Biological Phase Separation Research Articles

Liquid-Liquid Phase Separation in Biology: Specific Stoichiometric Molecular Interactions vs Promiscuous Interactions Mediated by Disordered Sequences.

Extensive studies in the past few years have shown that nonmembrane bound organelles are likely assembled via liquid-liquid phase separation (LLPS), a process that is driven by multivalent protein-protein and/or protein-nucleic acid interactions. Both stoichiometric molecular interactions and intrinsically disordered region (IDR)-driven interactions can promote the assembly of membraneless organelles, and the field is currently dominated by IDR-driven biological condensate formation. Here we discuss recent studies that demonstrate the importance of specific biomolecular interactions for functions of diverse physiological condensates. We suggest that phase separation based on combinations of specific interactions and promiscuous IDR-driven interactions is likely a general feature of biological condensation under physiological conditions.

Roles of Phase Separation for Cellular Redox Maintenance.

The oxidation reaction greatly alters characteristics of various cellular components. In exchange for efficient energy production, mitochondrial aerobic respiration substantially increases the risk of excess oxidation of cellular biomolecules such as lipids, proteins, nucleic acids, and numerous small molecules. To maintain a physiologically balanced cellular reduction-oxidation (redox) state, cells utilize a variety of molecular machineries including cellular antioxidants and protein degradation complexes such as the ubiquitin-proteasome system or autophagy. In the past decade, biomolecular liquid-liquid phase separation (LLPS) has emerged as a subject of great interest in the biomedical field, as it plays versatile roles in the maintenance of cellular homeostasis. With regard to redox homeostasis, LLPS arose as a major player in both well-characterized and newly emerging redox pathways. LLPS is involved in direct redox imbalance sensing, signal transduction, and transcriptional regulation. Also, LLPS is at play when cells resist redox imbalance through metabolic switching, translational remodeling, activating the DNA damage response, and segregation of vulnerable lipids and proteins. On the other hand, chronic accumulation of phase-separated molecular condensates such as lipid droplets and amyloid causes neurotoxic outcomes. In this review we enumerate recent progress on understanding how cells utilize LLPS to deal with oxidative stress, especially related to cell survival or pathogenesis, and we discuss future research directions for understanding biological phase separation in cellular redox regulation.

An interview with Mingjie Zhang: phase separation in biological systems.

Mingjie Zhang is a distinguished structural biologist whose research interest is the protein assemblies in cells, particularly those at the synapse, where two neurons meet and transmit nervous impulses from one to the other. In recent years, his group discovered the phenomenon of phase separation in both the pre- and post-synaptic sites of neurons and elucidated its regulation and physiological function. In this interview, NSR Executive Editor-in-Chief Mu-ming Poo talks with Prof. Zhang on the fast-developing research area of biological phase separation, as well as Zhang's new role as the Founding Dean of the School of Life Sciences at Southern University of Science and Technology (SUSTech) in Shenzhen.

Is transcriptional regulation just going through a phase?

The formation of nuclear structures such as Cajal bodies and nucleoli has been long accepted as arising from phase-separation processes. However, liquid-liquid phase separation is now being implicated in a multitude of additional nuclear processes. This includes transcriptional repression via heterochromatin and transcriptional activation via enhancer-promoter cooperation. The seemingly widespread presence of phase-separation in biology has generated much excitement in research and biotechnology.

Biological Phase Separation and Biomolecular Condensates in Plants.

A surge in research focused on understanding the physical principles governing the formation, properties, and function of membraneless compartments has occurred over the past decade. Compartments such as the nucleolus, stress granules, and nuclear speckles have been designated as biomolecular condensates to describe their shared property of spatially concentrating biomolecules. Although this research has historically been carried out in animal and fungal systems, recent work has begun to explore whether these same principles are relevant in plants. Effectively understanding and studying biomolecular condensates require interdisciplinary expertise that spans cell biology, biochemistry, and condensed matter physics and biophysics. As such, some involved concepts may be unfamiliar to any given individual. This review focuses on introducing concepts essential to the study of biomolecular condensates and phase separation for biologists seeking to carry out research in this area and further examines aspects of biomolecular condensates that are relevant to plant systems.

Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies

Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases—homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.

