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At least 19 recordsLinked to original sources

PubMed1975

Phase separations in phospholipd membranes.

Phase diagrams representing lateral phase separations in the plane of lipid bilayer membranes have been determined for binary mixtures containing dielaidoylphosphatidylcholine together with dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dipalmitoylphosphatidylethanolamine. The phase diagrams were deduced from observations of the temperature dependence of the paramagnetic resonance spectra of low concentrations of spin-labels incorporated in these bilayer membranes. In one case, the binary mixture of dipalmitoylphosphatidylethamine and dielaidoylphosphatidylcholine, evidence has been obtained for fluid-fluid immiscibility, in specified temperature and compoistion ranges. This immiscibility could give a lateral phase separation into fluid domains in the plane of the membrane, and/or a transverse phase separation into an asymmetrical bilayer membrane, and/or possibly disco ntinuous bilayer membranes of different composition. An asymmetrical bilayer membrane can be expected on theoretical grounds to form a nonplanar membrane.

Electron Spin Resonance Spectroscopy↗

PubMed2026

High-throughput identification of endogenous biomolecular condensates and phase-separating proteins.

Biomolecular condensates formed through liquid-liquid phase separation regulate cellular processes, and their dysregulation causes disease. Current methods for identifying endogenous phase-separating proteins have low throughput and cannot capture dynamic responses to stimuli. Here we present a protocol combining osmotic compression or transforming growth factor-β (TGF-β) treatment to induce condensation with sucrose density gradient centrifugation and quantitative mass spectrometry to enable systematic, high-throughput identification of endogenous condensates and phase-separating proteins. The method exploits the density changes that occur when phase-separating proteins undergo oligomerization during condensate formation. In H1975 cells, we identified over 1,500 phase-separating proteins under osmotic compression or TGF-β treatment; 538 of these candidates were not present in PhaSepDB, a database that compiles in vivo, in vitro and omics-derived proteins. The approach detects constitutive condensates and proteins that dynamically phase-separate in response to osmotic stress or TGF-β signaling. This protocol provides proteome-wide analysis of fractions of proteins having different densities and enables temporal resolution of phase-separation events. The procedure takes ~9 d and requires expertise in cell culture, biochemistry and mass spectrometry. This method enables systematic study of biomolecular condensates and disease-associated phase-separation mechanisms.

Phase Separation↗

PubMed1979

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current.

Disappearance of Ca2+-induced phase separation in phosphatidylserine-phosphatidylcholine membrane has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the pre parations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca2+-treated membrane was transferred to acidic salt solutions (less than or equal to pH 2.5) or to low ionic strength media (less than or equal to mM) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca2+ by proton, proton-induced separation, followed by disappearance of the phase separation in the buffered salt solution. Biological significance of the competition between Ca2+ and proton for the phase separation or domain formation in the membranes was emphasized.

Calcium↗

PubMed2026

Mixing chromatin fibers with different nucleosome repeat lengths changes dynamics of chromatin phase separation.

The eukaryotic genome is organized into chromatin at multiple lengths and timescales. Liquid-liquid phase separation has recently emerged as a mechanism for the dynamic compartmentalization of chromatin. However, it remains unclear how cells can locally alter phase separation behavior to condense, decondense, and segregate specific regions of their genome. Selective interactions between chromatin fibers with different nucleosome repeat lengths (NRLs), as well as their incorporation into existing condensates composed of different NRL chromatin fibers, may provide a pathway for such processes. Using fluorescence microscopy, we investigated how these mechanisms influence the formation, coalescence, and maturation of chromatin condensates. Our results show distinct NRL-dependent mixing behaviors of chromatin before and after condensate formation. 167 and 197 NRL fibers, known to fold into compact fibers by strong nucleosome stacking interactions, formed amorphous condensates. In contrast, 172 and 202 NRL fibers, which only allow for weak stacking, formed spherical condensates. When NRLs were mixed, amorphous condensates exhibited localized clustering of identical NRLs. In spherical condensates, however, both NRLs were homogeneously distributed, with a varying NRL ratio per condensate. In addition, incorporation of 167 NRLs into preexisting 172 NRL condensates resulted in a multiphase structure where 167 NRL fibers formed an outer layer. These findings present an intrinsic link between DNA sequence, nucleosome positioning, local chromatin configuration and multiscale phase separation behavior. More broadly, they contribute to a deeper understanding of the dynamic methods of genome organization employed by eukaryotic organisms.

chromatin↗

PubMed2025

BAV-LLPS: a database of bacterial, archaea, and virus liquid-liquid phase separation proteins.

