Liquid Phase Droplets Research Articles

Reaction-Driven Diffusiophoresis of Liquid Condensates: Potential Mechanisms for Intracellular Organization.

The cellular environment, characterized by its intricate composition and spatial organization, hosts a variety of organelles, ranging from membrane-bound ones to membraneless structures that are formed through liquid-liquid phase separation. Cells show precise control over the position of such condensates. We demonstrate that organelle movement in external concentration gradients, diffusiophoresis, is distinct from the one of colloids because fluxes can remain finite inside the liquid-phase droplets and movement of the latter arises from incompressibility. Within cellular domains diffusiophoresis naturally arises from biochemical reactions that are driven by a chemical fuel and produce waste. Simulations and analytical arguments within a minimal model of reaction-driven phase separation reveal that the directed movement stems from two contributions: Fuel and waste are refilled or extracted at the boundary, resulting in concentration gradients, which (i) induce product fluxes via incompressibility and (ii) result in an asymmetric forward reaction in the droplet's surroundings (as well as asymmetric backward reaction inside the droplet), thereby shifting the droplet's position. We show that the former contribution dominates and sets the direction of the movement, toward or away from fuel source and waste sink, depending on the product molecules' affinity toward fuel and waste, respectively. The mechanism thus provides a simple means to organize condensates with different composition. Particle-based simulations and systems with more complex reaction cycles corroborate the robustness and universality of this mechanism.

HSPA8 regulates anti-bacterial autophagy through liquid-liquid phase separation

ABSTRACT HSPA8 (heat shock protein family A (Hsp70) member 8) plays a significant role in the autophagic degradation of proteins, however, its effect on protein stabilization and anti-bacterial autophagy remains unknown. Here, it is discovered that HSPA8, as a binding partner of RHOB and BECN1, induce autophagy for intracellular bacteria clearance. Using its NBD and LID domains, HSPA8 physically binds to RHOB residues 1–42 and 89–118 as well as to BECN1 ECD domain, preventing RHOB and BECN1 degradation. Intriguingly, HSPA8 contains predicted intrinsically disordered regions (IDRs), and drives liquid-liquid phase separation (LLPS) to concentrate RHOB and BECN1 into HSPA8-formed liquid-phase droplets, resulting in improved RHOB and BECN1 interactions. Our study reveals a novel role and mechanism of HSPA8 in modulating anti-bacterial autophagy, and highlights the effect of LLPS-related HSPA8-RHOB-BECN1 complex on enhancing protein interaction and stabilization, which improves the understanding of autophagy-mediated defense against bacteria.

Combustion behavior of single iron particles, Part II: A theoretical analysis based on a zero-dimensional model

Following the ignition and solid-to-liquid phase transition of a fine (on the order of 10–100µm in diameter) iron particle, the self-sustained combustion of a liquid-phase droplet of iron and its oxides takes place. The objective of the current work is to develop an interpretive and explanatory model for the liquid-phase combustion of a single fine iron particle. A zero-dimensional physicochemical model is developed assuming fast internal processes, such that the combustion rate is limited by the rate of external oxygen (O2) transport. The model considers a particle covered by a shell of liquid-phase FeO enclosing a core of liquid-phase Fe. Stefan flow and diffusion are considered for the gas-transport of O2, while the gas-transport of gas-phase Fe and FeO are calculated via diffusion only. The outward gas-phase Fe and FeO consume inward-transported O2 to stoichiometrically form hematite (Fe2O3), and the remaining oxygen that reaches the particle surface is entirely consumed to form liquid-phase FeO. The time history of simulated particle temperature shows consistent overprediction of the peak particle temperature when compared to experimental temperature measurements, indicating that the assumption of fast internal kinetics may be incorrect. The model is also unable to capture the apparent slow cooling rate observed in experiments. A further analysis is performed through a heuristic model with a calibrated reaction-rate law, where the internal diffusion of reactive Fe and O ions may become rate-limiting. The calibration of the pre-exponential factor in the Arrhenius term to match the experimental peak temperature yielded good agreement of time to peak temperature, as well as the slow cooling rate. The heuristic model considering internal diffusion predicts a plateau in peak temperature with increasing oxygen concentration. Possible uncertainties of the models, as well as future work, are discussed.

