Phase-separated Droplets Research Articles

Complex Suspended Janus Droplets Constructed through Solvent Evaporation-Induced Phase Separation at the Air-Liquid Interface.

Phase separation technology has attracted extensive scientific interest because of its intriguing structure changes during the phase separation process. Phase separation inside emulsion droplets in continuous surroundings has been well studied in recent years. Many investigations have also been conducted to study the droplet phase separation phenomena in noncontinuous surroundings. However, studies on the phase separation phenomena and the spreading behavior of suspended droplets at the air-liquid interface were rarely reported. In this study, PEGDA-glycerol suspended Janus droplets with a patchy structure were produced by utilizing solvent evaporation-induced droplet phase separation at the air-liquid interface. By altering the glycerol/PEGDA volume ratio, the initial proportion of ethanol, and the concentration of surfactants, suspended droplets with different morphologies can be achieved, which include filbert-shaped droplets (FSDs), half lotus seedpod single-phase Janus droplets (HLSDs), lotus seedpod single-phase Janus droplets (LSDs), lotus seedpod-shaped droplets (LSSDs), multiple-bulge droplets (MBDs), and half gourd-shaped droplets (HGSDs). A patchy structure was generated at the air-droplet interface, which was attributed to the Marangoni stresses induced by nonuniform evaporation. Furthermore, a modified spreading coefficient theory was constructed and verified to illustrate the phase separation at the air-droplet interface, which was the first research to predict the phase separation phenomena at the air-liquid interface via spreading coefficients theory. Moreover, we studied the factors that led to the droplets being able to float by designing the combined parameters, including three interfacial tensions and the equilibrium contact angles. Therefore, a simple and versatile strategy for creating suspended Janus droplets has been developed for the first time, which holds significant potential in a variety of applications for material synthesis, such as the electrospinning solution behavior when sprayed from the nozzle into the air.

Tau liquid-liquid phase separation: At the crossroads of tau physiology and tauopathy.

Abnormal deposition of tau in neurons is a hallmark of Alzheimer's disease and several other neurodegenerative disorders. In the past decades, extensive efforts have been made to explore the mechanistic pathways underlying the development of tauopathies. Recently, the discovery of tau droplet formation by liquid-liquid phase separation (LLPS) has received a great deal of attention. It has been reported that tau condensates have a biological role in promoting and stabilizing microtubule (MT) assembly. Furthermore, it has been hypothesized that the transition of phase-separated tau droplets to a gel-like state and then to fibrils is associated with the pathology of neurodegenerative diseases. In this review, we outline LLPS, the structural disorder that facilitates tau droplet formation, the effects of posttranslational modification of tau on condensate formation, the physiological function of tau droplets, the pathways from droplet to toxic fibrils, and the therapeutic strategies for tauopathies that might evolve from toxic droplets. We expect a deeper understanding of tau LLPS will provide additional insights into tau physiology and tauopathies.

Liquid-Liquid Phase Separation of an Intrinsically Disordered Region of a Germ Cell-Specific Protein Modulates the Stability and Conformational Exchange Rate of SH3 Domain.

The phenomenon of liquid-liquid phase separation is found in numerous biological processes. The biomolecules enveloped in the phase-separated droplets experience an obviously different environment from those in cellular or aqueous solution. Herein, we quantitatively characterized the thermodynamics and exchange kinetics of a model protein SH3 domain in the condensed phase of an intrinsically disordered region of a germ cell-specific protein DDX4N1 by using 19F-NMR spectroscopy. The stability and exchange rate of the SH3 domain are different from those in buffer and macromolecular crowding conditions. Our finding indicates that the local transient ordered microstructure and heterogeneity in the condensates play significant roles in modulating the biophysical properties of the enveloped proteins, and this finding may be essential to further our understanding how phase separation regulates the function of proteins in cells.

Hijacking of transcriptional condensates by endogenous retroviruses

Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA polymerase II and the Mediator coactivator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and noncoding nascent RNAs, including those produced by retrotransposons, may facilitate ‘hijacking’ of transcriptional condensates in various developmental and disease contexts.