MOTIVATION: Liquid-liquid phase separation (LLPS) is a key process underlying the formation of biomolecular condensates, such as membrane-less organelles, that compartmentalize biochemical processes inside the cells. While LLPS has been extensively studied in eukaryotes, its role in bacteria, archaea, and viruses remains far less characterized. Recent studies in bacteria have revealed that LLPS-driven condensates play critical roles in RNA processing, stress response, and pathogenicity. Similarly, many viruses exploit LLPS to facilitate crucial steps in their infection cycles, including viral entry, genome replication, assembly, and host immune evasion. RESULTS: In this work, we introduce a hand-curated database of LLPS proteins from bacteria, archaea, and viruses (BAV-LLPS Database). This resource, extended through sequence similarity searches, comprises over 5000 proteins and integrates diverse data including biological annotations, sequence features, predicted disordered regions, LLPS per site probability, and AlphaFold2-based structural models. Additionally, our web server enables users to explore both the curated and homologous derived datasets, providing a platform to uncover evolutionary relationships and intrinsic and differential properties of LLPS proteins across various taxonomic groups. This work seeks to deepen our understanding of LLPS mechanisms beyond eukaryotic organisms, emphasizing their significance across diverse life forms. It also aims to foster the development of specialized predictive tools that will facilitate the exploration and characterization of LLPS processes in a wide array of living organisms, thereby contributing to advancements in both fundamental biological research and applied biomedical sciences. AVAILABILITY AND IMPLEMENTATION: BAV-LLPS DB is freely accessible at https://bav-llps-db.bioinformatica.org/. The data can be retrieved from the website. The source code of the database can be downloaded from https://bav-llps-db.bioinformatica.org/download.

Databases, Protein↗

PubMed1977

Lipid phase separation induced by a hydrophobic protein in phosphatidylserine--phosphatidylcholine vesicles.

Differential scanning calorimetry (DSC) was used to detect phase separation induced by hydrophobic myelin protein, lipophilin, in a mixture of phosphatidylserine (PS) and dipalmitoylphosphatidylcholine (DPPC). Preferential binding of PS to the boundary layer of lipophilin causes a decrease in the PS content of the remaining lamellar phase with a resultant shift in the phase-transition temperature to a higher temperature. The phase diagram for this mixture in the presence and absence of lipophilin is presented. From the phase diagram, it can be estimated that for an equimolar mixture of PS and DPPC, the boundary layer contains only PS, although for higher DPPC contents, some DPPC can also be found in the boundary layer. In the case where partial phase separation in induced in this mixture by Ca2+ alone, lipophilin increases the phase separation indicating that it also binds PS preferentially in the presence of Ca2+. Preferential binding of two other acidic lipids, phosphatidic acid and phosphatidyl-glycerol, to the boundary layer was also found, including a mixture where the acidic lipid was the higher melting component in the mixture.

Calcium↗

PubMed2026

Viral replication through phase separation: Cytosolic and nuclear condensates.

Replication of many RNA and DNA viruses occurs within specialized intracellular hubs organized as membraneless biomolecular condensates (BCs) driven by liquid-liquid phase separation. As obligate intracellular parasites, viruses depend on the host cell machinery to complete their replication cycles and therefore actively remodel the intracellular environment to favor viral genome replication, transcription, and assembly. Cytosolic and nuclear phase-separated replication compartments (RC) provide concentrated and dynamic platforms that promote efficient interactions between viral genomes and viral or host proteins essential for infection. The formation of viral replication BCs is typically facilitated by viral proteins enriched in intrinsically disordered regions and low-complexity domains, which enable multivalent interactions with viral nucleic acids and cellular factors. These interactions are mediated by diverse biophysical forces, including hydrophobic and π interactions, hydrogen bonding, molecular crowding, and osmotic effects. Throughout infection, viral BCs remain highly dynamic, allowing continuous exchange of components and functional maturation of replication hubs. Their properties and activities are further regulated by post-translational modifications of viral and host proteins, such as phosphorylation, acetylation, and methylation. In this review, we summarize current evidence supporting liquid-liquid phase separation as a central organizing principle of viral RCs. We focus on representative RNA and DNA viruses that replicate in the cytosol or nucleus, highlighting virus-specific strategies, conserved mechanisms, and the consequences of BC formation for viral replication efficiency, host antiviral responses, and therapeutic intervention.