Quantification of the Onset of Condensation in Negative Elongation Factors

Several biomolecules condensate in cells to form membraneless organelles via liquid-liquid phase separation, usually in response to changes in their environment. Recent studies suggest that biological phase separation for example regulates cell survival and pathology. The process of biological phase separation itself is not well understood. It is for example unclear how many proteins of a kind are necessary to induce a dense phase in cells – or if the local concentration is the limiting factor. Here we employ highly inclined and laminated optical sheet microscopy and single-molecule fluorescence methods to quantify early steps of phase separation of the negative elongation factor (NELF). NELF downregulates transcription and condensates upon heat and toxin stress. We treat the NELF as a model system to quantify the threshold concentration and rapidness of NELF condensation at low expression levels in fixed and live cells. by counting the number of NELF proteins in the dense phase in a time dependent manner we will contribute to understand the molecular mechanisms underlying liquid-liquid phase separation in cells.

Modeling Chromatin Condensation with Coarse-Grained Models

Insights into the detailed molecular interactions driving liquid-liquid phase separation (LLPS) in biology are difficult to obtain experimentally but crucial for understanding a wide variety of biomolecular interactions. Thus, understanding the driving forces and mechanics of LLPS are crucial, particularly as it applies to chromatin condensation. While experimental techniques for studying LLPS condensates are able to yield macroscopic information such as phase diagrams, teasing out the interactions stabilizing the condensed phase is challenging owing to its heterogeneous, disordered structure and high viscosity.

Interactions between Phase-Separated Liquids and Membrane Surfaces.

Liquid-liquid phase separation has recently emerged as an important fundamental organizational phenomenon in biological settings. Most studies of biological phase separation have focused on droplets that “condense” from solution above a critical concentration, forming so-called “membraneless organelles” suspended in solution. However, membranes are ubiquitous throughout cells, and many biomolecular condensates interact with membrane surfaces. Such membrane-associated phase-separated systems range from clusters of integral or peripheral membrane proteins in the plane of the membrane to free, spherical droplets wetting membrane surfaces to droplets containing small lipid vesicles. In this review, we consider phase-separated liquids that interact with membrane surfaces and we discuss the consequences of those interactions. The physical properties of distinct liquid phases in contact with bilayers can reshape the membrane, and liquid-liquid phase separation can construct membrane-associated protein structures, modulate their function, and organize collections of lipid vesicles dynamically. We summarize the common phenomena that arise in these systems of liquid phases and membranes.

Chemical Insights into Liquid-Liquid Phase Separation in Molecular Biology

Abstract Liquid-liquid phase separation (LLPS) in living organisms is a recently emerging biologic principle that may dramatically alter current perceptions of cellular systems. Various proteins, RNAs, and other biomolecules undergo LLPS, exhibiting various cellar functions. The field is still immature, however, there is no consensus regarding the basic experimental techniques used for characterizing the phenomenon, knowledge of the physicochemical basis driving and regulating LLPS in cells is insufficient, and very little is known about potential chemical interventions for LLPS. Addressing these deficiencies requires chemical approaches, and will markedly advance drug discovery, molecular biology, and medicine. Here, we introduce the basic biology of LLPS and present challenges in the field from a chemical viewpoint.

Biomolecular phase separation through the lens of sodium-23 NMR.

Phase separation is a fundamental physicochemical process underlying the spatial arrangement and coordination of cellular events. Detailed characterization of biomolecular phase separation requires experimental access to the internal environment of dilute and especially condensed phases at high resolution. In this study, we take advantage from the ubiquitous presence of sodium ions in biomolecular samples and present the potentials of 23Na NMR as a proxy to report the internal fluidity of biomolecular condensed phases. After establishing the temperature and viscosity dependence of 23Na NMR relaxation rates and translational diffusion coefficient, we demonstrate that 23Na NMR probes of rotational and translational mobility of sodium ions are capable of capturing the increasing levels of confinement in agarose gels in dependence of agarose concentration. The 23Na NMR approach is then applied to a gel‐forming phenylalanine‐glycine (FG)‐containing peptide, part of the nuclear pore complex involved in controlling the traffic between cytoplasm and cell nucleus. It is shown that the 23Na NMR together with the 17O NMR provide a detailed picture of the sodium ion and water mobility within the interior of the FG peptide hydrogel. As another example, we study phase separation in water‐triethylamine (TEA) mixture and provide evidence for the presence of multiple microscopic environments within the TEA‐rich phase. Our results highlight the potentials of 23Na NMR in combination with 17O NMR in studying biological phase separation, in particular with regards to the molecular properties of biomolecular condensates and their regulation through various physico‐ and biochemical factors.

