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

Biomimetics has inspired the development of various advanced technologies and novel materials, in which mussel adhesion has been regarded as a practical strategy to investigate the underwater adhesives with robust wet adhesion capacities attributed to the abundant post-translationally modified amino acid l-3,4-dihydroxyphenylalanine (Dopa) in mussel foot proteins (Mfps). Moreover, recent studies also reveal that the secretion of Mfps is conducted with a concentrated fluidic phase regulated by a liquid-liquid phase separation (LLPS) process. With the aim of exploring the phase behaviors of mussel plaque proteins and their Dopa-involved mechanisms, two mussel plaque proteins of different mussel species were chosen to gain insight into the natural secretion process and to have a deep understanding of the interaction information to the molecular level. The first study focuses on the mussel adhesive protein Pvfp-5 of the Asian green mussels with the attempt to examine the LLPS behaviors of mussel plaque proteins and figure out the decisive interactions within proteins at the molecular level. Based on the fact that only parts of Tyr residues are post-modified into Dopa residues within native Pvfp-5β, two Pvfp-5 variants, Pvfp-5-Tyr and Pvfp-5-Dopa, were expressed to investigate the exact roles of Dopa residues. Both Pvfp-5 variants exhibited comparable adhesion forces, which indicated the contribution of Dopa residues to form interfacial hydrogen bonds was negligible. Nonetheless, Pvfp-5-Dopa showed different phase behaviors compared to Pvfp-5-Tyr. Pvfp-5-Dopa could form coacervate microdroplets under seawater conditions, while Pvfp-5-Tyr could only form gel-like precipitates. Further characterization results using periodate oxidation, NMR and QC calculation found that Dopa residues could form a transient triangular hydrogen bonding network which is specific for Pvfp-5-Dopa. Pvfp-5-Dopa tended to form microdroplets through transient hydrogen bonds and π-π stacking, while Pvfp-5-Tyr preferred to form insoluble precipitates by the strong cation-π interactions. The second study investigated the GK-16 peptide derived from mussel adhesive protein Mfp-5 of the California blue mussels to identify the LLPS behaviors and to understand the molecular interactions involved in the phase separation process. Repeated three times within the Mfp-5 sequence, enzymatically modified GK-16* could undergo LLPS triggered by certain ionic strength at pH 3, in which the mechanical properties of coacervate droplets would be adjusted by salt concentration and urea concentration. CD and FTIR spectra also characterized the formation of localized β-sheet structures with the addition of salts. To further understand the specific roles of amino acids to the molecular level, several GK-16 variants were designed with a series of amino acid substituents. Observed from the phase diagrams, CD spectra and MD simulations, Dopa involved hydrogen bonds formed with a triangle configuration dominate within the LLPS process, followed by the side chain-main chain hydrogen bonds among Dopa residues and Gly residues. The thesis highlights the significance of Dopa residues, which could not only promote the wet adhesion by forming interfacial hydrogen bonds, but also facilitate the phase separation of mussel plaque proteins through intermolecular hydrogen bonds. By performing such fundamental studies, the natural mussel secretion procedure could be further decrypted and the detailed molecular interactions taking function could be acknowledged. Such results could provide sequence models according to their intrinsic properties to further design mussel-inspired materials with tailored mechanical properties. Especially for wet adhesives, the studies emphasize the multifunctional Dopa residues, which could construct the coacervate-based platform to promote the adhesion efficiency of Dopa residues.

While the liquid-liquid phase separation (LLPS) process in proteins has been studied in great detail, it has not been widely explored how the associated protein hydration changes during the process and how crucial its role is in the process itself. In this contribution, we experimentally explore the alteration of lysozyme hydration during its LLPS process using attenuated total reflection (ATR)-FTIR spectroscopy in the THz frequency region (1.5-21 THz). Additionally, we explore the role of excipients (l-arginine, sucrose, bovine albumin (BSA), and ubiquitin (Ubi)) in regulating the process and found that, while sucrose stabilizes the LLPS, BSA inhibits it. The effect of Arg in the LLPS is subtle, and that of Ubi is concentration dependent. We made a detailed analysis of the hydration profile of Lys in the presence of these excipients and observe that a change in hydration in terms of H-bond making/breaking is a definite signature regulating the process.