Biomolecular condensates under extreme martian salt conditions.

Biomolecular condensates formed by liquid–liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming non-membraneous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the sub-seafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.

Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus-ends

Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.

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.

Biomolecular Condensates under Extreme Martian Salt Conditions

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming nonmembranous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the subseafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch‐TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch‐TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress‐induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp‐1, suggesting gain‐of‐function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity.

A Droplet-Based Signal Reconstruction Approach to Channel Modeling in Molecular Communication

In this paper, a novel fluid dynamics-based approach to channel modeling, which considers liquid droplets as information carriers instead of molecules in the molecular communication (MC) channel, is proposed for practical macroscale MC systems. This approach considers a two-phase flow which is generated by the interaction of droplets in liquid phase with air molecules in gas phase. Two-phase flow is combined with the signal reconstruction (SR) of the receiver (RX) to propose a channel model. The SR part of the model quantifies how the accuracy of the sensed molecular signal in its reception volume depends on the sensitivity response of the RX and the adhesion/detachment process of droplets. The proposed channel model is validated by employing experimental data.

Synthesis and (some) applications of carbon-nanotube-supported pyrolytic carbon nanocones

Graphene-based cones with nanosized apex can be obtained by means of a high temperature pyrolytic carbon depositionprocess using methane and hydrogen as gaseous feedstock and single carbon nanotubes as deposition substrates. Aside thecones, micrometer-sized carbon beads or fibre segments are deposited meanwhile which are a key morphological componentfor allowing handling and mounting the carbon cones and then using them for various applications. Based on both theliterature dealing with pyrolytic carbon deposition processes and experimental observations, a peculiar depositionmechanism is proposed, involving the transient formation of pitch-like liquid phase droplets which deposit onto theindividual carbon nanotubes. In this picture, it is believed that a key parameter is the diameter ratio for the droplets and thenanotubes, respectively. The cone concentric texture and perfect nanotexture are shown by high resolution transmissionelectron microscopy, which allows interesting mechanical and conducting properties to be predicted. Correspondingly,applications of the carbon nanocones as electron emitters for cold-field electron sources on the one hand, and as probes forvarious modes of near-field microscopy on the other hand, have been tested.

Automated System for Small-Population Single-Particle Processing Enabled by Exclusive Liquid Repellency.

Exclusive liquid repellency (ELR) describes an extreme wettability phenomenon in which a liquid phase droplet is completely repelled from a solid phase when exposed to a secondary immiscible liquid phase. Earlier, we developed a multi-liquid-phase open microfluidic (or underoil) system based on ELR to facilitate rare-cell culture and single-cell processing. The ELR system can allow for the handling of small volumes of liquid droplets with ultra-low sample loss and biofouling, which makes it an attractive platform for biological applications that require lossless manipulation of rare cellular samples (especially for a limited sample size in the range of a few hundred to a few thousand cells). Here, we report an automated platform using ELR microdrops for single-particle (or single-cell) isolation, identification, and retrieval. This was accomplished via the combined use of a robotic liquid handler, an automated microscopic imaging system, and real-time image-processing software for single-particle identification. The automated ELR technique enables rapid, hands-free, and robust isolation of microdrop-encapsulated rare cellular samples.

Compositional adaptability in NPM1-SURF6 scaffolding networks enabled by dynamic switching of phase separation mechanisms

The nucleolus, the site for ribosome biogenesis contains hundreds of proteins and several types of RNA. The functions of many non-ribosomal nucleolar proteins are poorly understood, including Surfeit locus protein 6 (SURF6), an essential disordered protein with roles in ribosome biogenesis and cell proliferation. SURF6 co-localizes with Nucleophosmin (NPM1), a highly abundant protein that mediates the liquid-like features of the granular component region of the nucleolus through phase separation. Here, we show that electrostatically-driven interactions between disordered regions of NPM1 and SURF6 drive liquid-liquid phase separation. We demonstrate that co-existing heterotypic (NPM1-SURF6) and homotypic (NPM1-NPM1) scaffolding interactions within NPM1-SURF6 liquid-phase droplets dynamically and seamlessly interconvert in response to variations in molecular crowding and protein concentrations. We propose a mechanism wherein NPM1-dependent nucleolar scaffolds are modulated by non-ribosomal proteins through active rearrangements of interaction networks that can possibly contribute to the directionality of ribosomal biogenesis within the liquid-like nucleolus.

