systems and several future directions are also discussed, such as the stabilization of aqueous interfaces, the handling of low-volume samples, and exploration of suitable ATPSs compositions with the efficient protocol.

Diffusive Trends in Concentrated Oppositely-Charged Polyelectrolyte Solutions and Onset of Glassy Dynamics.

We utilize single particle tracking studies to investigate the diffusion of polylysine through concentrated matrices of cationic polylysine and anionic polyglutamic acid with no added salts. These studies show that diffusivity has a strong apparently exponential dependence on concentration in crowded systems that does not appear to be a function of the charge sign. These trends are consistent in both single-phase systems prepared at concentrated conditions and polymer-rich coacervate phases formed from dilute phase-separating systems. The likely origin of this behavior is the onset of glassy dynamics spurred by a decrease in plasticization by water and the large excluded volume associated with charge-bearing species. This effect can be contextualized through free volume-based theories such as the Vrentas-Duda model. Overall, we obtain dynamic behavior that is distinctly different from behavior observed in more dilute systems and warrants further investigation.

Preparation of thermally stable egg white protein microgel particles as assisted by differently charged polysaccharides: A comparatively study

Egg white proteins (EWP) were prone to thermal aggregation under heat sterilization conditions, thereby limiting their application in the field of protein beverages. In this study, EWP were microgel particleized via polysaccharide-protein phase separation to enhance their resistance to thermal aggregation during processing. Results indicated that the charge characteristics of polysaccharides were crucial factors influencing their interaction with EWP. Microstructural and physicochemical properties indicated that egg white protein microgel particles (EMGP) with the bilayer structure formation (polysaccharide shell layer and dense protein core) could only be formed in the phase separation system of EWP and anionic polysaccharides (carboxymethyl cellulose sodium salt (low viscosity), carboxymethyl cellulose sodium salt (high viscosity), High-methoxyl pectin). Additionally, the EMGP exhibited uniform size, high denaturation extent and dense structure that effectively prevented structural unfolding and exposure of active sites upon reheating of the EWP. The thermal denaturation temperatures of the EMGP were significantly higher (up to 155.8 °C, 163.5 °C, 165.7 °C), compared to natural proteins (88.8 °C). Thermal stability experiments confirmed that the suspension of 5% w/v microgel particles maintained good flow ability after heating at 100 °C for 30 min, demonstrating excellent thermal stability suitable for high-temperature pasteurization conditions. Moreover, its uniform particle size and adsorption of polysaccharides improved storage stability. In contrast, groups other than anionic polysaccharides exhibited aggregation and gelation upon heating. Overall, this study demonstrated that preparing heat-stable EMGP effectively reduces aggregation in EWP beverages during heat-pasteurization, enhanced product sensory attributes, extended shelf-life, and expanded the application scope of EWP.

Mesoscale molecular assembly is favored by the active, crowded cytoplasm

The mesoscale organization of molecules into membraneless biomolecular condensates is emerging as a key mechanism of rapid spatiotemporal control in cells. Principles of biomolecular condensation have been revealed through reconstitution. However, intracellular environments are much more complex than test-tube environments: they are viscoelastic, highly crowded at the mesoscale, and are far from thermodynamic equilibrium due to the constant action of energy-consuming processes. We developed synDrops, a synthetic phase separation system, to study how the cellular environment affects condensate formation. Three key features enable physical analysis: synDrops are inducible, bioorthogonal, and have well-defined geometry. This design allows kinetic analysis of synDrop assembly and facilitates computational simulation of the process. We compared experiments and simulations to determine that macromolecular crowding promotes condensate nucleation but inhibits droplet growth through coalescence. ATP-dependent cellular activities help overcome the frustration of growth. In particular, stirring of the cytoplasm by actomyosin dynamics is the dominant mechanism that potentiates droplet growth in the mammalian cytoplasm by reducing confinement and elasticity. Our results demonstrate that mesoscale molecular assembly is favored by the combined effects of crowding and active matter in the cytoplasm. These results move toward a better predictive understanding of condensate formation . Published by the American Physical Society 2024

Effect of konjac glucomannan on heat-induced pea protein isolate hydrogels: Evaluation of structure and formation mechanisms

The roles of increasing concentration of konjac glucomannan polysaccharide (KGM) in modifying the thermal, textural, and rheological properties of heat-induced pea protein isolate (PPI) hydrogels at neutral pH and low ionic strength were investigated in this study. Differential scanning calorimetry was first applied to investigate the effect of KGM addition on the thermal properties of pea proteins, finding that an addition of 0.5 % w/v increased the glass transition temperature of pea proteins (from 150 to 154 °C) while on the contrary, higher concentrations of 1.0–1.5 % w/v reduced glass transition temperatures (from 150 °C to ∼ 144 °C). Water holding capacity highlighted that KGM addition significantly increased water retention, reaching values up to 90 % for KGM concentration ≥ 1.0 % w/v. Confocal laser scanning microscopy showed the presence of a phase separation system with increasing concentrations of KGM. Molecular interactions and Fourier transform infrared spectroscopy analysis highlighted that the hydrogels were mainly stabilized by non-covalent intermolecular interactions. Texture analysis confirmed that KGM significantly influenced texture of the hydrogels, in which 1.5 % w/v KGM presented the highest hardness value of 1.34 N. Lastly, small amplitude rotational and oscillatory rheology was employed to evaluate the hydrogels viscosity and viscoelastic properties, which both were significantly increased with increasing KGM concentrations.

Transcription templated assembly of the nucleolus in the C. elegans embryo.

The nucleolus is a multicomponent structure made of RNA and proteins that serves as the site of ribosome biogenesis within the nucleus. It has been extensively studied as a prototype of a biomolecular condensate whose assembly is driven by phase separation. While the steady-state size of the nucleolus is quantitatively accounted for by the thermodynamics of phase separation, we show that experimental measurements of the assembly dynamics are inconsistent with a simple model of a phase-separating system relaxing to its equilibrium state. Instead, we show that the dynamics are well described by a model in which the transcription of ribosomal RNA actively drives nucleolar assembly. We find that our model of active transcription-templated assembly quantitatively accounts for the rapid kinetics observed in early embryos at different developmental stages, and for different RNAi perturbations of embryo size. Our model predicts a scaling of the time to assembly with the volume of the nucleus to the one-third power, which is confirmed by experimental data. Our study highlights the role of active processes such as transcription in controlling the placement and timing of assembly of membraneless organelles.