The role of macromolecule-macromolecule and macromolecule-H2O interactions and the resulting perturbation of the H-bonded network of H2O in the liquid-liquid phase separation (LLPS) process of biopolymers are well-known. However, the potential of the hydrated state of supramolecular structures (non-covalent analogs of macromolecules) of synthetic molecules is not widely recognized for playing a similar role in the LLPS process. Herein, LLPS occurred during the co-assembly of hydrated supramolecular vesicles (bolaamphiphile, BA1) with a net positive charge (zeta potential, ζ = +60 ± 2mV) and a dianionic chiral molecule (disodium l-[+]-tartrate) is reported. As inferred from cryo-transmission electron microscopy (TEM), the LLPS-formed droplets serve as the nucleation precursors, dictating the structure and properties of the co-assembly. The co-assembled structure formed by LLPS effectively integrates the counter anion's asymmetry, resulting in the formation of ultrathin free-standing, chiral 2D crystalline sheets. The significance of the hydrated state of supramolecular structures in influencing LLPS is unraveled through studies extended to a less hydrated supramolecular structure of a comparable system (BA2). The role of LLPS in modulating the hydrophobic interaction in water paves the way for the creation of advanced functional materials in an aqueous environment.

There is growing evidence showing that many critical biological processes are driven by biomolecule condensates through liquid-liquid phase separation (LLPS). Although the qualitative observation and description of LLPS have been well documented, quantitative simulations of the time-dependent progression of LLPS in live cells are generally lacking. In this work, we build a stochastic Monte Carlo model to simulate the dynamic LLPS process during the formation of bacterial aggresomes. We demonstrate that the size distribution of the protein condensates evolves from an exponential-like to a bimodal-like pattern, and the number of condensates increases at the beginning and then decreases after reaching a maximum. Incorporating diffusion and collision, our simplified model recapitulates the two-step LLPS process in which many smaller condensates are formed in the first step and then merged into a few larger ones. We further reveal that the condensation speed, which can be defined by the condensates formed in unit time during the first step, is mainly determined by both the collision energy barrier and the initial protein density, while the number of condensates at the equilibrium is mainly associated with the dissociation energy barrier. Moreover, the LLPS process is not sensitive to temperature changes ranging around physiological conditions. Additionally, we consider the effect of the nucleation energy barrier on LLPS. We find that a higher nucleation energy barrier brings a slower condensation speed. Overall, we simulate the spatiotemporal dynamics of the LLPS process and provide qualitative guidance for understanding the dynamics of LLPS in bacterial cells, which can faithfully recapitulate experimental observations and facilitate the design of future experimental tests.

We compare the process of Liquid-Liquid Phase Separation (LLPS) of flexible macromolecular solutions, with the Liquid-Liquid Crystalline Phase Separation (LLCPS) of semiflexible polymers and rigid filamentous colloids, which involves the formation of a liquid phase that possesses a directional alignment. Although the observed phase separation follows a similar dynamic path, namely nucleation and growth or spinodal decomposition separating two phases of dilute and concentrated compositions, the underlying physics that defines the theoretical framework of LLCPS is completely different from the one of LLPS. We review the main theories that describe the phase separation processes and relying on thermodynamics and dynamical arguments, we highlight the differences and analogies between these two phase separation phenomena, attempting to clarify the inner mechanisms that regulate those two processes. A particular focus is given to metastable phases, as these intermediate states represent a key element in understanding how phase separation works.

Mesoscale Liquid Model of Chromatin Recapitulates Nuclear Order of Eukaryotes

The limited options for treating patients with drug-resistant cancers have emphasized the need to identify alternative treatment targets. Tumor cells have large super-enhancers (SEs) in the vicinity of important oncogenes for activation. The physical process of liquid-liquid phase separation (LLPS) contributes to the assembly of several membrane-less organelles in mammalian cells. Intrinsically disordered regions (IDRs) of proteins induce LLPS formation by developing condensates. It was discovered that key transcription factors (TFs) undergo LLPS in SEs. In addition, TFs play critical roles in the epigenetic and genetic regulation of cancer progression. Recently, we revealed the essential role of disease-specific TF collaboration changes in advanced prostate cancer (PC). OCT4 confers epigenetic changes by promoting complex formation with TFs, such as Forkhead box protein A1 (FOXA1), androgen receptor (AR) and Nuclear respiratory factor 1 (NRF1), inducing PC progression. It was demonstrated that TF collaboration through LLPS underlying transcriptional activation contributes to cancer aggressiveness and drug resistance. Moreover, the disruption of TF-mediated LLPS inhibited treatment-resistant PC tumor growth. Therefore, we propose that repression of TF collaborations involved in the LLPS of SEs could be a promising strategy for advanced cancer therapy. In this article, we summarize recent evidence highlighting the formation of LLPS on enhancers as a potent therapeutic target in advanced cancers.

