High-fidelity reconstitution of stress granules and nucleoli in mammalian cellular lysate

Liquid–liquid phase separation (LLPS) is a mechanism of intracellular organization that underlies the assembly of a variety of RNP granules. Fundamental biophysical principles governing LLPS during granule assembly have been revealed by simple in vitro systems, but these systems have limitations when studying the biology of complex, multicomponent RNP granules. Visualization of RNP granules in cells has validated key principles revealed by simple in vitro systems, but this approach presents difficulties for interrogating biophysical features of RNP granules and provides limited ability to manipulate protein, nucleic acid, or small molecule concentrations. Here, we introduce a system that builds upon recent insights into the mechanisms underlying RNP granule assembly and permits high-fidelity reconstitution of stress granules and the granular component of nucleoli in mammalian cellular lysate. This system fills the gap between simple in vitro systems and live cells and allows for a variety of studies of membraneless organelles, including the development of therapeutics that modify properties of specific condensates.     

Sequence dependent phase separation of protein-polynucleotide mixtures elucidated using molecular simulations

Ribonucleoprotein (RNP) granules are membraneless organelles (MLOs), which majorly consist of RNA and RNA-binding proteins and are formed via liquid–liquid phase separation (LLPS). Experimental studies investigating the drivers of LLPS have shown that intrinsically disordered proteins (IDPs) and nucleic acids like RNA and other polynucleotides play a key role in modulating protein phase separation. There is currently a dearth of modelling techniques which allow one to delve deeper into how polynucleotides play the role of a modulator/promoter of LLPS in cells using computational methods. Here, we present a coarse-grained polynucleotide model developed to fill this gap, which together with our recently developed HPS model for protein LLPS, allows us to capture the factors driving protein-polynucleotide phase separation. We explore the capabilities of the modelling framework with the LAF-1 RGG system which has been well studied in experiments and also with the HPS model previously. Further taking advantage of the fact that the HPS model maintains sequence specificity we explore the role of charge patterning on controlling polynucleotide incorporation into condensates. With increased charge patterning we observe formation of structured or patterned condensates which suggests the possible roles of polynucleotides in not only shifting the phase boundaries but also introducing microscopic organization in MLOs.     

The physical forces mediating self-association and phase-separation in the C-terminal domain of TDP-43

The TAR DNA-binding protein of 43 kDa (TDP-43) has been identified as the main component of amyotrophic lateral sclerosis (ALS) cytoplasmic inclusions. The link between this proteinopathy and TDP-43′s intrinsically disordered C-terminal domain is well known, but recently also, this domain has been shown to be involved in the formation of the membraneless organelles that mediate TDP-43′s functions. The mechanisms that underpin the liquid-liquid phase separation (LLPS) of these membraneless organelles undergo remain elusive. Crucially though, these factors may be the key to understanding the delicate balance between TDP-43′s physiological and pathological functions. In this study, we used nuclear magnetic resonance spectroscopy and optical methods to demonstrate that an α-helical component in the centre (residues 320–340) of the C-terminal domain is related to the protein's self-association and LLPS. Systematically analysing ALS-related TDP-43 mutants (G298S, M337V, and Q331K) in different buffer conditions at different temperatures, we prove that this phase separation is driven by hydrophobic interactions but is inhibited by electrostatic repulsion. Based on these findings, we rationally introduced a mutant, W334G, and demonstrate that this mutant disrupts LLPS without disturbing this α-helical propensity. This tryptophan may serve as a key residue in this protein's LLPS.     

Initiation of stress granule assembly by rapid clustering of IGF2BP proteins upon osmotic shock

Stress granules (SGs) are membraneless organelles formed in the cytoplasm by liquid-liquid phase separation (LLPS) of translationally-stalled mRNA and RNA-binding proteins during stress response. Understanding the mechanisms governing SG assembly requires imaging SG formation in real time. Although numerous SG proteins have been identified, the kinetics of their recruitment during SG assembly has not been well established. Here we used live cell imaging and super-resolution imaging to visualize SG assembly in human cells. We found that IGF2BP proteins formed microscopically visible clusters in living cells almost instantaneously after osmotic stress, followed by fusion of clusters and the recruitment of G3BP1 and TIA1. Rapid clustering of IGF2BP1 was reduced in cells pretreated with emetine that stabilizes polysomes on mRNA. The KH3/4 di-domain and an intrinsically disordered region (IDR) of IGF2BP1 were found to mediate its clustering. Super-resolution imaging confirmed the formation of IGF2BP clusters associated with mRNA at 40 s after osmotic stress. In mature SGs, multiple clusters of poly(A) mRNA were found to associate with the periphery and the interior of a dense granule formed by IGF2BP1. Taken together, our findings revealed a novel, multi-stage LLPS process during osmotic stress, in which rapid clustering of IGF2BP proteins initiates SG assembly.     