Liquid-Liquid Phase Separation Bridges Physics, Chemistry, and Biology.

There is a fundamental gap between the inherent complexity of biology and the simplicity that physicists and chemists often seek. In this Perspective, we reason that liquid-liquid phase separation (LLPS) could be utilized to (partially) fill this gap and to bridge different disciplines because LLPS can produce condensed droplets with simplicity and complexity at the same time. Specifically, the droplets are often compositionally simple (made of, for example, proteins and polyelectrolytes) and structurally uniform (not so different from an oil droplet in water). Contrary to this simplicity is their functional complexity─the droplets can perform various physiological activities with subcellular precision. This spatiotemporal precision further stimulates an ongoing endeavor in the synthetic realm to develop regulatory strategies that may ultimately match or even surpass their biological counterparts. We envision the phase-separated droplets to open a window of simplicity for us to peek into the complexity of biology, and we foresee that joined forces across different disciplines would substantially advance our understanding of LLPS in biotic and abiotic contexts.

A unified statistical potential reveals that amino acid stickiness governs nonspecific recruitment of client proteins into condensates.

Membraneless organelles are cellular compartments that form by liquid–liquid phase separation of one or more components. Other molecules, such as proteins and nucleic acids, will distribute between the cytoplasm and the liquid compartment in accordance with the thermodynamic drive to lower the free energy of the system. The resulting distribution colocalizes molecular species to carry out a diversity of functions. Two factors could drive this partitioning: the difference in solvation between the dilute versus dense phase and intermolecular interactions between the client and scaffold proteins. Here, we develop a set of knowledge‐based potentials that allow for the direct comparison between stickiness, which is dominated by desolvation energy, and pairwise residue contact propensity terms. We use these scales to examine experimental data from two systems: protein cargo dissolving within phase‐separated droplets made from FG repeat proteins of the nuclear pore complex and client proteins dissolving within phase‐separated FUS droplets. These analyses reveal a close agreement between the stickiness of the client proteins and the experimentally determined values of the partition coefficients (R > 0.9), while pairwise residue contact propensities between client and scaffold show weaker correlations. Hence, the stickiness of client proteins is sufficient to explain their differential partitioning within these two phase‐separated systems without taking into account the composition of the condensate. This result implies that selective trafficking of client proteins to distinct membraneless organelles requires recognition elements beyond the client sequence composition.StatementEmpirical potentials for amino acid stickiness and pairwise residue contact propensities are derived. These scales are unique in that they enable direct comparison of desolvation versus contact terms. We find that partitioning of a client protein to a condensate is best explained by amino acid stickiness.

Camouflaged Fluorescent Silica Nanoparticles Target Aggregates and Condensates of the Amyloidogenic Protein Tau.

Intrinsically disordered proteins (IDPs) are increasingly found to be associated with irreversible neurodegenerative disorders. The protein tau is a prototypical IDP whose abnormal aggregation into insoluble filaments is a major hallmark of Alzheimer’s disease. The view has emerged that aggregation may proceed via alternative pathways involving oligomeric intermediates or phase-separated liquid droplets. Nanoparticles (NPs) offer significant potential for probing the mechanisms of protein fibrillation and may be capable of redirecting conformational transitions. Here, we camouflaged dye-doped silica NPs through functionalization with tau molecules to impart them the ability to associate with protein assemblies such as aggregates or condensates. The prepared NP–tau conjugates showed little influence on the aggregation kinetics and morphology of filamentous aggregates of tau but were found to associate with the filaments. Moreover, NP–tau conjugates were recruited and concentrated into polyanion-induced condensates of tau, driven by multivalent electrostatic interactions, thereby illuminating liquid droplets and their time-dependent transformation, as observed by fluorescence microscopy. NP–tau conjugates were capable of entering human neuroglioma cells and were not cytotoxic. Hence, we propose that NP–tau conjugates could serve as nanotracers for in vitro and in-cell studies to target and visualize tau assemblies and condensates, contributing to an explanation for the molecular mechanisms of abnormal protein aggregation.

Effect of Multivalency on Phase-Separated Droplets Consisting of Poly(PR) Dipeptide Repeats and RNA at the Solid/Liquid Interface.

