Driven Phase Separation Research Articles

A cytoplasmic osmosensing mechanism mediated by molecular crowding-sensitive DCP5.

Plants are frequently challenged by osmotic stresses. How plant cells sense environmental osmolarity changes is not fully understood. We report that Arabidopsis Decapping 5 (DCP5) functions as a multifunctional cytoplasmic osmosensor that senses and responds to extracellular hyperosmolarity. DCP5 harbors a plant-specific intramolecular crowding sensor (ICS) that undergoes conformational change and drives phase separation in response to osmotically intensified molecular crowding. Upon hyperosmolarity exposure, DCP5 rapidly and reversibly assembles to DCP5-enriched osmotic stress granules (DOSGs), which sequestrate plenty of mRNA and regulatory proteins, and thus adaptively reprograms both the translatome and transcriptome to facilitate plant osmotic stress adaptation. Our findings uncover a cytoplasmic osmosensing mechanism mediated by DCP5 with plant-specific molecular crowding sensitivity and suggest a stress sensory function for hyperosmotically induced stress granules.

SERRATE drives phase separation behaviours to regulate m6A modification and miRNA biogenesis.

The methyltransferase complex (MTC) deposits N6-adenosine (m6A) onto RNA, whereas the microprocessor produces microRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B tends to form insoluble condensates with poor activity, with its level monitored by the 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promote the solubility and stability of the MTC subunit B, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behaviour, exhibit reduced m6A levels. Reciprocally, MTC can recruit the microprocessor to the MIRNA loci, prompting co-transcriptional cleavage of primary miRNA substrates. Additionally, primary miRNA substrates carrying m6A modifications at their single-stranded basal regions are enriched by m6A readers, which retain the microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.

Effect of anion α-functionalization on the water affinity of thermo-responsive phosphonium acetate-derived ionic liquids

This study presents a facile method for assessing the effect of anion functional group substitution on the water affinity of phosphonium-acetate ionic liquids. By combining the same cation with three different α-substituted carboxylate anions, the effect of anion functional group substitution on the IL miscibility with water and hygroscopicity was experimentally evaluated and rationalized using COSMO-RS sigma profile analysis. Lower critical solution temperature (LCST) phase separation experiments and water vapor partial pressure measurements revealed that there is a stark α-substitution dependency on the affinity of water for acetate [AcO], pivalate [PivO], and trifluoroacetate [TFA] derived tributyl(octyl)phosphonium [P4448] ILs. It was found that the IL water affinities varied as: [P4448][AcO] > [P4448][PivO] > [P4448][TFA]. With the lowest water partial pressure and no LCST driven phase separation, [P4448][AcO] exhibited a high affinity for water. On the other hand, [P4448][PivO] and [P4448][TFA] exhibited LCST-driven phase separation and room temperature immiscibility, respectively, with higher water partial pressures, as a result of relatively weak water affinities. COSMO-RS sigma profile analysis revealed that the aliphatic carboxylate anions should incorporate apolar interactions commensurate with or greater than the polar hydrogen bond acceptor interactions for phase immiscibility of the corresponding IL with water. It was found that the relative magnitudes of apolar and polar interactions, assessed using the anion sigma profile peak intensity ratio (Ir), are directly correlated with the IL water affinity, and inversely correlated with the solution water activity coefficient. Therefore, it was concluded that Ir forms a quantitative link between the structural changes at the molecular level and the bulk water affinity exhibited by the ionic liquid as a whole.

VAMP2 regulates phase separation of α-synuclein

α-Synuclein (αSYN), a pivotal synaptic protein implicated in synucleinopathies such as Parkinson’s disease and Lewy body dementia, undergoes protein phase separation. We reveal that vesicle-associated membrane protein 2 (VAMP2) orchestrates αSYN phase separation both in vitro and in cells. Electrostatic interactions, specifically mediated by VAMP2 via its juxtamembrane domain and the αSYN C-terminal region, drive phase separation. Condensate formation is specific for R-SNARE VAMP2 and dependent on αSYN lipid membrane binding. Our results delineate a regulatory mechanism for αSYN phase separation in cells. Furthermore, we show that αSYN condensates sequester vesicles and attract complexin-1 and -2, thus supporting a role in synaptic physiology and pathophysiology.