Phase Separation↗

PubMed1977

A unitary theory of anesthesia based on lateral phase separations in nerve membranes.

This paper relates research on anesthetic effects on lipid membrane systems to mechanisms of neural function. A unitary theory of anesthesia based on anesthetic-induced changes in fluid-solid-phase separations in the lipid region of nerve membranes is presented. It is suggested that anesthetics act by fluidizing nerve membranes to a point where critical lipid regions no longer contain phase separations. As a consequence, the membranes are less able to facilitate the conformational changes in proteins that may be the basis for such membrane events as ion gating, synaptic transmitter release, and transmitter binding to receptors. It is proposed that the anesthetic-modified phase separation behavior of the membrane may alter neural function by a combination of the following effects: inhibition of conformational changes of intrinsic membrane proteins; prevention of the association of protein subunits to form polymeric ion channels; depression of transmitter release by preventing fusion of vesicles containing synaptic transmitter with the membrane of the presynaptic terminal.

Anesthesia, Inhalation↗

PubMed1975

Chemically induced lipid phase separation in model membranes containing charged lipids: a spin label study.

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.

Calcium↗

PubMed2025

Phase separation of hnRNPA1 and TERRA regulates telomeric stability.

Telomeres are the complexes composed of repetitive DNA sequences and associated proteins located at the end of chromatin. As a result of the DNA replication ending issue, telomeric DNA shortens during each cell cycle. The shelterin protein complex caps telomeric ends and forms a high-order protein-DNA structure to protect telomeric DNA. The stability of telomeres is critical for cellular function and related to the progression of many human diseases. Telomeric repeat-containing RNA (TERRA) is a noncoding RNA transcribed from telomeric DNA regions. TERRA plays an essential role in regulating and maintaining the stability of telomeres. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-binding proteins associated with complex and diverse biological processes. hnRNPA1 can recognize both TERRA and telomeric DNA. Previous research reported that hnRNPA1, TERRA, and POT1, a component of the shelterin complex, work coordinately and displace replication protein A from telomeric single-stranded DNA after DNA replication, promoting telomere capping to preserve genomic integrity. However, the detailed molecular mechanism has remained unclear for >20 years. Here, our study revealed the molecular structure through which the hnRNPA1 UP1 domain interacts with TERRA and identified critical residues on the interacting surface between UP1 and TERRA. Furthermore, we proved that nucleic acids significantly increase the phase-separating ability of hnRNPA1, while disrupting the UP1-TERRA interaction extraordinarily affects hnRNPA1 droplet formation both in vitro and in vivo. Taken together, these data reveal the molecular mechanism of the phase separation of hnRNPA1 and TERRA and the potential contribution of the droplets to maintaining genomic stability.

Heterogeneous Nuclear Ribonucleoprotein A1↗

PubMed1978

Membrane lateral phase separations and chlortetracycline transport by Bacillus megaterium.

Chlortetracycline, a fluorescent probe of its own active transport, has been used to study lateral phase separations of membrane lipid in Bacillus megaterium cells. Arrhenius plots of initial accumulation rates are triphasic, with transitions or characteristic temperatures of 20 degrees and 9.5 degrees . At the higher temperature, the mobility of the chloretracycline, as measured by fluorescence polarization, is markedly altered. Chlortetracycline transport exhibits saturation kinetics, and fluorescence energy transfer from protein to bound antibiotic can be observed. N-Phenyl-1-naphthylamine, a lipophilic fluorescent probe, responds to changes in the hydrophobic regions of the membrane that are distinct from membrane protein. The fluorescent properties of N-phenylnaphthylamine in partitioning and polarization experiments are altered most significantly at the lower characteristic temperature. No fluorescence energy transfer between N-phenylnaphthylamine and membrane protein or bound tetracycline can be detected. In correlative electron spin resonance experiments on the partitioning of a lipid-soluble spin label, the same characteristic temperatures detected in the fluorescence studies were measured. These data suggest that different probes may respond to either or both of the characteristic temperatures describing the lateral phase separation. Between these characteristic temperatures the chlortetracycline transport system is most intimately associated with relatively immobile lipids that are surrounded by a more mobile lipid phase.