Maintaining homeostasis is a fundamental characteristic of living systems. In cells, this is contributed to by the assembly of biochemically distinct organelles, many of which are not membrane bound but form by the physical process of liquid-liquid phase separation (LLPS). By analogy with LLPS in binary solutions, cellular LLPS was hypothesized to contribute to homeostasis by facilitating "concentration buffering," which renders the local protein concentration within the organelle robust to global variations in the average cellular concentration (e.g., due to expression noise). Interestingly, concentration buffering was experimentally measured in vivo in a simple organelle with a single solute, while it was observed not to be obeyed in one with several solutes. Here, we formulate theoretically and solve analytically a physical model of LLPS in a ternary solution of two solutes (ϕ and ψ) that interact both homotypically (ϕ-ϕ attractions) and heterotypically (ϕ-ψ attractions). Our physical theory predicts how the coexisting concentrations in LLPS are related to expression noise and thus, generalizes the concept of concentration buffering to multicomponent systems. This allows us to reconcile the seemingly contradictory experimental observations. Furthermore, we predict that incremental changes of the homotypic and heterotypic interactions among the molecules that undergo LLPS, such as those that are caused by mutations in the genes encoding the proteins, may increase the efficiency of concentration buffering of a given system. Thus, we hypothesize that evolution may optimize concentration buffering as an efficient mechanism to maintain LLPS homeostasis and suggest experimental approaches to test this in different systems.

Cofactor-dependent structural transition of SOD1 modulates its ability to undergo liquid-liquid phase separation.

Nanoscale materials, when compared to their bulk components, possess unique properties. In particular, shifts in phase transitions can occur for submicrometer particles. For instance, small particles do not undergo the process of liquid-liquid phase separation (LLPS). LLPS has applications in emulsions such as Janus particles, controllable morphology to create drug-rich phases during drug delivery, and is often observed in atmospheric aqueous aerosol particles. In atmospheric particles, LLPS is tracked as a function of particle water activity, which is equivalent to the relative humidity (RH) at equilibrium. We probed three organic/inorganic aerosol systems in the range of RH over which phase separation occurs (SRH). Our findings indicate that SRH for submicrometer aerosol particles is lower than for micrometer-sized droplets. These findings show that it may be necessary to update the representation of phase transitions in aerosol particles in climate models. The vast majority of organic/inorganic aerosol particles have submicrometer diameters, and a decrease in SRH for submicrometer particles indicates that the current estimation of phase-separated aerosols may be overestimated. Furthermore, understanding the properties of LLPS at the nanoscale can provide key parameters to describe these systems and may lead to better control of phase separation in submicrometer particles.

The physical process of liquid-liquid phase separation (LLPS), where the drive to minimize global free energy causes a solution to demix into dense and light phases, plays many important roles in biology. It is implicated in the formation of so-called "membraneless organelles" such as nucleoli, nuclear speckles, promyelocytic leukemia protein bodies, P bodies, and stress granules along with the formation of biomolecular condensates involved in transcription, signaling, and transport. Quantitative studies of LLPS in vivo are complicated by the out-of-equilibrium, multicomponent cellular environment. While in vitro experiments with purified biomolecules are inherently an oversimplification of the cellular milieu, they allow probing of the rich physical chemistry underlying phase separation. Critically, with the application of suitable models, the thermodynamics of equilibrium LLPS can inform on the nature of the intermolecular interactions that mediate it. These same interactions are likely to exist in out-of-equilibrium condensates within living cells. Phase diagrams map the coexistence points between dense and light phases and quantitatively describe LLPS by mapping the local minima of free energy versus biomolecule concentration. Here, we describe a light scattering method that allows one to measure coexistence points around a high-temperature critical region using sample volumes as low as 10μl.