Molecular dynamics simulation on regulation of liquid–liquid phase separation of repetitive peptides

Understanding the intricate interactions governing protein and peptide behavior in liquid–liquid phase separation (LLPS) is crucial for unraveling biological functions and dysfunctions. This study employs a residue-leveled coarse-grained molecular dynamics approach to simulate the phase separation of repetitive polyproline and polyarginine peptides (poly PR) with varying lengths and sequences in solution, considering different concentrations and temperatures. Our findings highlight the crucial role of sequence order in promoting LLPS in peptides with identical lengths of repetitive sequences. Interestingly, repetitive peptides containing fewer than 10 polyarginine repeats exhibit no LLPS, even at salt concentrations up to 3 M. Notably, our simulations align with experimental observations, pinpointing a salt concentration of 2.7 M for PR25-induced LLPS. Utilizing the same methodology, we predict the required salt concentrations for LLPS induction as 1.2 M, 1.5 M, and 2.7 M for PR12, PR15, and PR35, respectively. These predictions demonstrate good agreement with experimental results. Extending our investigation to include the peptide glutamine and arginine (GR15) in DNA solution, our simulations mirror experimental observations of phase separation. To unveil the molecular forces steering peptide phase separation, we introduce a dielectric constant modifier and hydrophobicity disruptor into poly PR systems. Our coarse-grained analysis includes an examination of temperature effects, leading to the inference that both hydrophobic and electrostatic interactions drive phase separation in peptide systems.

In-situ engineering catalytically active surfaces for tribocatalysis with layered double hydroxide nanoparticles

Ensuring long-lasting lubrication is vital for sustainable machinery operation, made possible by self-regenerating carbon-based tribofilms via tribocatalysis. Conventional methods use expensive catalytic coatings, posing challenges for replacement and maintenance in practice. Here, we are proposing catalytic layered double hydroxide (LDH) nanoparticles as cost-effective and easily replenished lubricant additives to engineer catalytically active surfaces in situ where binary and ternary LDHs with Ni2+, Co2+, and/or Cu2+ divalent cations alongside Al3+ trivalent cations are investigated for lubrication performance. Under 100 °C sliding condition equivalent to the lubricating temperature in an internal combustion engine, NiCoAl–CO3 LDH exhibits the lowest wear losses alongside the durable low-friction regime. This excellent performance is attributed to Co-containing spinel and oxide phases in the catalytic tribo-oxide layer which help stabilize and maintain the microstructures of the tribo-oxide layer. In contrast, deterioration in lubrication performance at this temperature was observed for copper-containing LDHs, especially NiCuAl–CO3 LDHs, which is due to the reduction of metallic oxides that drive phase separation in the catalytic oxide tribo-layers. The more stable tribo-oxide layers can result in thick, durable carbon-based tribofilm during sliding along with higher resistance to plastic deformation bulk interlayer. This study offers valuable insight into the synergy of catalytic oxide materials, opening avenues for a rational design of innovative catalytic nano-materials for tribocatalysis processes.

Membrane Tilt Drives Phase Separation of Adhesion Receptors.

Cell adhesion receptors are transmembrane proteins that bind cells to their environment. These proteins typically cluster into disk-shaped or linear structures. Here, we show that such clustering patterns spontaneously emerge when the receptor senses the membrane deformation gradient, for example, by reaching a lower-energy conformation when the membrane is tilted relative to the underlying binding substrate. Increasing the strength of the membrane gradient-sensing mechanism first yields isolated disk-shaped clusters and then long linear structures. Our theory is coherent with experimental estimates of the parameters, suggesting that a tilt-induced clustering mechanism is relevant in the context of cell adhesion.

