A framework for morphological evolution vis-à-vis phase transitions in polymer solutions

When a polymer crystallizes from solution, it is well known that the resulting morphology depends on whether any liquid–liquid phase separation (LLPS) has preceded crystallization. In addition to the dense morphology that results when crystallization occurs directly from a homogeneous solution, at least three other distinctly different morphologies are produced if crystallization follows LLPS. Although much work has been reported in this regard, a framework that can relate the path that a process might follow across a phase diagram to the consequent morphology is lacking. We report here the fundamental elements of a simple thermodynamic framework that serves to identify the driving forces that produce these different morphologies. It is based on identification of the nucleating phase, if any, in LLPS and coupling it with the domain in which nucleation of crystallization occurs. The essential elements of the framework for morphological evolution are demonstrated by relating the sequence of phase transitions to the morphology which can result in the crystallized polymer when a polymer solution is cooled from a homogeneous state at a high temperature. Four distinctly different morphologies are shown to evolve, depending on whether crystallization occurs (a) directly from a homogeneous solution (dense); (b) following binodal liquid–liquid phase separation, LLPS, with nucleation of the polymer-rich phase (GMP—globular microporous); (c) following spinodal LLPS (FMP—fibrillar microporous); or (d) following binodal LLPS with nucleation of the solvent-rich phase (CTMP—cell-tunnel microporous). An important implication of the framework is that a predictable sequence of “dense → GMP → FMP → CTMP → dense” morphologies has to arise with increase in overall polymer concentration in such solutions. The framework also serves to identify conditions, such as passage through specific temperature/concentration regions in the phase diagram, that would increase the likelihood of forming mixed or coexisting morphologies. However, it is still necessary to develop appropriate kinetic models to predict sizes of the morphological components within each of the four morphologies. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 1343–1355, 1999     

Phase-Separated Subcellular Compartmentation and Related Human Diseases

In live cells, proteins and nucleic acids can associate together through multivalent interactions, and form relatively isolated phases that undertake designated biological functions and activities. In the past decade, liquid–liquid phase separation (LLPS) has gradually been recognized as a general mechanism for the intracellular organization of biomolecules. LLPS regulates the assembly and composition of dozens of membraneless organelles and condensates in cells. Due to the altered physiological conditions or genetic mutations, phase-separated condensates may undergo aberrant formation, maturation or gelation that contributes to the onset and progression of various diseases, including neurodegenerative disorders and cancers. In this review, we summarize the properties of different membraneless organelles and condensates, and discuss multiple phase separation-regulated biological processes. Based on the dysregulation and mutations of several key regulatory proteins and signaling pathways, we also exemplify how aberrantly regulated LLPS may contribute to human diseases.     

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

Living cells have long been a source of inspiration for chemists. Their capacity of performing complex tasks relies on the spatiotemporal coordination of matter and energy fluxes. Recent years have witnessed growing interest in the bottom-up construction of cell-like models capable of reproducing aspects of such dynamic organisation. Liquid–liquid phase-separation (LLPS) processes in water are increasingly recognised as representing a viable compartmentalisation strategy through which to produce dynamic synthetic cells. Herein, we highlight examples of the dynamic properties of LLPS used to assemble synthetic cells, including their biocatalytic activity, reversible condensation and dissolution, growth and division, and recent directions towards the design of higher-order structures and behaviour.     

Integration of Data from Liquid–Liquid Phase Separation Databases Highlights Concentration and Dosage Sensitivity of LLPS Drivers

Liquid–liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles, representing functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integrating the data on LLPS-associated proteins from dedicated databases revealed only modest agreement between them and yielded a high-confidence dataset of 89 human LLPS drivers. Analysis of the supporting evidence for our dataset uncovered a systematic and potentially concerning difference between protein concentrations used in a good fraction of the in vitro LLPS experiments, a key parameter that governs the phase behavior, and the proteomics-derived cellular abundance levels of the corresponding proteins. Closer scrutiny of the underlying experimental data enabled us to offer a sound rationale for this systematic difference, which draws on our current understanding of the cellular organization of the proteome and the LLPS process. In support of this rationale, we find that genes coding for our human LLPS drivers tend to be dosage-sensitive, suggesting that their cellular availability is tightly regulated to preserve their functional role in direct or indirect relation to condensate formation. Our analysis offers guideposts for increasing agreement between in vitro and in vivo studies, probing the roles of proteins in LLPS.     