The Role of Post-Translational Modifications on Prion-Like Aggregation and Liquid-Phase Separation of FUS.

Subcellular mislocalization and aggregation of the human FUS protein occurs in neurons of patients with subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. FUS is one of several RNA-binding proteins that can functionally self-associate into distinct liquid-phase droplet structures. It is postulated that aberrant interactions within the dense phase-separated state can potentiate FUS’s transition into solid prion-like aggregates that cause disease. FUS is post-translationally modified at numerous positions, which affect both its localization and aggregation propensity. These modifications may influence FUS-linked pathology and serve as therapeutic targets.

Direct Surface and Droplet Microsampling for Electrospray Ionization Mass Spectrometry Analysis with an Integrated Dual-Probe Microfluidic Chip.

Ambient mass spectrometry (MS) has revolutionized the way of MS analysis and broadened its application in various fields. This paper describes the use of microfluidic techniques to simplify the setup and improve the functions of ambient MS by integrating the sampling probe, electrospray emitter probe, and online mixer on a single glass microchip. Two types of sampling probes, including a parallel-channel probe and a U-shaped channel probe, were designed for dry-spot and liquid-phase droplet samples, respectively. We demonstrated that the microfabrication techniques not only enhanced the capability of ambient MS methods in analysis of dry-spot samples on various surfaces, but also enabled new applications in the analysis of nanoliter-scale chemical reactions in an array of droplets. The versatility of the microchip-based ambient MS method was demonstrated in multiple different applications including evaluation of residual pesticide on fruit surfaces, sensitive analysis of low-ionizable analytes using postsampling derivatization, and high-throughput screening of Ugi-type multicomponent reactions.

Genetic visualization of protein interactions harnessing liquid phase transitions

Protein-protein interactions (PPIs) are essential components of cellular function. Current fluorescence-based technologies to measure PPIs have limited dynamic range and quantitative reproducibility. Here, we describe a genetically-encoded PPI visualization system that harnesses the dynamics of condensed liquid-phase transitions to analyze protein interactions in living cells. The fluorescent protein Azami-Green and p62-PB1 domain when fused to PPI partners triggered a rapid concatenation/oligomerization process that drove the condensation of liquid-phase droplets for real-time analysis of the interaction with unlimited dynamic range in the fluorescence signal. Proof-of-principle studies revealed novel insights on the live cell dynamics of XIAP-Smac and ERK2-dimer interactions. A photoconvertible variant allowed time-resolved optical highlighting for PPI kinetic analysis. Our system, called Fluoppi, demonstrates the unique signal amplification properties of liquid-phase condensation to detect PPIs. The findings introduce a general method for discovery of novel PPIs and modulators of established PPIs.

A new method for sampling low-volume AMCs in a front opening unified pod

The Front Opening Unified Pod (FOUP) is widely utilized in advanced semiconductor production. However, the airborne molecular contaminants (AMCs) inside FOUPs are difficult to monitor by sampling because of the limited volume and the low concentrations of contaminants. To reduce cross contamination and to help identify the causes of product failures, high-throughput screening of FOUP AMC surveying is essential. In order to acquire an effective sample in a FOUP with a limited 30-liter volume, a rapid, simple, and reliable method of chelating and chilled impinger sampling is proposed. A chemical approach first produces AMC derivatives of gaseous reactions, and then heterogeneous over-saturation nucleation transforms the AMC derivatives into condensation nuclei so as to precipitate liquid phase droplets. Thus, the AMCs inside a purged FOUP can be captured rapidly as dissolved acid/alkaline crystalline salts. Experimental results have shown that the proposed technique is a promising method of surveying AMCs inside a purged FOUP that bypasses the constraints of the limited volume and low concentration conditions.