Dipeptide repeat proteins (DRPs) are considered a significant cause of amyotrophic lateral sclerosis (ALS), and their liquid–liquid phase separation (LLPS) formation with other biological molecules has been studied both in vitro and in vivo. The immobilization and wetting of the LLPS droplets on glass surfaces are technically crucial for the measurement with optical microscopy. In this work, we characterized the surface diffusion of LLPS droplets of the DRPs with different lengths to investigate the multivalent effect on the interactions of their LLPS droplets with the glass surface. Using fluorescence microscopy and the single-particle tracking method, we observed that the large multivalency drastically changed the surface behavior of the droplets. The coalescence and wetting of the droplets were accelerated by increasing the multivalency of peptides in the LLPS system. Our findings on the effect of multivalency on interactions between droplets and glass surfaces could provide a new insight to enhance the understanding of LLPS formation and biophysical properties related to the solid/liquid interface.

Computational DNA Droplets Recognizing miRNA Sequence Inputs Based on Liquid–Liquid Phase Separation

AbstractPhase‐separated biomolecular droplets are formed in cells to regulate various biological processes. This phenomenon can be applied to constructing self‐assembled dynamic molecular systems such as artificial cells and molecular robots. Recently, programmable phase‐separated droplets called DNA droplets have been reported as a possible method to construct such dynamic molecular systems. This study reports a computational DNA droplet that can recognize a specific combination of tumor biomarker microRNAs (miRNAs) as molecular inputs and output a DNA logic computing result by physical DNA droplet phase separation. A mixed DNA droplet consisting of three DNA nanostructures with orthogonal sticky‐end sequences and two linker DNAs to cross‐bridge the orthogonal DNA nanostructures is proposed. By the hybridization of miRNAs with the linkers, the cross‐bridging ability is lost, causing the phase‐separation of the mixed DNA droplet into three DNA droplets, resulting in executing a miRNA pattern recognition described by a logical expression ((miRNA‐1 ∧ miRNA‐2) ∧ (miRNA‐3 ∧ ¬miRNA‐4)). This experimentally demonstrates that the computational DNA droplets recognize the above specific pattern of chemically synthesized miRNA sequences as a model experiment. In the future, this method will provide potential applications such as diagnosis and therapy with integration to biomolecular robots and artificial cells.

Characterization of RNA content in individual phase-separated coacervate microdroplets

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

Evolution of α-synuclein conformation ensemble toward amyloid fibril via liquid-liquid phase separation (LLPS) as investigated by dynamic nuclear polarization-enhanced solid-state MAS NMR

Protein fibrillation and human neurodegenerative diseases, with a profound underlying connection suggested between them, have been the subject of intense investigations in the medical, biophysical and bio-engineering sciences. For gaining the molecular mechanistic insights into such connection, i.e., the cause and effect, atomic-resolution molecular structure information especially on the initial oligomeric states is of paramount importance, not only that on the mature amyloid fibrils. α-Synuclein (αSyn) and its amyloid fibril has a direct relevance to the Parkinson's disease and other synucleinopathies, but what triggers the fibrillation is still not entirely clear. We here describe the liquid-liquid phase separation (LLPS) of αSyn and investigate its conformational evolution from its monomeric state into oligomer state within the early-stage of the phase-separated droplets, mainly using solution and magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopies, aided with optical and fluorescent microscopies and CD spectroscopy. Based on the analysis of the intricately broadened shapes of the MAS NMR peaks observed for isotopically 13C-labeled His-50 of αSyn, we show that the distribution of the αSyn conformation is skewed from the initial completely random state to a loose β-rich ensembles at/around His-50 as early as day-3 (d3) within the droplet. This intra-droplet loose β-rich assembly showed a very slow progression until d8, and eventually maturated into ThT-positive, long and unbranched amyloid fibrils after 8 weeks. The obtained information on the evolution of the distribution of the conformation ensemble is unique, and difficult to obtain with X-ray crystallography and cryo-electron microscopy (cryoEM). In particular, the sensitivity-enhanced MAS NMR based on the low-temperature dynamic nuclear polarization (DNP) technique was proven to be a key tool in characterizing the conformational ensemble with dilute protein samples such as the liquid-phase droplets.

