Liquid-liquid Phase Separation Process Research Articles

RNA stores tau reversibly in complex coacervates.

Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils.

Integrating the genomic architecture of human nucleolar organizer regions with the biophysical properties of nucleoli.

Nucleoli are the sites of ribosome biogenesis and the largest membraneless subnuclear structures. They are intimately linked with growth and proliferation control and function as sensors of cellular stress. Nucleoli form around arrays of ribosomal gene (rDNA) repeats also called nucleolar organizer regions (NORs). In humans, NORs are located on the short arms of all five human acrocentric chromosomes. Multiple NORs contribute to the formation of large heterochromatin-surrounded nucleoli observed in most human cells. Here we will review recent findings about their genomic architecture. The dynamic nature of nucleoli began to be appreciated with the advent of photodynamic experiments using fluorescent protein fusions. We review more recent data on nucleoli in Xenopus germinal vesicles (GVs) which has revealed a liquid droplet-like behavior that facilitates nucleolar fusion. Further analysis in both XenopusGVs and Drosophila embryos indicates that the internal organization of nucleoli is generated by a combination of liquid-liquid phase separation and active processes involving rDNA. We will attempt to integrate these recent findings with the genomic architecture of human NORs to advance our understanding of how nucleoli form and respond to stress in human cells.

Effect of melt superheating treatment on solidification structures of Al75Bi9Sn16 immiscible alloy

In this paper, the liquid-liquid phase separation (L-LPS) and solidification process of the Al75Bi9Sn16 immiscible alloy were studied with calorimetric and resistivity methods to ascertain the melt superheated treatment process. The impact of melt superheating treatment (MST) on the phase constitution and solidification microstructures were investigated using X-ray diffraction (XRD) and field emission scanning electron microscope (FESEM) to determine the structural sensitivity to the melt superheated degree, and find a new strategy for improving the forming ability of the core-shell structure of the Al75Bi9Sn16 alloy. The results show that: resistivity method is a more sensitive, intuitive, and effective method to investigate the liquid-liquid phase separation, and the liquid-liquid phase separation and precipitation of primary (Sn) phase occur in 1039K–880K and 460K–403K respectively. In addition, the core-shell structure with Sn-Bi-rich core and Al-rich shell can be formed under conventional casting conditions; the melt superheating treatment (MST) can promote the formation of core-shell structure by increasing solidification time t0 and decreasing the average solidification rate v and viscosity η.

Structure and Self-Assembly of Elastin-Like Peptides: A Joint Molecular Dynamics and NMR Study

Elastin endows tissues such as skin, arterial walls, lung alveoli, and the uterus with extensibility and elasticity. Elastin and elastin-like peptides self-aggregate via a liquid-liquid phase separation process. Although elastin has been the object of biophysical investigation for over eighty years, the structural basis for the self-assembly and the mechanical properties of elastin remains controversial. Elastin is structurally heterogeneous and must be described by an ensemble of structures. As an essential step towards elucidating the structural ensemble of elastin, we combine molecular dynamics simulations and nuclear magnetic resonance (NMR) spectroscopy to study an elastin-like peptide modelled after the sequence of alternating hydrophobic and cross-linking domains of elastin. We perform extensive all-atom molecular dynamics simulations totalling hundreds of microseconds and obtain detailed structural and dynamic data by NMR on the same peptide sequences under the same thermodynamic conditions. Simulation and NMR results are in excellent agreement and show that although the peptide is highly disordered, it possesses a significant propensity for secondary structure. The cross-linking domains are characterized by fluctuating helical structure, whereas the hydrophobic domains adopt fluctuating hydrogen-bonded turns. The latter secondary structure elements are sparse, local, and transient. As a result, the individual domains do not form extensive interactions; they are collapsed but not compact, and they remain disordered and hydrated despite their predominantly hydrophobic character. Taken together, these findings resolve long-standing discrepancies between previous models of the structure and function of elastin. Importantly, these results also afford unprecedented insight into the physical and structural basis for the liquid-liquid phase separation of disordered proteins.

Droplet organelles?

Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular activities, and they have been variously described as bodies, granules, or organelles. Recent evidence suggests that a liquid-liquid phase separation (LLPS) process may drive their formation, possibly justifying the unifying term "droplet organelle". A veritable deluge of recent publications points to the importance of low-complexity proteins and RNA in determining the physical properties of phase-separated structures. Many of the proteins linked to such structures are implicated in human diseases, such as amyotrophic lateral sclerosis (ALS). We provide an overview of the organizational principles that characterize putative "droplet organelles" in healthy and diseased cells, connecting protein biochemistry with cell physiology.

