Driven Phase Separation Research Articles

Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation.

RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro- and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA- and DNA-related processes by concentrating them in membraneless organelles. The general function and wide distribution of PUN motifs across species suggest that in an ancient 'RNA world' PUN-like motifs may have supported the correct folding of early ribozymes.

Entropically driven phase separation and effective multibody interactions in block copolymers

Entropically driven phase separation and effective multibody interactions in block copolymers

Direct and indirect salt effects on homotypic phase separation

The low-complexity domain of hnRNPA1 (A1-LCD) phase separates in a salt-dependent manner. Unlike many intrinsically disordered proteins (IDPs) whose phase separation is suppressed by increasing salt concentrations, the phase separation of A1-LCD is promoted by >100 mM NaCl. To investigate the atypical salt effect on A1-LCD phase separation, we carried out all-atom molecular dynamics simulations of systems comprising multiple A1-LCD chains at NaCl concentrations from 50 to 1000 mM NaCl. The ions occupy first shell as well as more distant sites around the IDP chains, with Arg sidechains and backbone carbonyls the favored partners of Cl– and Na+, respectively. They play two direct roles in driving A1-LCD condensation. The first is to neutralize the high net charge of the protein (+9) by an excess of bound Cl– over Na+; the second is to bridge between A1-LCD chains, thereby fortifying the intermolecular interaction networks in the dense phase. At high concentrations, NaCl also indirectly strengthens π–π, cation–π, and amino–π interactions, by drawing water away from the interaction partners. Therefore, at low salt, A1-LCD is prevented from phase separation by net charge repulsion; at intermediate concentrations, NaCl neutralizes enough of the net charge while also bridging IDP chains to drive phase separation. This drive becomes even stronger at high salt due to strengthened π-type interactions. Based on this understanding, four classes of salt dependence of IDP phase separation can be predicted from amino-acid composition.

Direct and indirect salt effects on homotypic phase separation.

The low-complexity domain of hnRNPA1 (A1-LCD) phase separates in a salt-dependent manner. Unlike many intrinsically disordered proteins (IDPs) whose phase separation is suppressed by increasing salt concentrations, the phase separation of A1-LCD is promoted by >100 mM NaCl. To investigate the atypical salt effect on A1-LCD phase separation, we carried out all-atom molecular dynamics simulations of systems comprising multiple A1-LCD chains at NaCl concentrations from 50 to 1000 mM NaCl. The ions occupy first shell as well as more distant sites around the IDP chains, with Arg sidechains and backbone carbonyls the favored partners of Cl- and Na+, respectively. They play two direct roles in driving A1-LCD condensation. The first is to neutralize the high net charge of the protein (+9) by an excess of bound Cl- over Na+; the second is to bridge between A1-LCD chains, thereby fortifying the intermolecular interaction networks in the dense phase. At high concentrations, NaCl also indirectly strengthens π-π, cation-π, and amino-π interactions, by drawing water away from the interaction partners. Therefore, at low salt, A1-LCD is prevented from phase separation by net charge repulsion; at intermediate concentrations, NaCl neutralizes enough of the net charge while also bridging IDP chains to drive phase separation. This drive becomes even stronger at high salt due to strengthened π-type interactions. Based on this understanding, four classes of salt dependence of IDP phase separation can be predicted from amino-acid composition.

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.