Quantitative fuel vapor/air mixing imaging in droplet/gas regions of an evaporating spray flow using filtered Rayleigh scattering.

This Letter demonstrates the application of filtered Rayleigh scattering (FRS) for quantitative two-dimensional fuel vapor/air mixing measurements in an evaporating hydrocarbon fuel spray flow. Using the FRS approach, gas-phase measurements are made in the presence of liquid-phase droplets without interference. Effective suppression of the liquid-phase droplet scattering using FRS is enabled by the high spectral purity of the current Nd:YAG laser system. Simultaneous Mie-scattering imaging is used to visualize the droplet field and illustrate the droplet loading under which the FRS imaging is applied in the current spray flows. The initial quantification of the FRS imaging is based on calibration measurements from a flow cell of known fuel vapor/air mixtures, while future work targets the utilization of a Rayleigh-Brillouin spectral model for quantification of the FRS signals.

Characteristic Modification of Catalysts by Use of a Chloride Source

Structural control and morphological modification of a series of Si-based nanostructures were studied from the viewpoint of modifying the catalyst’s characteristics. The catalyst was modified from a liquid to a solid during its growth. The growth evolution of the faceted Si nanowires occurred via a vapor–liquid–solid mechanism followed by a silicide vapor–solid–solid mechanism. The shapes of the catalysts defined the shapes of the nanowires during the vapor–solid–solid growth. The catalyst was further modified by the deposition of MnCl2. Only irregularly shaped Si particles or MnCl2 particles were observed on top of the Si nanowires. The characteristic modification of catalysts by liquid-phase crystal nucleation and deposition of liquid-phase droplets was discussed. In addition, the synthesis of a CrSi2 nanowire bundle by the formation of dense nanoparticles was studied.

Three-Dimensional Graphene-Based Microbarriers for Controlling Release and Reactivity in Colloidal Liquid Phases.

Two-dimensional materials are of great interest as high-performance molecular barriers. Graphene in particular is atomically thin, is impermeable to all molecules, and in some forms can be easily deposited over large areas into planar multilayer films that have been shown to suppress molecular transport. Graphene and graphene oxide sheets are also known to spontaneously self-assemble at liquid-liquid interfaces on the surfaces of dispersed droplets, but much less is known about the barrier properties of these ultrathin films in 3D curved microgeometries. This article demonstrates that 3D films self-assembled from graphene oxide or reduced graphene oxide sheets can be exploited to control the release of small molecules from dispersed liquid phase droplets by evaporation. The release rate and containment time can be tuned by addition of multivalent cations that recruit additional sheets from the bulk liquid to the interface, which is shown by molecular dynamics to occur by an electrostatic bridging mechanism. 3D graphene-based films on droplet surfaces can also be used to control the release and transport of soluble molecules from the droplet to surrounding bulk solvent phases. In some cases, the release can be effectively stopped to produce unique kinetically trapped emulsion phases consisting of two fully miscible but segregated liquids. Finally, interfacial graphene-based films are also shown to control interfacial chemical reaction processes by serving as transport barriers between the phases or by intercepting reactive cross-phase molecular collisions. This reaction control is demonstrated by using 3D graphene-based microbarriers to protect oxidation-sensitive oils from attack by aqueous-phase reactive oxygen species, which is an undesirable pathway implicated in many chemical product degradation and spoilage processes.

INVITED] Laser-induced forward transfer: A high resolution additive manufacturing technology

Among the additive manufacturing techniques, laser-induced forward transfer addresses the challenges of printing thin films in solid phase or small volume droplets in liquid phase with very high resolution. This paper reviews the physics of this process and explores the pros and cons of this technology versus other digital printing technologies. The main field of applications are printed electronics, organic electronics and tissue engineering, and the most promising short terms ones concern digital laser printing of sensors and conductive tracks. Future directions and emerging areas of interest are discussed such as printing solid from a liquid phase and 3D digital nanomanufacturing.