Two-dimensional droplet ripening in a concentration gradient

The discovery of various membraneless subcellular structures (biological condensates) in the cytoplasm and nucleus of cells has generated considerable interest in the effects of non-equilibrium chemical reactions on liquid–liquid phase separation and droplet ripening. Examples include the suppression of droplet ripening due to ATP-driven protein phosphorylation and the spatial segregation of droplets due to regulation by protein concentration gradients. Most studies of biological phase separation have focused on 3D droplet formation, for which mean field methods can be applied. However, mean field theory breaks down in the case of 2D systems, since the concentration around a droplet varies as ln R rather than R−1, where R is the distance from the center of a droplet. In this paper we use the asymptotic theory of diffusion in domains with small holes or exclusions (strongly localized perturbations) to study the segregation of circular droplets in gradient systems. We proceed by partitioning the region outside the droplets into a set of inner regions around each droplet together with an outer region where mean-field interactions occur. Asymptotically matching the inner and outer solutions, we derive dynamical equations for the position-dependent growth and drift of droplets. We thus show how a gradient of regulatory proteins leads to the segregation of droplets to one end of the domain, as previously found for 3D droplets.

Phase Separation in Membrane Biology: The Interplay between Membrane-Bound Organelles and Membraneless Condensates.

In eukaryotic cells, various membrane-bound organelles compartmentalize diverse cellular activities in a spatially and temporally controlled manner. Numerous membraneless organelles assembled via liquid-liquid phase separation (LLPS), known as condensates, also facilitate compartmentalization of cellular functions. Emerging evidence shows that these two organelle types interact in many biological processes. Membranes modulate the biogenesis and dynamics of phase-separated condensates by serving as assembly platforms or by forming direct contacts. Phase separation of membrane-associated proteins participates in various trafficking events, such as clustering of vesicles for temporally controlled fusion and storage, and transport of membraneless condensates on membrane-bound organelles. Phase separation also acts in cargo trafficking pathways by sorting and docking cargos for translocon-mediated transport across membranes, by shuttling cargos through the nuclear pore complex, and by triggering the formation of surrounding autophagosomes for delivery to lysosomes. The coordinated actions of membrane-bound and membraneless organelles ensure spatiotemporal control of various cellular functions.

Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases.

Cells are compartmentalized by numerous membrane-enclosed organelles and membraneless compartments to ensure that a wide variety of cellular activities occur in a spatially and temporally controlled manner. The molecular mechanisms underlying the dynamics of membrane-bound organelles, such as their fusion and fission, vesicle-mediated trafficking and membrane contactmediated inter-organelle interactions, have been extensively characterized. However, the molecular details of the assembly and functions of membraneless compartments remain elusive. Mounting evidence has emerged recently that a large number of membraneless compartments, collectively called biomacromolecular condensates, are assembled via liquid-liquid phase separation (LLPS). Phase-separated condensates participate in various biological activities, including higher-order chromatin organization, gene expression, triage of misfolded or unwanted proteins for autophagic degradation, assembly of signaling clusters and actin- and microtubule-based cytoskeletal networks, asymmetric segregations of cell fate determinants and formation of pre- and post-synaptic density signaling assemblies. Biomacromolecular condensates can transition into different material states such as gel-like structures and solid aggregates. The material properties of condensates are crucial for fulfilment of their distinct functions, such as biochemical reaction centers, signaling hubs and supporting architectures. Cells have evolved multiple mechanisms to ensure that biomacromolecular condensates are assembled and disassembled in a tightly controlled manner. Aberrant phase separation and transition are causatively associated with a variety of human diseases such as neurodegenerative diseases and cancers. This review summarizes recent major progress in elucidating the roles of LLPS in various biological pathways and diseases.

Biomolecular Phase Separation: From Molecular Driving Forces to Macroscopic Properties.

Biological phase separation is known to be important for cellular organization, which has recently been extended to a new class of biomolecules that form liquid-like droplets coexisting with the surrounding cellular or extracellular environment. These droplets are termed membraneless organelles, as they lack a dividing lipid membrane, and are formed through liquid-liquid phase separation (LLPS). Elucidating the molecular determinants of phase separation is a critical challenge for the field, as we are still at the early stages of understanding how cells may promote and regulate functions that are driven by LLPS. In this review, we discuss the role that disorder, perturbations to molecular interactions resulting from sequence, posttranslational modifications, and various regulatory stimuli play on protein LLPS, with a particular focus on insights that may be obtained from simulation and theory. We finally discuss how these molecular driving forces alter multicomponent phase separation and selectivity.

Interfacial solvation can explain attraction between like-charged objects in aqueous solution.