Exploiting the Phenomenon of Liquid-Liquid Phase Separation for Enhanced and Sustained Membrane Transport of a Poorly Water-Soluble Drug.

Recent studies on aqueous supersaturated lipophilic drug solutions prepared by methods including antisolvent addition, pH swing, or dissolution of amorphous solid dispersions (ASDs) have demonstrated that when crystallization is slow, these systems undergo liquid-liquid phase separation (LLPS) when the concentration of the drug in the medium exceeds its amorphous solubility. Following LLPS, a metastable equilibrium is formed where the concentration of drug in the continuous phase corresponds to the amorphous solubility while the dispersed phase is composed of a nanosized drug-rich phase. It has been reasoned that the drug-rich phase may act as a reservoir, enabling the rate of passive transport of the drug across a membrane to be maintained at the maximum value for an extended period of time. Herein, using clotrimazole as a model drug, and a flow-through diffusion cell, the reservoir effect is demonstrated. Supersaturated clotrimazole solutions at concentrations below the amorphous solubility show a linear relationship between the maximum flux and the initial concentration. Once the concentration exceeds the amorphous solubility, the maximum flux achieved reaches a plateau. However, the duration for which the high flux persists was found to be highly dependent on the number of drug-rich nanodroplets present in the donor compartment. Macroscopic amorphous particles of clotrimazole did not lead to the same reservoir effect observed with the nanodroplets formed through the process of LLPS. A first-principles mathematical model was developed which was able to fit the experimental receiver concentration-time profiles for concentration regimes both below and above amorphous solubility, providing support for the contention that the nanodroplet phase does not directly diffuse across the membrane but, instead, rapidly replenishes the drug in the aqueous phase that has been removed by transport across the membrane. This study provides important insight into the properties of supersaturated solutions and how these might in turn impact oral absorption through effects on passive membrane transport rates.

Liquid-liquid phase separation of freely falling undercooled ternary Fe-Cu-Sn alloy.

The active modulation and control of the liquid phase separation for high-temperature metallic systems are still challenging the development of advanced immiscible alloys. Here we present an attempt to manipulate the dynamic process of liquid-liquid phase separation for ternary Fe47.5Cu47.5Sn5 alloy. It was firstly dispersed into numerous droplets with 66 ~ 810 μm diameters and then highly undercooled and rapidly solidified under the containerless microgravity condition inside drop tube. 3-D phase field simulation was performed to explore the kinetic evolution of liquid phase separation. Through regulating the combined effects of undercooling level, phase separation time and Marangoni migration, three types of separation patterns were yielded: monotectic cell, core shell and dispersive structures. The two-layer core-shell morphology proved to be the most stable separation configuration owing to its lowest chemical potential. Whereas the monotectic cell and dispersive microstructures were both thermodynamically metastable transition states because of their highly active energy. The Sn solute partition profiles of Fe-rich core and Cu-rich shell in core-shell structures varied only slightly with cooling rate.

Easy fabrication and high electrochemical capacitive performance of hierarchical porous carbon by a method combining liquid-liquid phase separation and pyrolysis process

A hierarchical porous carbon membrane was designed and prepared through a method combining liquid-liquid phase separation and then pyrolysis process using polyacrylonitrile (PAN) as precursor. The results of scan electron microscopy, transmission electron microscope and Brunauer-Emmett-Teller characterization reveal that the 3D nanoscaled architecture with hierarchical porous structure was achieved, which not only provide a continuous electron pathway to ensure good electrical contact, but also facilitate ion transport by shortening diffusion pathways. The effect of PAN concentration in casting solution on structure feature of carbon membrane was also studied, indicating that the membrane thickness with different porous structure can be mediated by PAN concentration. As the electrode material for supercapacitor, a high specific capacitance of 277.0Fg−1 was attained at a current density of 5mAcm−2 and long cycle life of 90.0% capacity retention was obtained after 2000 charge-discharge cycles in 2molL−1 KOH solution.