Get closer and make hotspots: liquid–liquid phase separation in plants

Liquid–liquid phase separation (LLPS) facilitates the formation of membraneless compartments in a cell and allows the spatiotemporal organization of biochemical reactions by concentrating macromolecules locally. In plants, LLPS defines cellular reaction hotspots, and stimulus‐responsive LLPS is tightly linked to a variety of cellular and biological functions triggered by exposure to various internal and external stimuli, such as stress responses, hormone signaling, and temperature sensing. Here, we provide an overview of the current understanding of physicochemical forces and molecular factors that drive LLPS in plant cells. We illustrate how the biochemical features of cellular condensates contribute to their biological functions. Additionally, we highlight major challenges for the comprehensive understanding of biological LLPS, especially in view of the dynamic and robust organization of biochemical reactions underlying plastic responses to environmental fluctuations in plants.     

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.     

A predictive coarse-grained model for position-specific effects of post-translational modifications

Biomolecules undergo liquid-liquid phase separation (LLPS), resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post-translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed-charge atomistic force fields, we parameterize the size and atomistic hydropathy of the coarse-grained-modified amino acid beads and, hence, the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the protein’s single-chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of Fused in Sarcoma (FUS) and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution.     

Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble

Liquid–liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.     

Controlling Liquid–Liquid Phase Separation of Cold-Adapted Crystallin Proteins from the Antarctic Toothfish

Liquid–liquid phase separation (LLPS) of proteins is important to a variety of biological processes both functional and deleterious, including the formation of membraneless organelles, molecular condensations that sequester or release molecules in response to stimuli, and the early stages of disease-related protein aggregation. In the protein-rich, crowded environment of the eye lens, LLPS manifests as cold cataract. We characterize the LLPS behavior of six structural γ-crystallins from the eye lens of the Antarctic toothfish Dissostichus mawsoni, whose intact lenses resist cold cataract in subzero waters. Phase separation of these proteins is not strongly correlated with thermal stability, aggregation propensity, or cross-species chaperone protection from heat denaturation. Instead, LLPS is driven by protein–protein interactions involving charged residues. The critical temperature of the phase transition can be tuned over a wide temperature range by selective substitution of surface residues, suggesting general principles for controlling this phenomenon, even in compactly folded proteins.     

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization,phase separation,and RNA splicing

TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro, fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.     

Modulation of biomolecular phase behavior by metal ions

Liquid–liquid phase separation (LLPS) appears to be a newly appreciated aspect of the cellular organization of biomolecules that leads to the formation of membraneless organelles (MLOs). MLOs generate distinct microenvironments where particular biomolecules are highly concentrated compared to those in the surrounding environment. Their thermodynamically driven formation is reversible, and their liquid nature allows them to fuse with each other. Dysfunctional biomolecular condensation is associated with human diseases. Pathological states of MLOs may originate from the mutation of proteins or may be induced by other factors. In most aberrant MLOs, transient interactions are replaced by stronger and more rigid interactions, preventing their dissolution, and causing their uncontrolled growth and dysfunction. For these reasons, there is great interest in identifying factors that modulate LLPS. In this review, we discuss an enigmatic and mostly unexplored aspect of this process, namely, the regulatory effects of metal ions on the phase behavior of biomolecules.     

Liquid-liquid phase separation as a common organizing principle of intracellular space and biomembranes providing dynamic adaptive responses

This work is devoted to the phenomenon of liquid-liquid phase separation (LLPS), which has come to be recognized as fundamental organizing principle of living cells. We distinguish separation processes with different dimensions. Well-known 3D-condensation occurs in aqueous solution and leads to membraneless organelle (MLOs) formation. 2D-films may be formed near membrane surfaces and lateral phase separation (membrane rafts) occurs within the membranes themselves. LLPS may also occur on 1D structures like DNA and the cyto- and nucleoskeleton. Phase separation provides efficient transport and sorting of proteins and metabolites, accelerates the assembly of metabolic and signaling complexes, and mediates stress responses. In this work, we propose a model in which the processes of polymerization (1D structures), phase separation in membranes (2D structures), and LLPS in the volume (3D structures) influence each other. Disordered proteins and whole condensates may provide membrane raft separation or polymerization of specific proteins. On the other hand, 1D and 2D structures with special composition or embedded IDRs can nucleate condensates. We hypothesized that environmental change may trigger a LLPS which can propagate within the cell interior moving along the cytoskeleton or as an autowave. New phase propagation quickly and using a low amount of energy adjusts cell signaling and metabolic systems to new demands. Cumulatively, the interconnected phase separation phenomena in different dimensions represent a previously unexplored system of intracellular communication and regulation which cannot be ignored when considering both physiological and pathological cell processes.     