Compatibility versus reaction diffusion: Factors that determine the heterogeneity of polymerized adhesive networks

ObjectivesHeterogeneity and phase separation during network polymerization is a major issue contributing to the failure of dental adhesives. This study investigates how the ratio of hydrophobic crosslinkers to hydrophilic comonomer (C/H ratio), as well as cosolvent fraction (ethanol/water) influences the degree of heterogeneity and proclivity for phase separation in a series of model adhesive formulations. MethodsTwelve formulations were investigated, with 4 different C/H ratios (7:1, 2.2:1, 1:1, 0.5:1) and 3 different overall cosolvent fractions (0, 10 and 20 wt%). The heterogeneity and phase behavior were characterized using Fourier Transform Infrared Spectroscopy (FT-IR), dynamic mechanical analysis (DMA), small-angle x-ray scattering (SAXS) and atomic force microscopy (AFM). ResultsIn resins without cosolvent, all characterizations confirm reduced heterogeneity as C/H ratio decreases. However, when 10 or 20 wt% of cosolvent is included in the adhesive formulation, a higher degree of heterogeneity and even distinct phase separation with domains ranging from a few hundreds of nanometers to a few micrometers in size form. This is particularly noticeable at lower C/H ratios, which is surprising as HEMA is commonly considered a compatibilizer between hydrophobic crosslinkers and aqueous (co)solvents. SignificanceOur experiments demonstrate that formulations with lower C/H ratio and thus a lower viscosity experience later onsets of diffusion limitations during polymerization, which favors thermodynamically driven phase separation. Therefore, to determine or predict the resulting phase structure of adhesive materials, it is necessary to consider the kinetics and diffusion constraints during the formation of the polymer network and not just the compatibility of resin constituents.

Influence of Ionic Liquid and Light Intensity on a Polymer Nanostructure Formed in Lyotropic Liquid Crystalline Templates.

Utilizing self-assembled lyotropic liquid crystal (LLC) templates with radical photopolymerization shows promise in controlling polymer structure on the nanometer scale This control of nanostructure allows tailoring and enhancement of material properties not attainable in traditional polymerization in applications including hydrogels and stimuli-responsive systems. However, thermodynamically driven phase separation between the polymer and LLC templates often hinders the control of local polymer order and resultant polymer properties. This study investigates an alternative method to control the hydrogel nanostructure and avoid phase separation using imidazolium ionic liquids (ILs) in the LLC template while modulating the light intensity used in photopolymerization. The addition of the IL improves the thermodynamic stability and enhances the polymerization rate in the LLC system. The degree of LLC nanostructure retention is increased by increasing light intensities during polymerization. In addition, intermediate concentrations of cross-linker allow a balance between phase stability and cross-linking to lock in LLC morphology. With enhanced retention, the maximum water uptake is significantly higher compared with isotropic controls. These results demonstrate a method to increase the structure on the nanometer scale of a polymer by combining the addition of ILs with the proper selection of light intensity and cross-link density that allows access to unique hydrogel properties. These templated polymers demonstrate enhanced swelling and a stimuli response that show promise in applications ranging from drug delivery to water remediation.

Transient helices with functional roles

Intrinsically disordered regions (IDRs) of proteins contribute to various cellular functions, including signal transduction, gene regulation, and subcellular organization (1). One of the most important aspects of IDRs is their contribution to biomolecular condensate formation within cells. IDRs can undergo multivalent intermolecular interactions that drive phase separation. For the multivalent interactions, IDRs may have preferential interaction sites ('stickers') flanked by inert sequences ('spacers') (1). However, how these interactions occur between conformationally flexible polypeptide chains is not well understood.

Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

The male pachynema-specific protein MAPS drives phase separation in vitro and regulates sex body formation and chromatin behaviors in vivo

Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation invitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps-/- pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. MapsΔ2-9/Δ2-9 male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps-/- mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.

Segregative phase separation of strong polyelectrolyte complexes at high salt and high polymer concentrations.

The phase behavior of polyelectrolyte complexes and coacervates (PECs) at low salt concentrations has been well characterized, but their behavior at concentrations well above the binodal is not well understood. Here, we investigate the phase behavior of stoichiometric poly(styrene sulfonate)/poly(diallyldimethylammonium) mixtures at high salt and high polymer concentrations. Samples were prepared by direct mixing of PSS/PDADMA PECs, water, and salt (KBr). Phase separation was observed at salt concentrations approximately 1 M above the binodal. Characterization by thermogravimetric analysis, FTIR, and NMR revealed that both phases contained significant amounts of polymer, and that the polymer-rich phase was enriched in PSS, while the polymer-poor phase was enriched in PDADMA. These results suggest that high salt concentrations drive salting out of the more hydrophobic polyelectrolyte (PSS), consistent with behavior observed in weak polyelectrolyte systems. Interestingly, at the highest salt and polymer concentrations studied, the polymer-rich phase contained both PSS and PDADMA, suggesting that high salt concentrations can drive salting out of partially-neutralized complexes as well. Characterization of the behavior of PECs in the high concentration limit appears to be a fruitful avenue for deepening fundamental understanding of the molecular-scale factors driving phase separation in these systems.