The Liquid State of (Elastomeric) Proteins

Self-assembled elastomeric proteins make up an important and unusual class of structural proteins endowing biological tissues as diverse as spider silks and lung alveoli with extensibility and elastic recoil. In humans, elastin is the polymeric extracellular matrix protein responsible for the elasticity of lungs, skin, the bladder, the uterus, and large blood vessels. Elastin self-assembly involves a process of liquid-liquid phase separation called coacervation. Although the structure of elastin in the resulting matrix is unknown, the source of elastic recoil is known to be primarily entropic. Two different sources of entropy have been proposed in seemingly-contradictory models: rubber-like models attribute the elasticity of elastin to the entropy of the polypeptide chain in random-coil conformation, whereas “liquid drop” models ascribe the recoil of this highly-hydrophobic protein to the hydrophobic effect. Although its self-organization and mechanical properties have spurred interest in elastin as a model for useful biomimetic polymers, the molecular determinants of these properties are poorly understood.We use atomistic molecular dynamics simulations in explicit solvent to examine the structural basis for the self-assembly and mechanical properties of elastin-like polypeptides in water. Massive sampling over simulation times totalling 0.3 millisecond indicates that the peptides remain highly disordered (though not random) even in the aggregated state, and provides an ensemble description of phase-separated peptide aggregates in which the polypeptide chains exhibit liquid-like properties approaching those of a polymer melt. In this phase-separated state, the peptides are largely solvated by one another, although the polypeptide backbone remains significantly hydrated. Crucially, both conformational entropy and hydrophobic burial drive the formation of this protein-rich phase, which may be called the liquid state of proteins. These findings support a unified model of self-assembled elastomeric proteins in which these two entropic forces play essential roles in both self-assembly and elastic recoil.

Microscopic Evidence for Liquid-Liquid Separation in Supersaturated CaCO 3 Solutions

Recent experimental observations of the onset of calcium carbonate (CaCO3) mineralization suggest the emergence of a population of clusters that are stable rather than unstable as predicted by classical nucleation theory. This study uses molecular dynamics simulations to probe the structure, dynamics, and energetics of hydrated CaCO3 clusters and lattice gas simulations to explore the behavior of cluster populations before nucleation. Our results predict formation of a dense liquid phase through liquid-liquid separation within the concentration range in which clusters are observed. Coalescence and solidification of nanoscale droplets results in formation of a solid phase, the structure of which is consistent with amorphous CaCO3. The presence of a liquid-liquid binodal enables a diverse set of experimental observations to be reconciled within the context of established phase-separation mechanisms.

Structural and morphological development in polypropylene/poly(propylene‐1‐octene) in‐reactor alloy due to the competition between liquid–liquid phase separation and crystallization

AbstractRelationship between phase separation and crystallization of polypropylene/poly(propylene‐1‐octene) in‐reactor alloy (iPP/PPOc) were studied using optical microscopy, scanning electron microscopy, and differential scanning calorimetry. Optical microscopy was used to monitor nuclei density and spherulite growth rates, providing complementary information about the effect of liquid–liquid phase separation (LLPS) on crystallization behavior. We found that LLPS process had a retardation effect on crystallization rate and had a dominant effect on the final crystalline morphology of the iPP/PPOc alloy. By simply changing the LLPS time or temperature, we could control the size and the distribution of the elastomer phase that dispersed in the iPP spherulites. The growth rate of the spherulites significantly depended on the degree of LLPS. Higher degree of phase separation reduced nuclei density and the growth rate of spherulites. However, it was helpful to the formation of more perfect spherulites. But surprisingly, there seemed to be little variation of crystallinity between the two quenching procedures (i.e., single quench vs. double quench). Overall, the competition between LLPS and crystallization significantly influenced the structural and morphological development of the iPP/PPOc alloy. By controlling the interplay between LLPS and crystallization of iPP/PPOc alloy, it was possible to control the structure and morphology as needed in applications. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

A Homogeneous Solidification Criterion for Undercooled Ni-20at%Pb Hypermonotectic Melts

The kinetic process of liquid-liquid phase separation in the undercooled Ni-20at%Pb hypermonotectic alloy melts was analyzed theoretically. The results showed that liquid-liquid separation could not be inhibited due to smaller nucleation barrier and bigger nucleation rate of Pb-rich droplets. In the course of liquid-liquid phase separation, the volume fraction of Pb-rich droplets was thought as a function of time or temperature. At a certain cooling rate, the volume fraction was mainly controlled by the undercooling of Ni-Pb hypermonotectic melts. Based on the above results, a homogeneous solidification criterion for the undercooled Ni-20at%Pb hypermonotectic alloy melts was developed. Such a criterion predicted that the homogeneous microstructure could be obtained at the undercooling 263K, and the experimental results accorded with the predicting ones on the whole.