The DEAD-box RNA helicase DDX3X is a multifunctional protein involved in RNA metabolism and stress responses. In this study, we investigated the role of RG/RGG motifs in the dynamic process of liquid-liquid phase separation (LLPS) of DDX3X using cell-free assays and explored their potential link to cancer development through bioinformatic analysis. Our results demonstrate that the number, location, and composition of RG/RGG motifs significantly influence the ability of DDX3X to undergo phase separation and form self-aggregates. Mutational analysis revealed that the spacing between RG/RGG motifs and the number of glycine residues within each motif are critical factors in determining the extent of phase separation. Furthermore, we found that DDX3X is co-expressed with the stress granule protein G3BP1 in several cancer types and can undergo co-phase separation with G3BP1 in a cell-free system, suggesting a potential functional interaction between these proteins in phase-separated structures. DDX3X and G3BP1 may interact through their RG/RGG domains and subsequently exert important cellular functions under stress situation. Collectively, our findings provide novel insights into the role of RG/RGG motifs in modulating DDX3X phase separation and their potential contribution to cancer pathogenesis.

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.

RNA viruses hijack host cell functions to promote viral replication. During infection, viral components enter membrane-bound and membrane-less cellular organelles, including the phase-separated nucleolus, where they perturb nucleolar proteins to aid virus replication. Recently, viruses have been shown to utilize the process of liquid-liquid phase separation (LLPS) to package their genome and promote viral RNA synthesis. The viral nucleocapsid (N) protein is critically involved in several aspects of virus replication. Importantly, the nucleolar phosphoprotein, Nucleophosmin (NPM1), with roles in ribosome biogenesis, was reported to interact with the N protein of several different RNA viruses and facilitate viral replication. However, the impact of this interaction and SARS CoV-2 N protein (NCoV-2) nucleolar localization on ribosome biogenesis and viral life cycle remains poorly understood. Our in vitro studies show that NPM1 undergoes heterotypic phase separation with NCoV-2, causing the formation of liquid-like droplets or condensates with restricted NPM1 mobility. Sequence analysis of NCoV-2 revealed enrichment of positively charged (Arg and Lys) amino acids, an important “sequence feature” that governs nuclear and nucleolar localization. Importantly, our cellular studies reveal the colocalization of NCoV-2 with NPM1 in the nucleoli of Vero E6 cells. These results suggest that there is a direct interaction between NPM1 and NCoV-2. Strikingly, NCoV-2 displaces NPM1 from reconstituted, granular component-like condensates and from nucleoli. Based on our collective findings, we propose that displacement of regulatory proteins (e.g., NPM1) from nucleoli by NCoV-2 through interactions of its Arg residues with ribosomal RNA (rRNA), may promote protein synthesis and the viral life cycle. These mechanistic insights will provide new avenues for the development of novel antiviral therapies for inhibition of nucleolar localization of NCoV-2.

Biomolecular condensates (BCs) are crucial membraneless organelles formed through the process of liquid-liquid phase separation (LLPS) involving proteins and nucleic acids. These LLPS processes are tightly linked with essential cellular activities. Stress granules (SGs), functioning as cytoplasmic BCs, play indispensable roles in maintaining cellular homeostasis and are implicated in diseases like cancers and neurodegenerative disorders. However, devices that can regulate SG LLPS are lacking. Herein, a triangular prism-shaped DNA nanostructure containing polythymidine (ΔDNA(polyT)) is presented as a nanodevice to investigate the LLPS process of in vitro reconstructed SGs (rSGs), a mixture of marker protein G3BP1 and total RNAs. Our observations reveal that the concentration threshold required for rSG LLPS decreases upon addition of ΔDNA(polyT), suggesting an enhancement in SG LLPS efficiency. It is speculated that ΔDNA(polyT) can concentrate mRNAs onto its surface via polyT hybridization with poly-adenosine sequences (polyA) in mRNAs. This alteration in the spatial distribution of mRNAs subsequently affects the multivalency interactions between G3BP1 and mRNAs. Furthermore, ΔDNA(polyT) exhibits excellent colocalization with cytoplasmic SGs under stressed conditions. This DNA-based nanodevice presents a new artificial approach for the targeted regulation of BC LLPS and holds promise for future studies focusing on BCs.