Modeling Lithium Plating Onset on Porous Graphite Electrodes Under Fast Charging with Hierarchical Multiphase Porous Electrode Theory

Lithium plating during fast charging of porous graphite electrodes in lithium-ion batteries accelerates degradation and raises safety concerns. Predicting lithium plating is challenging due to the close redox potentials of lithium reduction and intercalation, obscured by the nonlinear dynamics of electrochemically driven phase separation in hierarchical pore structures. To resolve dynamical resistance of realistic porous graphite electrodes, we introduce a model of porous secondary graphite particles to the multiphase porous electrode theory (MPET), based on electrochemical nonequilibrium thermodynamics and volume averaging. The resulting computational framework of “hierarchical MPET” is validated and tested against experimental data over a wide range of fast charging conditions and capacities. With all parameters estimated from independent sources, the model is able to quantitatively predict the measured cell voltages, and, more importantly, the experimentally determined capacity for lithium plating onset at fast 2C to 6C rates. Spatial and temporal heterogeneities in the lithiation of porous graphite electrodes are revealed and explained theoretically, including key features, such as idle graphite particles and non-uniform plating, which have been observed experimentally.

PHF1 compartmentalizes PRC2 via phase separation.

Polycomb repressive complex 2 (PRC2) is central to polycomb repression as it trimethylates lysine 27 on histone H3 (H3K27me3). How PRC2 is recruited to its targets to deposit H3K27me3 remains an open question. Polycomb-like (PCL) proteins, a group of conserved PRC2 accessory proteins, can direct PRC2 to its targets. In this report, we demonstrate that a PCL protein named PHF1 forms phase-separated condensates at H3K27me3 loci that recruit PRC2. Combining cellular observation and biochemical reconstitution, we show that the N-terminal domains of PHF1 cooperatively mediate target recognition, the chromo-like domain recruits PRC2, and the intrinsically disordered region (IDR) drives phase separation. Moreover, we reveal that the condensates compartmentalize PRC2, DNA, and nucleosome arrays by phase separation. Luciferase reporter assays confirm that PHF1 phase separation promotes transcription repression, further supporting a role of the condensates in polycomb repression. Based on our findings, we propose that these condensates create favorable microenvironments at the target loci for PRC2 to function.

Reduction of oligomer size modulates the competition between cluster formation and phase separation of the tumor suppressor SPOP

Phase separation compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of soluble complexes below the saturation concentration, the contribution of condensates vs complexes to function is sometimes unclear. Here, we characterized several new cancer-associated mutations of the tumor suppressor Speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase (CRL3). These pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to its substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX but unexpectedly enhance the poly-ubiquitination activity of SPOP towards DAXX. Enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of complexes versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate activity and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.

“Boundary residues” between the folded RNA recognition motif and disordered RGG domains are critical for FUS–RNA binding

Fused in sarcoma (FUS) is an abundant RNA-binding protein, which drives phase separation of cellular condensates and plays multiple roles in RNA regulation. The RNA-binding ability of FUS protein is crucial to its cellular function. Here, our molecular simulation study on the FUS-RNA complex provides atomic resolution insights into the observations from biochemical studies and also illuminates our understanding of molecular driving forces that mediate the structure, stability, and interaction of the RNA recognition motif (RRM) and RGG domains of FUS with a stem-loop junction RNA. We observe clear cooperativity and division of labor among the ordered (RRM) and disordered domains (RGG1 and RGG2) of FUS that leads to an organized and tighter RNA binding. Irrespective of the length of RGG2, the RGG2-RNA interaction is confined to the stem-loop junction and the proximal stem regions. On the other hand, the RGG1 interactions are primarily with the longer RNA stem. We find that the C terminus of RRM, which make up the "boundary residues" that connect the folded RRM with the long disordered RGG2 stretch of the protein, plays a critical role in FUS-RNA binding. Our study provides high-resolution molecular insights into the FUS-RNA interactions and forms the basis for understanding the molecular origins of full-length FUS interaction with RNA.