Bacillus megaterium↗

PubMed2025

Proteins driving liquid-liquid phase separation and histone modifications cooperatively associate with chromatin looping and transcriptional regulation.

BACKGROUND: Although liquid-liquid phase separation (LLPS) proteins are known to participate in genome organization and transcriptional regulation through the formation of biomolecular condensates, their functional interplay with other regulatory proteins and histone modifications in chromatin loop formation remains poorly characterized. By combining Hi-C chromatin interaction data with ChIP-seq profiles of 12, 27, and 24 LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively, we identified chromatin loops associated with LLPS proteins and systematically analysed patterns of cooperative protein binding and histone modification enrichment within these loop-associated peaks. RESULTS: We identified 162, 313, and 431 chromatin loops associated with LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively. These loops were relatively small in size and predominantly anchored at enhancer regions. Examination of cooperative binding of proteins within loop-associated peaks revealed that transcriptional repressor IKZF1, HDAC1, and SAP130 most frequently co-localized with LLPS proteins in GM12878, K562, and HepG2 cells, respectively. Further analysis of histone modification enrichment patterns revealed that active histone modifications, such as H3K4me2, H3K4me3, H3K9ac, and H3K27ac, co-localized at loop-associated peaks, with H3K4me1 exhibiting additional specific co-localization with these four histone modifications at enhancer-localized loop-associated peaks. Notably, bivalent chromatin domains where H3K27me3 co-localized with active histone modifications were identified at promoter-localized loop-associated peaks in HepG2 cells, and elevated H3K27me3 occupancy at these peaks was associated with transcriptional repression of target genes. Moreover, quantitative RNA-seq analysis revealed that the expression of target genes associated with enhancer-promoter loops was correlated with both the binding of LLPS proteins and the enrichment patterns of histone modifications within their ChIP-seq peaks at loop anchors. CONCLUSIONS: Our study suggests that LLPS proteins may cooperate with transcriptional repressors to facilitate chromatin looping. Furthermore, local enrichment of histone modifications at loop-associated peaks provides additional regulatory control over chromatin architecture and gene transcription.

Humans↗

PubMed1979

Effects of lipid-phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells, as studied by freeze-fracture electron microscopy.

The effects of lipid-phase separation on the filipin action on pellicle membranes of ergosterol-replaced Tetrahymena pyriformis cells were studied by freeze-fracture electron microscopy. The pellicle membranes with phase separations induced by chilling from 34 degrees C (growth temperature) to lower temperatures (30, 22 and 15 degrees C) were treated with filipin. This produced filipin-induced lesions ("pits") only in the particulated (liquid) regions along the margin between solid and liquid domains, while they were produced in the particle-free (solid) areas when membranes were chilled to 15 degrees C. The pellicle membranes with lesions induced by filipin at 34 degrees C were chilled to 22 degrees C. This chilling raised larger particle-free areas and more condensed particle-aggregations on the membranes than on the membranes without the filipin treatment. These results suggest that the membrane fluidity affects induction and development of the ergosterol-filipin complex in the membrane.

Animals↗

PubMed1979

Phase separations in membranes of Anacystis nidulans grown at different temperatures.

Freeze fracture electron microscopy studies were performed on samples of Anacystis nidulans quenched from different temperatures. Membrane lipid phase separations were observed to take place over the ranges 15--30 degrees C, 5--25 degrees C and -5--15 degrees C for cultures grown at 38, 28 and 18 degrees C, respectively. Differential scanning calorimetry heating curves showed endotherms which coincided with these temperature ranges. Variations of phase separation temperatures with growth temperature, and hysteresis effects in the calorimetric measurements, were related to changes in the fatty acid composition of membrane lipids.

