Phase Separation In Systems Research Articles

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.

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.

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.

Anisotropic remixing of a phase separated binary colloidal system with particles of different sizes in an external modulation.

We explore the phase behavior of a binary colloidal system under external spatially periodic modulation. We perform Monte Carlo simulations on a binary mixture of big and small repulsive Lennard-Jones particles with a diameter ratio of 2:1. We characterize structure by isotropic and anisotropic pair correlation functions, cluster size distribution, bond angle distribution, order parameter, and specific heat. We observe the demixing of the species in the absence of external modulation. However, the mixing of the species gets enhanced with increasing potential strength along with the alignment of the particles transverse to the modulation. The de-mixing order parameter shows discontinuity with increasing modulation strength, characterizing a first order phase transition. The peak in specific heat increases linearly with the size of the system. We also look into the dynamical behavior of the system via computing Mean Square Displacement (MSD) along both parallel and perpendicular directions to the modulation. We observe a decrease in the diffusion coefficient for both types of particles as we increase the strength of the modulation.

Completely recyclable and reusable automatic phase-separable catalytic system for the reductive debromination of polybrominated diphenyl ethers

An automatic phase-separable catalytic system was developed for the reductive debromination of polybrominated diphenyl ethers (PBDEs) using thiol-modified Pd nanoparticles (thiol-Pd NPs) as the catalyst, H2 as the reducing agent, and n-butanol (oil phase) and water (water phase) as solvents. This system achieved efficient and complete debromination of various PBDEs with 1–10 bromine atoms and other bromo-organic pollutants at concentrations ranging from 15 to 1500 mg L−1. Typically, this system achieved a debromination efficiency of 100 % within 15 min for all the tested bromo-organic pollutants. Importantly, the debromination reaction was conducted in a microemulsion state under stirring; upon stopping the stirring, the microemulsion automatically separated into an upper oil phase, a down water phase, and a thiol-Pd NP self-assembled membrane phase at the oil/water interface. These three phases could be easily separated from each other, allowing for their reuse across multiple debromination cycles with good reusability. The thiol modifier enhanced the stability and reusability of Pd NPs and improved the adsorption of PBDEs, facilitating their reaction with atomic hydrogen (the dominant reactive species). This method successfully eliminated PBDEs in soils by integrating with a pre-extraction process.

Synthetic Image Analysis for Accurate Agglomerate Characterization and Control in Liquid–Liquid Phase Separation Systems

Synthetic Image Analysis for Accurate Agglomerate Characterization and Control in Liquid–Liquid Phase Separation Systems

Verteporfin inhibits TGF-β signaling by disrupting the Smad2/3-Smad4 interaction.

Transforming growth factor-β (TGF-β) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion. Targeting TGF-β signaling is a promising therapeutic approach, but nonspecific inhibition may result in adverse effects. In this study, we focus on the Smad2/3-Smad4 complex, a key component in TGF-β signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3-Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-β signaling, but not the interaction between Smad1 and Smad4 in bone morphogenetic protein (BMP) signaling. Furthermore, VP exhibited inhibitory effects on TGF-β-induced EMT and cell migration. Our findings indicate a novel approach to develop protein-protein interaction inhibitors of the canonical TGF-β signaling pathway for treatments of related diseases.

Water Hydrogen-Bond Mediated Layer by Layer Alignment of Lipid Rafts as a Precursor of Intermembrane Processes.

Lipid rafts, which are dynamic nanodomains in the plasma membrane, play a crucial role in intermembrane processes by clustering together and growing in size within the plane of the membrane while also aligning with each other across different membranes. However, the physical origin of layer by layer alignment of lipid rafts remains to be elucidated. Here, by using fluorescence imaging and synchrotron X-ray reflectivity in a phase-separated multilayer system, we find that the alignment of raft-mimicking Lo domains is regulated by the distance between bilayers. Molecular dynamics simulations reveal that the aligned state is energetically preferred when the intermembrane distance is small due to its ability to minimize the volume of surface water, which has fewer water hydrogen bonds (HBs) compared to bulk water. Our results suggest that water HB-driven alignment of lipid rafts plays a role as a precursor of intermembrane processes such as cell-cell fusion, virus entry, and signaling.