BIAPSS: A Comprehensive Physicochemical Analyzer of Proteins Undergoing Liquid–Liquid Phase Separation

The liquid–liquid phase separation (LLPS) of biomolecules is a phenomenon which is nowadays recognized as the driving force for the biogenesis of numerous functional membraneless organelles and cellular bodies. The interplay between the protein primary sequence and phase separation remains poorly understood, despite intensive research. To uncover the sequence-encoded signals of protein capable of undergoing LLPS, we developed a novel web platform named BIAPSS (Bioinformatics Analysis of LLPS Sequences). This web server provides on-the-fly analysis, visualization, and interpretation of the physicochemical and structural features for the superset of curated LLPS proteins.     

Liquid–Liquid Phase Separation in the Presence of Macromolecular Crowding and State-dependent Kinetics

Biomolecular condensates formed via liquid–liquid phase separation (LLPS) are increasingly being shown to play major roles in cellular self-organization dynamics in health and disease. It is well established that macromolecular crowding has a profound impact on protein interactions, particularly those that lead to LLPS. Although synthetic crowding agents are used during in vitro LLPS experiments, they are considerably different from the highly crowded nucleo-/cytoplasm and the effects of in vivo crowding remain poorly understood. In this work, we applied computational modeling to investigate the effects of macromolecular crowding on LLPS. To include biologically relevant LLPS dynamics, we extended the conventional Cahn–Hilliard model for phase separation by coupling it to experimentally derived macromolecular crowding dynamics and state-dependent reaction kinetics. Through extensive field-theoretic computer simulations, we show that the inclusion of macromolecular crowding results in late-stage coarsening and the stabilization of relatively smaller condensates. At a high crowding concentration, there is an accelerated growth and late-stage arrest of droplet formation, effectively resulting in anomalous labyrinthine morphologies akin to protein gelation observed in experiments. These results not only elucidate the crowder effects observed in experiments, but also highlight the importance of including state-dependent kinetics in LLPS models, and may help in designing further experiments to probe the intricate roles played by LLPS in self-organization dynamics of cells.     

The Patterning and Proportion of Charged Residues in the Arginine-Rich Mixed-Charge Domain Determine the Membrane-Less Organelle Targeted by the Protein

Membrane-less organelles (MLOs) are formed by biomolecular liquid–liquid phase separation (LLPS). Proteins with charged low-complexity domains (LCDs) are prone to phase separation and localize to MLOs, but the mechanism underlying the distributions of such proteins to specific MLOs remains poorly understood. Recently, proteins with Arg-enriched mixed-charge domains (R-MCDs), primarily composed of R and Asp (D), were found to accumulate in nuclear speckles via LLPS. However, the process by which R-MCDs selectively incorporate into nuclear speckles is unknown. Here, we demonstrate that the patterning of charged amino acids and net charge determines the targeting of specific MLOs, including nuclear speckles and the nucleolus, by proteins. The redistribution of R and D residues from an alternately sequenced pattern to uneven blocky sequences caused a shift in R-MCD distribution from nuclear speckles to the nucleolus. In addition, the incorporation of basic residues in the R-MCDs promoted their localization to the MLOs and their apparent accumulation in the nucleolus. The R-MCD peptide with alternating amino acids did not undergo LLPS, whereas the blocky R-MCD peptide underwent LLPS with affinity to RNA, acidic poly-Glu, and the acidic nucleolar protein nucleophosmin, suggesting that the clustering of R residues helps avoid their neutralization by D residues and eventually induces R-MCD migration to the nucleolus. Therefore, the distribution of proteins to nuclear speckles requires the proximal positioning of D and R for the mutual neutralization of their charges.     

Effects of liquid–liquid phase separation on crystallization kinetics and morphology of isotactic polypropylene/poly (ethylene-co-octene) in-reactor alloy

In this work, we investigated the effects of liquid–liquid phase separation (LLPS) on the crystallization kinetics and morphology of isotactic polypropylene/poly (ethylene-co-octene) (iPP/PEOc) in-reactor alloy with polarized optical microscopy (POM), differential scanning calorimeter (DSC), scanning electron microscopy (SEM), wide angle X-ray scattering (WAXS) and small angle X-ray scattering (SAXS) methods. Based on crystallization kinetics analysis by Avrami equation, we found that the overall crystallization rate was almost independent on LLPS time, whereas was strongly dependent on crystallization temperature. However, by combination with POM, we found that the LLPS played two opposite roles on the overall crystallization rate, i.e. the nucleation rate decreased and the spherulite growth rate increased as increasing LLPS time. It is due to the nucleation rate was dominated by fluctuation-assisted nucleation mechanism and the growth rate was dominated by diffusion-controlled growth. Furthermore, the spherulite size and PEOc domain size of iPP/PEOc in-reactor alloy were significantly dependent on LLPS time; however, the crystallinity was almost not dependent on LLPS time.     