PPAR\u03b3 phase separates with RXR\u03b1 at PPREs to regulate target gene expression

Peroxisome proliferator-activated receptor (PPAR)-γ is a key transcription activator controlling adipogenesis and lipid metabolism. PPARγ binds PPAR response elements (PPREs) as the obligate heterodimer with retinoid X receptor (RXR) α, but exactly how PPARγ orchestrates the transcriptional response is unknown. This study demonstrates that PPARγ forms phase-separated droplets in vitro and solid-like nuclear condensates in cell, which is intriguingly mediated by its DNA binding domain characterized by the zinc finger motif. Furthermore, PPARγ forms nuclear condensates at PPREs sites through phase separation to compartmentalize its heterodimer partner RXRα to initiate PPARγ-specific transcriptional activation. Finally, using an optogenetic approach, the enforced formation of PPARγ/RXRα condensates leads to preferential enrichment at PPREs sites and significantly promotes the expression of PPARγ target genes. These results define a novel mechanism by which PPARγ engages the phase separation principles for efficient and specific transcriptional activation.

Multivalent Peptide Ligands To Probe the Chromocenter Microenvironment in Living Cells.

Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid-liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.

Condensation of Ede1 promotes the initiation of endocytosis

Clathrin-mediated endocytosis is initiated by a network of weakly interacting proteins through a poorly understood mechanism. Ede1, the yeast homolog of mammalian Eps15, is an early-arriving endocytic protein and a key initiation factor. In the absence of Ede1, most other early endocytic proteins lose their punctate localization and endocytic uptake is decreased. We show that in yeast cells, cytosolic concentration of Ede1 is buffered at a critical level. Excess amounts of Ede1 form large condensates which recruit other endocytic proteins and exhibit properties of phase-separated liquid droplets. We demonstrate that the central region of Ede1, containing a coiled-coil and a prion-like region, is essential for both the condensate formation and the function of Ede1 in endocytosis. The functionality of Ede1 mutants lacking the central region can be partially rescued by an insertion of heterologous prion-like domains. Conversely, fusion of a heterologous lipid-binding domain with the central region of Ede1 can promote clustering into stable plasma membrane domains. We propose that the ability of Ede1 to form condensed networks supports the clustering of early endocytic proteins and promotes the initiation of endocytosis.

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state

SummaryThe recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.

Reproduction of phase-separated celadon glazes with Zijin clay

The preparation of R2O-RO-Al2O3-SiO2-P2O5 system phase-separated celadon glaze by using Zijin clay is investigated, which with the potential of environmental protection. Si and SiC were introduced to reduce iron oxide (Fe2O3) in an oxidizing environment. UV–vis–NIR, XRD, Raman, XPS, SEM, and TEM were used to explore the effects of reducing agents on the glaze color, microstructure, and phase structures. The results indicated that the introduction of 0.5 wt% Si and 0.5 wt% SiC will change the chemical state of iron ions. More concretely, the ratio of Fe2+/Fe3+ changed from 22.9/77.1–72.2/27.8 and 60.2/39.8, respectively, and the Si4+-O-Fe2+ was the main coloring factor, which made glazes appear blue green. Among them, the Si4+ produced by the introduction of Si contributed to the formation of the fancy patterns, but phase separation droplets formed the weak structural color. The glazes with SiC had the Rayleigh scattering effect, which was combined with the chemical color to improve the blue-green lightness of glazes.