Particle levitation tensiometry (PLT) for probing interfaces of liquid–liquid phase separation systems (LLPSs)

Particle levitation tensiometry (PLT) for probing interfaces of liquid–liquid phase separation systems (LLPSs)

A delayed decoupling methyl-TROSY pulse sequence for atomic resolution studies of folded proteins and RNAs in condensates

Solution NMR spectroscopy has tremendous potential for providing atomic resolution insights into the interactions between proteins and nucleic acids partitioned into condensed phases of phase-separated systems. However, the highly viscous nature of the condensed phase challenges applications, and in particular, the extraction of quantitative, site-specific information. Here, we present a delayed decoupling-based HMQC pulse sequence for methyl-TROSY studies of ‘client’ proteins and nucleic acids partitioned into ‘scaffold’ proteinaceous phase-separated solvents. High sensitivity and excellent quality spectra are recorded of a nascent form of superoxide dismutase and of a small RNA fragment partitioned into CAPRIN1 condensates.

Engineering heterogeneous hierarchical hydrogels based on Artemisia sphaerocephala Krasch polysaccharide/whey protein isolate fibrils aqueous two-phase emulsion

Emulsion-templating techniques have been used to develop structurally anisotropic hydrogels. This paper proposes a method to couple biopolymer assembly behavior with a phase-separated aqueous two-phase system (ATPS) to construct a hierarchical structure on protein-polysaccharide hydrogels. Based on the phase separation of Artemisia sphaerocephala Krasch polysaccharide (ASKP) and whey protein isolate (WPI) fibrils, a bi-continuous hydrogel was fabricated through spontaneous iron-facilitated molecular chelation. The impact of WPI fibrils under ultrasonic treatment for varying durations on gel structurization and physicochemical properties was also exploited. Shorter length of WPI fibrils facilitated stepwise cross-linking gelation during the oxidation of ferrous ions to ferric ions. Specifically, ultrasonic treatment for 6 min and 8 min promoted the accumulation of shorter fibrils, resulting in the formation of a more intensive clockwise-oriented hierarchical network. Compared to the hydrogels with longer linear aggregates without the ultrasonic treatment, the incorporation of the shorter aggregates resulting from ultrasonic treatment for 6 min and 8 min significantly increased the gel's water holding capacity and mechanical properties. Additionally, the fibrils that underwent ultrasonic treatment for 6 min resulted in a more stable three-dimensional network with optimal elastic performance. This study presents a new water-in-water emulsion-templated strategy for structural regulation and stabilization of multiform hydrogel.

Multifunctional Polyoxometalates-Based Ionohydrogels toward Flexible Electronics.

Multifunctional flexible electronics present tremendous opportunities in the rapidly evolving digital age. One potential avenue to realize this goal is the integration of polyoxometalates (POMs) and ionic liquid-based gels (ILGs), but the challenge of macrophase separation due to poor compatibility, especially caused by repulsion between like-charged units, poses a significant hurdle. Herein, the possibilities of producing diverse and homogenous POMs-containing ionohydrogels by nanoconfining POMs and ionic liquids (ILs) within an elastomer-like polyzwitterionic hydrogel using a simple one-step random copolymerization method, are expanded vastly. The incorporation of polyzwitterions provides a nanoconfined microenvironment and effectively modulates excessive electrostatic interactions in POMs/ILs/H2O blending system, facilitating a phase transition from macrophase separation to a submillimeter scale worm-like microphase-separation system. Moreover, combining POMs-reinforced ionohydrogels with a developed integrated self-powered sensing system utilizing strain sensors and Zn-ion hybrid supercapacitors has enabled efficient energy storage and detection of external strain changes with high precision. This work not only provides guidelines for manipulating morphology within phase-separation gelation systems, but also paves the way for developing versatile POMs-based ionohydrogels for state-of-the-art smart flexible electronics.