Lattice Boltzmann modelling of liquid-liquid phase separation of monotectic alloys

本文建立了一个模拟在弥散相液滴的扩散长大、碰撞凝并和Ostwald熟化等因素的作用下偏晶合金液-液相分离过程的二维格子玻尔兹曼方法 (lattice Boltzmann method, LBM) 模型.该模型结合了Shan-Chen的两相流模型和Qin的介观粒子相互作用势模型的优点，并在LB演化方程中引入了反映相变的源项.应用该模型模拟研究了偏晶合金液-液相分离过程中单液滴的生长、两液滴的合并和多液滴的生长规律.结果表明在两液相区中第二相单个液滴的生长是一个通过扩散从非平衡态到平衡态过渡的过程.两液滴合并

Coupling effects of spinodal decomposition and crystallization on mechanical properties of polyolefin blends

The influences of preferentially occurred liquid–liquid phase separation (LLPS) and following crystallization processes on the mechanical properties of statistical copolymer blends of poly(ethylene-co-hexene) (PEH) and poly(ethylene-co-butene) (PEB) have been investigated in detail through tensile deformation tests with a relatively high extension rate to avoid the effect of interfacial properties of the blends. Crystallinity and lamellar thickness of the samples are estimated by using the wide-angle X-ray diffraction and small-angle X-ray scattering techniques, respectively. The tensile modulus and yield stress are found to increase with LLPS time up to 6h, but decrease afterwards, under the conditions of temperature of 120°C and isothermal crystallization time of 10min. It is considered that the instantaneous tensile properties are substantially largely affected by the much perfect lamellar structures formed during crystallization with a long time prior LLPS step. This finding is further experimentally substantiated by the scanning electron microscope observation. Whereas the strain-hardening modulus described by a simple neo-Hookean relation increases with LLPS time and reaches a plateau after 6h, which can be accounted for by the cooperation effect between amorphous entanglement density, insensitive to LLPS time, and crystallinity redistribution. The similarity of the results observed on the blends experiencing the spinodal decomposition (SD) process supports that the redistribution of crystallizable components contributes to the tensile stress increase, which is primarily controlled by the development of LLPS process. This simple relationship gives us a new insight of what controls the mechanical properties of the phase separated polymer blends and of how we might be able to predict the mechanical properties of as yet unmixed polymer pairs.

Nascent Phase Separation and Crystallization Kinetics of an iPP/PEOc Polymer Alloy Prepared on a Single Multicatalyst Reactor Granule

The crystalline morphology and the liquid−liquid phase separation (LLPS) of an isotactic polypropylene/poly(ethylene-co-octene) (iPP/PEOc) polymer alloy prepared on a single multicatalyst reactor granule were studied by polarized optical microscope, phase contrast optical microscope, and scanning electron microscopy (SEM). We found that the LLPS process had a dominant effect on the final crystalline morphology of the iPP/PEOc polymer alloy. As the LLPS time was increased at 160 °C, more and more elastomer component was dispersed uniformly in the iPP spherulites as droplets, instead of being rejected to the outside and concentrated at the boundaries of the spherulites, when the sample was isothermally crystallized at 140 °C. The morphologies can be explained by the phase separation and crystallization kinetics. By simply changing the LLPS time, we could control the size of the elastomer droplets (for example between 1 and 10 μm) that dispersed in the iPP spherulites. Meanwhile, a special structural feature...

Size and number density of precrystalline aggregates in lysozyme crystallization process

Using dynamic light scattering, we investigated supersaturated aqueous solutions of hen egg white lysozyme. We could observe the formation of aggregates only in solutions, from which crystals grew within a few days. The aggregates were grouped into smaller “units” and larger “clusters.” The units consisted of a few molecules, whereas the clusters grew from about 100 nm to 1 μm. At the beginning of aggregation, the number density of the units decreased, while that of the clusters increased. At this stage, unit-cluster aggregation proceeded. At the next stage, the number density of the units became constant, while that of the clusters began to decrease, which means that the units stopped aggregating and cluster-cluster aggregation started. The aggregation mechanism for the clusters fit well with the diffusion limited cluster aggregation model, but this model alone could not explain that the aggregates separated into two groups, corresponding to units and clusters, and that the units stopped aggregating during the aggregation process. We find that the observed aggregation process has several similarities to the liquid-liquid phase separation process, which occurs metastably in protein solution. Furthermore, using both models for diffusion limited aggregation and the liquid-liquid phase separation together, we could naturally explain the process of the cluster formation.

Liquid-Liquid Phase Separation Process in the Two-Liquid Regions of Binary Borate Melts

Liquid-Liquid Phase Separation Process in the Two-Liquid Regions of Binary Borate Melts

Eeg brain mapping in diagnosis and treatment of SDAT and MID : B. Saletu, E. Paulus, P. Anderer. Department of Psychiatry, School of Medicine, University of Vienna, A-1090 Vienna, Austria.