Abstract TP250: N-3 Fatty Acid Diglyceride Emulsions As A Novel Acute Treatment For Ischemic Brain Injury

Introduction: Omega-3 (n-3) fatty acids (FAs), specifically docohexaenoic acid (DHA) and eicosapentaenoic acid (EPA), act as bioactive unsaturated lipids with pleiotropic effects, affording neuroprotection in ischemic brain injury. Hypothesis: We reported that n-3 FAs injected acutely as triglyceride (TG) emulsions provide neuroprotection after ischemic brain injury. We now questioned whether novel lipid emulsions made from n-3 diglycerides (DG) would improve the delivery and effectiveness of n-3 FAs in brain after injury. Methods: We evaluated in vitro interactions of DG (DG-DHA) vs TG (TG-DHA) in phosphatidylcholine (PC) bilayer liposomes, as a model membrane system, by NMR spectroscopy. We compared the in vitro kinetics of DG vs TG hydrolysis by lipoprotein lipase. We investigated the neuroprotective effects of DG emulsions in a Vannucci murine model of hypoxic-ischemic (HI) brain injury. Results: NMR spectra of PC liposomes incubated with DG-DHA showed an additional peak, adjacent to the phospholipid carbonyl region, indicating a higher incorporation into PC bilayers and a narrower peak at almost the same position in a more fluid phase. In contrast, spectra of liposomes incubated with TG-DHA showed narrow peaks well-separated from PC resonances, representing phase-separated oil droplets. In lipolysis assays, DG emulsions had more efficient hydrolysis than TGs. Neonatal mice treated with DG-EPA, DG-DHA, or the combination of both (DG-DHA+EPA) after HI injury showed up to 3X better reduction in infarct volumes compared to TGs (p<0.05). Conclusions: Our data demonstrate that DG molecules incorporate in membrane bilayers more efficiently than TG. We postulate that the faster hydrolysis of DGs contributes to higher neuroprotection compared with TGs. Our findings indicate that n-3 DG emulsions represent a novel and much more efficient modality than n-3 TG for improving ischemic brain injury outcomes.

Intelligent fluorescence image analysis of giant unilamellar vesicles using convolutional neural network

BackgroundFluorescence image analysis in biochemical science often involves the complex tasks of identifying samples for analysis and calculating the desired information from the intensity traces. Analyzing giant unilamellar vesicles (GUVs) is one of these tasks. Researchers need to identify many vesicles to statistically analyze the degree of molecular interaction or state of molecular organization on the membranes. This analysis is complicated, requiring a careful manual examination by researchers, so automating the analysis can significantly aid in improving its efficiency and reliability.ResultsWe developed a convolutional neural network (CNN) assisted intelligent analysis routine based on the whole 3D z-stack images. The programs identify the vesicles with desired morphology and analyzes the data automatically. The programs can perform protein binding analysis on the membranes or state decision analysis of domain phase separation. We also show that the method can easily be applied to similar problems, such as intensity analysis of phase-separated protein droplets. CNN-based classification approach enables the identification of vesicles even from relatively complex samples. We demonstrate that the proposed artificial intelligence-assisted classification can further enhance the accuracy of the analysis close to the performance of manual examination in vesicle selection and vesicle state determination analysis.ConclusionsWe developed a MATLAB based software capable of efficiently analyzing confocal fluorescence image data of giant unilamellar vesicles. The program can automatically identify GUVs with desired morphology and perform intensity-based calculation and state decision for each vesicle. We expect our method of CNN implementation can be expanded and applied to many similar problems in image data analysis.

Pattern Formation and Information Processing Based on Phase-separated DNA Droplets

Pattern Formation and Information Processing Based on Phase-separated DNA Droplets

Controlled evaporation-induced phase separation of droplets containing nanogels and salt molecules.

Droplets without protection from surfactants or surfactant-like objects normally experience merging or a coalescence process since it is thermodynamically favored. However, division or replication of droplets is thermodynamically unfavored and comparably more difficult to realize. Herein, we demonstrate that a population of droplets that are composed of nanogels and salt spontaneously undergo a separation process under a slow solvent evaporation condition. Each individual droplet underwent changes in size, shape and eventually developed into two domains, which was caused by the screening effect due to the increased salt concentration as a result of solvent evaporation. The two domains gradually separated into nanogel-rich and salt-rich parts. These two parts eventually evolved into nanogel aggregates and branched structures, respectively. This separation was mainly due to the salting out effect and dewetting. Comparison studies indicate that both the nanogels and salt are indispensable ingredients for the phase separation. These discoveries may have profound applications in the fields of biomimetics and offer new routes for self-replication systems.