ParSe 2.0: A web tool to identify drivers of protein phase separation at the proteome level.

We have developed an algorithm, ParSe, which accurately identifies from the primary sequence those protein regions likely to exhibit physiological phase separation behavior. Originally, ParSe was designed to test the hypothesis that, for flexible proteins, phase separation potential is correlated to hydrodynamic size. While our results were consistent with that idea, we also found that many different descriptors could successfully differentiate between three classes of protein regions: folded, intrinsically disordered, and phase-separating intrinsically disordered. Consequently, numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. Built from that finding, ParSe 2.0 uses an optimal set of property scales to predict domain-level organization and compute a sequence-based prediction of phase separation potential. The algorithm is fast enough to scan the whole of the human proteome in minutes on a single computer and is equally or more accurate than other published predictors in identifying proteins and regions within proteins that drive phase separation. Here, we describe a web application for ParSe 2.0 that may be accessed through a browser by visiting https://stevewhitten.github.io/Parse_v2_FASTA to quickly identify phase-separating proteins within large sequence sets, or by visiting https://stevewhitten.github.io/Parse_v2_web to evaluate individual protein sequences.

Repeated Microphase Separation and Heating-free Distillation-like Behavior of Miscible Binary Liquid Mixtures Using Nanoconfined Grafted Polymer Layers.

Nanoconfinement is known to drive phase separation (often denoted as microphase separation) of two highly miscible liquids by subjecting the two liquids to disparate influences. Here, we propose a paradigm shift to this problem: we introduce the idea of "repeatability" in nanoconfinement-driven microphase separation. A drop consisting of two highly miscible liquids (A and B) is made to pass through a nanochannel grafted with a collapsed layer of polymer that is philic to A but phobic to B. Subsequently, a significant number of molecules of liquid A get imbibed into the polymer layer and the polymer layer partially swells, while the molecules of liquid B mostly remain out of the polymeric layer and are carried away, emerging as a drop on the other side of the polymer bilayer. This passage of drop (of liquids A and B) is continued, and each time liquids A and B get separated with liquid A imbibing into the polymer layer and liquid B being carried away with the drop. This scenario, therefore, points to the repeated occurrence of the microphase separation of miscible binary liquid mixtures, enabling the processing of a much larger volume of liquid, given the fact that the presence of a grafted polymer layer continues to provide a dynamically increasing space where liquid A can get localized after being separated from liquid B. We quantify such repeated microphase separation by noting the extent of separation (of liquid A) and extent of recovery (of liquid B) as functions of nanochannel height and number of passes. Interestingly, we establish that this process also leads to a distillation-like behavior (without any heat addition), where the concentration of liquid B (equivalent to the "less volatile" liquid in a standard distillation process) progressively increases inside the drop after its passage through the nanochannel.

Predicting disordered regions driving phase separation of proteins under variable salt concentration

We investigate intrinsically disordered regions (IDRs) of phase separating proteins regarding their impact on liquid-liquid phase separation (LLPS) of the full protein. Our theoretical approach uses a mean-field theory that accounts for sequence-dependent electrostatic interactions via a Random Phase Approximation (RPA) and in addition allows for variable salt concentration for the condensed and dilute protein phases. The numerical solution of the complete phase diagrams together with the tie lines that we derive for this model system leaves two parameters to be determined by fitting experimental data on concentrations of all species involved in the system. For our comparisons, we focus on two proteins, PGL-3 and FUS, known to undergo LLPS. For PGL-3 we predict that its long IDR near the C-terminus promotes LLPS, which we validate through direct comparison with in vitro experimental results under the same physiological conditions. For the structurally more complex protein FUS the role of the low complexity (LC) domain in LLPS has been intensively studied. Apart from the LC domain we here investigate theoretically two IDRs, one near the N-terminus and another near the C-terminus. Our theoretical analysis of these domains predict that the IDR at the N-terminus (aa 1-285) is the main driver of LLPS of FUS by comparison to in vitro experiments of the full length protein under the same physiological temperature and salt conditions.