Cell Fractionation↗

PubMed2024

Rabies Virus Phosphoprotein Exhibits Thermoresponsive Phase Separation with a Lower Critical Solution Temperature.

Rabies virus (RABV) generates membrane-less liquid organelles (Negri bodies) in the cytoplasm of its host cell, where genome transcription and replication and nucleocapsid assembly take place, but the mechanisms of their assembly and maturation remain to be explained. An essential component of the viral RNA synthesizing machine, the phosphoprotein (P), acts as a scaffold protein for the assembly of these condensates. This intrinsically disordered protein forms star-shaped dimers with N-terminal negatively charged flexible arms and C-terminal globular domains exhibiting a large dipole moment. Our study shows that in vitro self-association of RABV P drives a complex thermoresponsive phase separation with a lower critical solution temperature. Protein dimers assemble already below the saturation concentration, and condensation is driven by attractive conformation-specific interactions leading to reentrant liquid phase separation over a narrow range of salt concentration. We propose a minimal molecular model in which P can adopt three limit conformational states and the disordered N-terminal arms control the interactions between giant dipoles that is consistent with our observations.

Rabies virus↗

PubMed1976

The evolutionary significance of phase-separated microsystems.

The source, preparation, and properties of phase-separated systems such as lipid layers, coacervate droplets, sulphobes, and proteinoid microspheres are reviewed. These microsystems are of interest as partial models for the cell and as partial or total models for the protocell. Conceptual benefits from study of such models are: clues to experiments on origins, insights into principles of action and, in some instances, presumable models of the origin of the protocell. The benefits to evolution of organized chemical units are many, and can in part be analyzed. Ease of formation suggests that such units would have arisen early in primondiae organic evolution. Integration of these various concepts and the results of consequent experiments have contributed to the developing theory of the origins of primordial and of contemporary life.

Cells↗

PubMed1976

Cytochrome c induced lateral phase separation in a diphosphatidylglycerol-steroid spin-label model membrane.

The extrinsic membrane protein cytochrome c binds to lipid mixtures containing negatively charged phospholipids such as diphosphatidylglycerol (DPG). In this study the effect of cytochrome c on the lipid distribution in a DPG-steroid spin-label (3-doxyl-5alpha-cholestane) model membrane system is examined. The electron spin resonance (ESR) line-shape changes indicate that cytochrome c induces lateral phase separation at room temperature. The resulting two-dimensional lipid distribution is nonrandom, consisting of clusters of phospholipids bound to cytochrome c and patches of steroid spin-label molecules. Phase separations are also observed in the three-component system: DPG, phosphatidylcholine, and 3-doxyl-5alpha-cholestane.

Animals↗

PubMed1975

Synthesis of a new phosphatidylserine spin-label and calcium-induced lateral phase separation in phosphatidylserine-phosphatidylcholine membranes.

A new phosphatidylserine spin label with nitroxide stearate attached at the 2 position has been synthesized by the reaction of spin-labeled CDP-diglyceride with L-serine under the catalytic action of phosphatidylserine synthetase. Some structural properties of pure phosphatidylserine (PS) and binary PS-phosphatidylcholine (PC) membranes were studied with the spin label. PS membrane became solidified on lowering solution pH, 50% solidification being attained at pH 3.5. The membrane was also solidified by addition of Ca-2+. The effect of Ba-2+,Sr-2+, and Mg-2+ was smaller than that of Ca-2+. The calcium-induced lateral phase separation in the binary membrane was studied from the side of the calcium-receiving lipid. The results confirmed and extended our previous conclusion drawn with PC spin label. The phase diagram of the binary membrane in the presence of Ca-2+ was determined. Not all PS molecules were aggregated to form the solid patches but some remained dissolved in the fluid PC matrix. The fluid PS fraction was larger for the membranes containing more PC. The membrane with 10% PS still had a significant fraction of solid phase. The rate of calcium-induced aggregation was greatly dependent on the PS content. The aggregation was almost complete within 5 min in the membrane containing 67% PS, while it was still proceeding after several hours in the membrane with 20% PS. The rate-limiting step was suggested to be in the formation of "stable" nuclei consisting of larger aggregates. The possible biological significance of the ionotropic phase separation was discussed whereby a transient density fluctuation was emphasized.

Barium↗