Phase separation in amino acid mixtures is governed by composition

Macromolecular phase separation has recently come to immense prominence as it is central to the formation of membraneless organelles, leading to a new paradigm of cellular organization. This type of phase transition, often termed liquid-liquid phase separation (LLPS), is mediated by molecular interactions between biomolecules, including nucleic acids and both ordered and disordered proteins. In the latter case, the separation between protein-dense and -dilute phases is often interpreted using models adapted from polymer theory. Specifically, the “stickers and spacers” model proposes that the formation of condensate-spanning networks in protein solutions originates from the interplay between two classes of residues and that the main determinants for phase separation are multivalency and sequence patterning. The duality of roles of stickers (aromatics like Phe and Tyr) and spacers (Gly and polar residues) may apply more broadly in protein-like mixtures, and the presence of these two types of components alone may suffice for LLPS to take place. In order to explore this hypothesis, we use atomistic molecular dynamics simulations of capped amino acid residues as a minimal model system. We study the behavior of pure amino acids in water for three types of residues corresponding to the spacer and sticker categories and of their multicomponent mixtures. In agreement with previous observations, we find that the spacer-type amino acids fail to phase separate on their own, while the sticker is prone to aggregation. However, ternary amino acid mixtures involving both types of amino acids phase separate into two phases that retain intermediate degrees of compaction and greater fluidity than sticker-only condensates. Our results suggest that LLPS is an emergent property of amino acid mixtures determined by composition.     

Metals and inorganic molecules in regulating protein and nucleic acid phase separation

The realization that liquid–liquid phase separation (LLPS) underlies the formation of membraneless compartments in cells has motivated efforts to modulate the condensation process of biomolecules. Increasing evidence shows that metals and inorganic molecules abundantly distributed in cells play important roles in the regulation of biomolecular condensation. Herein, we briefly reviewed the background of biomacromolecular phase separation and summarized the recent research progress on the roles of metals and inorganic molecules in regulating protein and nucleic acid phase separation in vitro and in cells.     

The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid–liquid phase separation

Liquid–liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.     

生物大分子相分离在植物中的研究进展

细胞中存在种类繁多的无膜细胞器,在感知环境信号,基因表达调控,RNA加工等过程中发挥了重要的作用,而生物大分子相分离被证明是无膜细胞器形成的主要方式。文章介绍了生物大分子相分离的概念与特征,总结了有关相分离在植物对环境信号响应中的研究进展,并对相分离在植物中的生物学功能进行了分类,以期解析相分离在植物生长发育和逆境适应中的作用机理,揭示植物无膜细胞器的本质与功能。     

Electrostatic modulation of hnRNPA1 low‐complexity domain liquid–liquid phase separation and aggregation

Membrane‐less organelles and RNP granules are enriched in RNA and RNA‐binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear‐cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C‐terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid–liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane‐less organelles, aggregation within phase‐separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS‐mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS‐mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.     

Liquid-Liquid Phase Separation of Histone Proteins in Cells: Role in Chromatin Organization

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids has emerged as an important phenomenon in membraneless intracellular organization. We demonstrate that the linker histone H1 condenses into liquid-like droplets in the nuclei of HeLa cells. The droplets, observed during the interphase of the cell cycle, are colocalized with DNA-dense regions indicative of heterochromatin. In vitro, H1 readily undergoes LLPS with both DNA and nucleosomes of varying lengths but does not phase separate in the absence of DNA. The nucleosome core particle maintains its structural integrity inside the droplets, as demonstrated by FRET. Unexpectedly, H2A also forms droplets in the presence of DNA and nucleosomes in vitro, whereas the other core histones precipitate. The phase diagram of H1 with nucleosomes is invariant to the nucleosome length at physiological salt concentration, indicating that H1 is capable of partitioning large segments of DNA into liquid-like droplets. Of the proteins tested (H1, core histones, and the heterochromatin protein HP1α), this property is unique to H1. In addition, free nucleotides promote droplet formation of H1 nucleosome in a nucleotide-dependent manner, with droplet formation being most favorable with ATP. Although LLPS of HP1α is known to contribute to the organization of heterochromatin, our results indicate that H1 also plays a role. Based on our study, we propose that H1 and DNA act as scaffolds for phase-separated heterochromatin domains.     

Liquid-liquid Phase Separation in Viral Function

An emerging set of results suggests that liquid-liquid phase separation (LLPS) is the basis for the formation of membrane-less compartments in cells. Evidence is now mounting that various types of virus-induced membrane-less compartments and organelles are also assembled via LLPS. Specifically, viruses appear to use intracellular phase transitions to form subcellular microenvironments known as viral factories, inclusion bodies, or viroplasms. These compartments - collectively referred to as viral biomolecular condensates - can be used to concentrate replicase proteins, viral genomes, and host proteins that are required for virus replication. They can also be used to subvert or avoid the intracellular immune response. This review examines how certain DNA or RNA viruses drive the formation of viral condensates, the possible biological functions of those condensates, and the biophysical and biochemical basis for their assembly.