Over the past few decades, the experimental literature has consistently reported observations of attraction between like-charged colloidal particles and macromolecules in aqueous solution. Examples include nucleic acids and colloidal particles in the bulk solution and under confinement, and biological liquid-liquid phase separation. This observation is at odds with the intuitive expectation of an interparticle repulsion that decays monotonically with distance. Although attraction between like-charged particles can be rationalized theoretically in the strong-coupling regime, e.g., in the presence of multivalent counterions, recurring accounts of long-range attraction in aqueous solution containing monovalent ions at low ionic strength have posed an open conundrum. Here, we show that the behavior of molecular water at an interface-traditionally disregarded in the continuum electrostatics picture-provides a mechanism to explain the attraction between like-charged objects in a broad spectrum of experiments. This basic principle will have important ramifications in the ongoing quest to better understand intermolecular interactions in solution.

Phase separation at the synapse.

Emerging evidence indicates that liquid-liquid phase separation, the formation of a condensed molecular assembly within another diluted aqueous solution, is a means for cells to organize highly condensed biological assemblies (also known as biological condensates or membraneless compartments) with very broad functions and regulatory properties in different subcellular regions. Molecular machineries dictating synaptic transmissions in both presynaptic boutons and postsynaptic densities of neuronal synapses may be such biological condensates. Here we review recent developments showing how phase separation can build dense synaptic molecular clusters, highlight unique features of such condensed clusters in the context of synaptic development and signaling, discuss how aberrant phase-separation-mediated synaptic assembly formation may contribute to dysfunctional signaling in psychiatric disorders, and present some challenges and opportunities of phase separation in synaptic biology.

Contribution of organic composition and loading to enhance nutrient removal in biological spatial-temporal phase separation

A biological spatial-temporal phase separation system without an additional anaerobic reactor was devised for simultaneous removal of nitrogen and phosphorus. The process was operated at hydraulic retention time (HRT) of 8 h, solids retention time (SRT) of 10 days, and cycle time of 4 h (Anoxic/Anaerobic periods : Aerobic period = 1.5 : 2.5). Three types of influents were examined, namely glucose, glucose/acetate/propionate, and domestic wastewater. They were used to evaluate the contribution of organic composition and loading on the system performance. Nitrogen removal was strongly affected by the organic composition upon low influent C/N ratio. However, for influent C/N of 9.2–11.7 and C/P of 53.0–88.1, nitrogen and phosphorus removal noticeably improved regardless of the organic composition. Biological reactions including nitrification, denitrification, phosphate release and uptake, and PHA synthesis and utilization were clearly observed during the cycle with high organic loading, without any decline on organics removal. Furthermore, significant phosphate release and uptake were successfully accomplished without any nitrate interference and carbon competition under high organic loading. Therefore, organic loading is essential for the simultaneous removal of nitrogen and phosphorus in biological spatial-temporal phase separation.

Phase separation in biology and disease-a symposium report.

Phase separation of multivalent protein and RNA molecules enables cells the formation of reversible nonstoichiometric, membraneless assemblies. These assemblies, referred to as biomolecular condensates, help with the spatial organization and compartmentalization of cellular matter. Each biomolecular condensate is defined by a distinct macromolecular composition. Distinct condensates have distinct preferential locations within cells, and they are associated with distinct biological functions, including DNA replication, RNA metabolism, signal transduction, synaptic transmission, and stress response. Several proteins found in biomolecular condensates have also been implicated in disease, including Huntington's disease, amyotrophic lateral sclerosis, and several types of cancer. Disease-associated mutations in these proteins have been found to affect the material properties of condensates as well as the driving forces for phase separation. Understanding the intrinsic and extrinsic forces driving the formation and dissolution of biomolecular condensates via spontaneous and driven phase separation is an important step in understanding the processes associated with biological regulation in health and disease.

Cellular consequences of arginine methylation

Arginine methylation is a ubiquitous post-translational modification. Three predominant types of arginine-guanidino methylation occur in Eukarya: mono (Rme1/MMA), symmetric (Rme2s/SDMA), and asymmetric (Rme2a/ADMA). Arginine methylation frequently occurs at sites of protein-protein and protein-nucleic acid interactions, providing specificity for binding partners and stabilization of important biological interactions in diverse cellular processes. Each methylarginine isoform-catalyzed by members of the protein arginine methyltransferase family, Type I (PRMT1-4,6,8) and Type II (PRMT5,9)-has unique downstream consequences. Methylarginines are found in ordered domains, domains of low complexity, and in intrinsically disordered regions of proteins-the latter two of which are intimately connected with biological liquid-liquid phase separation. This review highlights discoveries illuminating how arginine methylation affects genome integrity, gene transcription, mRNA splicing and mRNP biology, protein translation and stability, and phase separation. As more proteins and processes are found to be regulated by arginine methylation, its importance for understanding cellular physiology will continue to grow.