Liquid-liquid phase separation (LLPS) brings together functionally related proteins through the intrinsic biophysics of proteins in a process that is driven by reducing free energy and maximizing entropy. The process of LLPS allows proteins to form structures, termed membrane-less organelles. These diverse, dynamic organelles are active in a wide range of processes in the nucleus, cytoplasm, mitochondria and synapse, and ranging from bacteria to plants to eukaryotes. RNA and DNA present long chained charged polymers that promote LLPS. Consequently, many RNA binding proteins (RBPs) and DNA binding proteins form membrane-less organelles. However, the highly concentrated phase separated state creates conditions that also promote formation of irreversible protein aggregates. Mutations in RNA and DNA binding proteins that increase the stability of irreversible aggregates also increase the accumulation of irreversible aggregates directly and from membrane-less organelles. Many of the RBPs that exhibit disease-linked mutations carry out cytoplasmic actions through stress granules, which are a pleiotropic type of RNA granule that regulates the translational response to stress. Phosphorylation and oligomerization of tau facilitates its interactions with RBPs and ribosomal proteins, affecting RNA translation; we propose that this is a major reason that tau becomes phosphorylated with stress. Persistent stress leads to the accumulation of irreversible aggregates composed of RBPs or tau, which then cause toxicity and form many of the hallmark pathologies of major neurodegenerative diseases. This pathophysiology ultimately leads to multiple forms of neurodegenerative diseases, the specific type of which reflects the temporal and spatial accumulation of different aggregating proteins.

Structural instability and relaxation in liquid and glassy phases near the fragile liquid limit

We consider two sorts of structural instability of glassy substances, each of which may be released by a relaxation process with its own characteristic relaxation time, and specific kinetic features. The first of these is the instability against relaxation out of the amorphous state into the crystalline state, while the second is the instability against relaxation within the amorphous state itself. The latter may often involve relaxation out of a homogeneous amorphous phase into a two-phase amorphous structure, but we will not specifically consider this liquid-liquid phase separation process here. In most glasses, the former (which is no more than the characteristic nucleation time) is much longer than the latter time. However, there are important classes of glasses, for instance the metallic glasses, in which the former is in fact the shorter time, a fact which is responsible for the inability to observe the glass transition phenomenon in such substances. In this paper we will be considering the relation between these two times and the specific kinetics of each. The nucleation time has been the subject of theoretical developments over a number of decades, and details will be omitted in order to concentrate on experimental studies of this phenomenon. We will described briefly the recently developed DSC techniques for determining the classical time-temperature-transformation curves for a variety of supercooled liquids, and the relation of these to the nucleation curves. The relaxation process within the amorphous state, which can be observed for cases where the nucleation time is relatively long, has a number of features which currently lack a complete explanation. In most cases the relaxation process is non-Arrhenius in its temperature dependence, nonexponential in its time dependence, and nonlinear in its structural state dependence. Some examples taken from glasses at the “fragile” edge of the deduced viscosity-temperature pattern for glassforming liquids are dealt with in detail, and the distinction between shear stress relaxation and thermodynamic stress relaxation is made. The possibility that near T g the latter relaxation time remains Vogel-Fulcher in form with T 0 ≡ T K (the Kauzmann temperature), in contrast with the common observations for (the decoupled) shear relaxation, is raised. Strong support for this notion is found in the current “specific heat spectroscopy” results of Nagel and co-workers. Microscopic relaxation processes, as observed using spectroscopic probes and neutron scattering techniques, are reviewed, and the difference in non-exponentiality from macroscopic relaxation are examined in the light of current theories. Finally, secondary relaxations in ionic and molecular glasses, and their relation to the fastest of all glassy state relaxation processes, the tunnelling modes (TLS), are briefly considered.

Liquid-liquid phase separation and crystallization in binary polymer systems

The crystalline entities formed upon crystallization of semidilute ( ca. 1 to 5%) solutions of polyethylene in poor solvents were examined, mainly by scanning electron microscopy. Due to a liquid-liquid phase separation process, these entities are small ( ca. 1 to 10 μm), more or less spherical globules. Homogeneous and heterogeneous nucleation result in smooth and rough globules, respectively. Different thermal cycles were also investigated. The polymer globules offer a potential means of investigating the mechanism and kinetics of liquid-liquid phase separation. This phenomenom is also offered as an explanation for several unusual crystalline morphologies of polymers reported in the literature.