A method to crystallize substances that oil out

A new approach to crystallize oily substances is described. The tendency for liquid–liquid phase separation (LLPS) is reduced by decreasing the kinetics of self-association via the formation of an intermediate amorphous network. The path to initial crystal formation followed a sequence of first freeze-drying an emulsion of solute in the solvent system followed by suspending the dried solid in water to obtain a hydrated crystalline form. This new procedure was applied successfully to a pharmaceutical organic substance that was previously isolated only as a viscous oil. Once isolated, crystals of the drug were utilized as seeds to allow the successful transformation of an emulsion of the substance into a suspension of crystalline drug solid thus avoiding the freeze-drying step. The isolated crystalline solid retained its physical and chemical purity at room temperature for at least 3 months.     

Tau N-Terminal Inserts Regulate Tau Liquid-Liquid Phase Separation and Condensates Maturation in a Neuronal Cell Model

The microtubule-associated protein tau can undergo liquid–liquid phase separation (LLPS) to form membraneless condensates in neurons, yet the underlying molecular mechanisms and functions of tau LLPS and tau droplets remain to be elucidated. The human brain contains mainly 6 tau isoforms with different numbers of microtubule-binding repeats (3R, 4R) and N-terminal inserts (0N, 1N, 2N). However, little is known about the role of N-terminal inserts. Here we observed the dynamics of three tau isoforms with different N-terminal inserts in live neuronal cell line HT22. We validated tau LLPS in cytoplasm and found that 2N-tau forms liquid-like, hollow-shell droplets. Tau condensates became smaller in 1N-tau comparing with 2N-tau, while no obvious tau accumulated dots were shown in 0N-tau. The absence of N-terminal inserts significantly affected condensate colocalization of tau and p62. The results reveal insights into the tau LLPS assembly mechanism and functional effects of N-terminal inserts in tau.     

Effects of liquid–liquid phase separation on crystallization of poly(ethylene glycol) in blends with isotactic poly(methyl methacrylate)

To analyze the interplay between crystallization and liquid–liquid phase separation (LLPS), isothermal crystallization behavior of poly(ethylene glycol) (PEG) in blends with isotactic poly(methyl methacrylate) (i-PMMA) was investigated by differential scanning calorimetry (DSC). The blend system had an upper critical solution temperature (UCST) type phase diagram. When the crystallization occurred simultaneously with LLPS, the overall crystallization rate was enhanced at high crystallization temperatures T_c, relatively compared with that of neat PEG. This behavior was interpreted by the combination of the effects of spinodal quench depth ?T_s and usual supercooling degree ?T_c, according to the theory of Mitra and Muthukumar, namely, the crystallization rate is enhanced by the concentration fluctuation-assisted nucleation at high T_c. In the crystallization after LLPS proceeded, on the other hand, the overall crystallization rate was slow and less dependent on the blend composition. In addition, it was revealed by small-angle X-ray scattering measurements that amorphous i-PMMA was excluded from the interlamellar region of PEG crystals in SQ as well as WQ.     

Determination of Aqueous Two-Phase System Binodals and Tie-Lines by Electrowetting-on-Dielectric Droplet Manipulation

Handling the aqueous two-phase systems (ATPSs) formed by liquid–liquid phase separation (LLPS) relies on the accurate construction of binodal curves and tie-lines, which delineate the polymer concentrations required for phase separation and depict the properties of the resulting phases, respectively. Various techniques to determine the binodal curves and tie-lines of ATPSs exist, but most rely on manually pipetting relatively large volumes of fluids in a slow and tedious manner. We describe a method to determine ATPS binodals and tie-lines that overcomes these disadvantages: microscale droplet manipulation by electrowetting-on-dielectric (EWOD). EWOD enables automated handling of droplets in an optically transparent platform that allows for in situ droplet observation. Separated phases are clearly visible, and the volumes of each phase are readily determined. Additionally, in considering the molecular crowding present in living cells, this work examines the role of a macromolecule in prompting LLPS. These results show that EWOD-driven droplet manipulation effectively interrogates the phase dynamics of ATPSs and macromolecular crowding in LLPS.     

Seeing Keratinocyte Proteins through the Looking Glass of Intrinsic Disorder

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid–liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.     

正渗透水肥一体化灌溉中化肥驱动液的筛选

选择尿素溶液、磷酸二氢钾溶液、硝酸钾溶液、硝酸铵溶液4种化肥溶液作为汲取液,重金属镉溶液作为原料液,CTA膜作为正渗透膜,进行化肥正渗透驱动液的筛选试验。以NaCl-纯水为对照组,设置3次重复试验。定期观测记录质量、EC值、TDS值、体积、流量、进出压力值、温度及pH等变化,计算正向水通量、反向盐通量及正向截留率,并测定溶液中重金属及氮磷钾含量变化。结果表明:性能最佳的化肥汲取液是KH_2PO_4,其水通量高而且溶质返混运动营养损失最低。     

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 6 h, but decrease afterwards, under the conditions of temperature of 120 °C and isothermal crystallization time of 10 min. 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 6 h, 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.     

Residual stress versus microstructural effects on the strength and toughness of phase-separated PbO–B2O3–Al2O3 glasses

A few authors have reasonably proposed that liquid–liquid phase-separated (LLPS) glasses could show improved fracture strength, S_f, and toughness, K_Ic, as the second phase could provide a barrier to crack propagation via deflection, bowing, trapping, or bridging. Due to the associated tensile or compressive residual stresses, the second phase could also act as a toughening or a weakening mechanism. In this work, we investigated five glasses of the PbO–B₂O₃–Al₂O₃ system spanning across the miscibility gap: Four of them undergo LLPS—three are binodal (two B₂O₃-rich and one PbO-rich) and one is spinodal—and one does not show LLPS (composition outside the miscibility gap). Their compositions were designed in such a way that the amorphous particles are under compressive residual stresses in some and under tensile residual stresses in others. The following mechanical properties were determined: the Vickers hardness, ball on three balls (B3B) strength, and toughness, K_Ic-SEVNB (single-edge V-notch beam [SEVNB]). The microstructures and compositions were analyzed using scanning electron microscopy with energy-dispersive X-ray spectrometry. The spinodal glass showed, by far, the best mechanical properties. Its K_Ic-SEVNB = 1.6 ± 0.1 MPa m^1/2, which embodies an increase of almost 50% over the B₂O₃-rich binodal composition, and 90% considering the PbO-rich binodal composition. Moreover, its fracture strength, S_f = 166 ± 7 MPa, is one of the highest ones ever reported for an LLPS glass. Fracture analyses evidenced that the spinodal composition exhibited the lowest net stress at the fracture point. Moreover, calculations indicate that the internal residual stress level is the lowest in the spinodal glass. The overall results indicate that the microstructural effect of the spinodal glass is the most significant factor for its superior mechanical properties. This work corroborates the idea that LLPS provides a feasible and stimulating solution to improve the mechanical properties of glasses.     

Crystallization and phase separation kinetics in blends of linear low-density polyethylene copolymers

We have systematically studied the crystallization and liquid-liquid phase separation (LLPS) kinetics in statistical copolymer blends of poly(ethylene-co-hexene) (PEH) and poly(ethylene-co-butene) (PEB) using primarily optical microscopy. The PEH/PEB blends exhibit upper critical solution temperature (UCST) in the melt and crystallization temperature below the UCST. The time evolution of the characteristic morphology for both crystallization and LLPS is recorded for blends at various compositions and following a quench from initial homogenous melts at high temperature to various lower temperatures. The crystallization kinetics is measured as the linear growth rate of the super structural crystals, whereas the LLPS kinetics is measured as the linear growth rate of the characteristic length of the late-stage spinodal decomposition. The composition dependence crystallization kinetics, G, shows very different characteristics at low and high isothermal crystallization temperature. Below 116 °C, G decreases with increasing PEB content in the blend, implying primarily the composition effect on materials transport; whereas at above 116 °C, G shows a minimum at about the critical composition for LLPS, implying the influence of the LLPS. On the other hand, LLPS kinetics at 130 °C is relatively invariant at different compositions in the two-phase regime. The length scale at which domains are kinetically pinned, however, depends strongly on the composition. In a blend near critical composition, a kinetics crossover is shown to separate the crystallization dominant and phase separation dominant morphology as isothermal temperature increases.     

Kinetic and thermodynamic study of the transfer of anionic polyamidoamine dendrimers across two immiscible liquids

The kinetics and thermodynamics for the phase transfer of carboxyl-terminated polyamidoamine (PAMAM) dendrimers across the water/dichloroethane interface were analyzed by cyclic voltammetry and electrochemical impedance spectroscopy. A three phase junction was employed by inserting a cylindrical gold electrode through the liquid–liquid interface. The reversible redox species decamethylferrocene (DMFc) was used in the organic phase in order to promote dendrimer transfer. It was found that the electrochemical behaviour of DMFc at the gold/dichloroethane interface depends on the generation and concentration of the dendrimer species in the aqueous phase. In addition, it was observed that the electrochemically driven transfer of these macromolecules corresponds to a quasi-reversible process. The data obtained from thermodynamic studies indicate that dendrimers are transferred between the two phases under study by an entropy controlled process.