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The phase separation of N 6 -methyladenosine (m 6 A) binding protein YTHDF2 plays a vital role in arsenic-induced skin damage, and YTHDF2 can bind to m 6 A-methylated mRNA of tumor suppressor PTEN. However, whether and how YTHDF2 phase separation regulates PTEN involved in arsenic-induced malignant transformation of keratinocytes remains blank. Here, we established arsenite-induced transformation models with stable expression of wild-type YTHDF2 or mutant YTHDF2 protein in vitro and in vivo. We found that the YTHDF2 protein underwent phase separation during arsenite-induced malignant transformation of keratinocytes, and YTHDF2 phase separation promoted the malignant phenotype of keratinocytes. Mechanically, YTHDF2 phase separation reduced PTEN protein levels, which in turn activated the pro-survival AKT signal. The binding of YTHDF2 to multiple m 6 A sites on PTEN mRNA drove YTHDF2 phase separation, inhibiting PTEN translation initiation and thus reducing PTEN protein levels. YTHDF2 phase separation recruited translation-initiation-factor kinase EIF2AK1 to phosphorylate eIF2α, thereby inhibiting translation initiation of poly-m 6 A-methylated PTEN mRNA. Furthermore, arsenite-induced oxidative stress triggered YTHDF2 phase separation by increasing m 6 A levels of PTEN mRNA. Our results demonstrated that YTHDF2 phase separation promotes arsenite-induced malignant transformation by inhibiting PTEN translation in a poly-m 6 A-dependent manner. This study sheds light on arsenic carcinogenicity from the novel aspect of m 6 A-mediated YTHDF2 phase separation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The SARS-CoV-2 Nucleocapsid protein (N) performs several functions during the viral lifecycle, including transcription regulation and viral genome encapsulation. We hypothesized that N toggles between these functions via phosphorylation-induced conformational change, thereby altering N interactions with membranes and RNA. We found that phosphorylation changes how biomolecular condensates composed of N and RNA interact with membranes: phosphorylated N (pN) condensates form thin films, while condensates with unmodified N are engulfed. This partly results from changes in material properties, with pN forming less viscous and elastic condensates. The weakening of protein-RNA interaction in condensates upon phosphorylation is driven by a decrease in binding between pN and unstructured RNA. We show that phosphorylation induces a conformational change in the serine/arginine-rich region of N that increases interaction between pN monomers and decreases nonspecific interaction with RNA. These findings connect the conformation, material properties, and membrane-associated states of N, with potential implications for COVID-19 treatment., Competing Interests: Declaration of Interests The authors declare no competing interests.

Liquid-liquid phase separation (LLPS) regulates many biological processes including RNA metabolism, chromatin rearrangement, and signal transduction. Aberrant LLPS potentially leads to serious diseases. Therefore, the identification of the LLPS proteins is crucial. Traditionally, biochemistry-based methods for identifying LLPS proteins are costly, time-consuming, and laborious. In contrast, artificial intelligence-based approaches are fast and cost-effective and can be a better alternative to biochemistry-based methods. Previous research methods employed word2vec in conjunction with machine learning or deep learning algorithms. Although word2vec captures word semantics and relationships, it might not be effective in capturing features relevant to protein classification, like physicochemical properties, evolutionary relationships, or structural features. Additionally, other studies often focused on a limited set of features for model training, including planar π contact frequency, pi-pi, and β-pairing propensities. To overcome such shortcomings, this study first constructed a reliable dataset containing 1206 protein sequences, including 603 LLPS and 603 non-LLPS protein sequences. Then a computational model was proposed to efficiently identify the LLPS proteins by perceiving semantic information of protein sequences directly; using an ESM2-36 pre-trained model based on transformer architecture in conjunction with a convolutional neural network. The model could achieve an accuracy of 85.68% and 89.67%, respectively on training data and test data, surpassing the accuracy of previous studies. The performance demonstrates the potential of our computational methods as efficient alternatives for identifying LLPS proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Some negative-sense RNA viruses, including measles virus (MeV), share the characteristic that during their infection cycle, cytoplasmic inclusion bodies (IBs) are formed where components of the viral replication machinery are concentrated. As a foci of viral replication, how IBs act to enhance the efficiency of infection by affecting virus-host interactions remains an important topic of investigation. We previously established that upon MeV infection, the epigenetic host protein, WD repeat-containing protein 5 (WDR5), translocates to cytoplasmic viral IBs and facilitates MeV replication. We now show that WDR5 is recruited to IBs by forming a complex with IB-associated MeV phosphoprotein via a conserved binding motif located on the surface of WDR5. Furthermore, we provide evidence that WDR5 promotes viral replication by suppressing a major innate immune response pathway, the double-stranded RNA-mediated activation of protein kinase R and integrated stress response., Importance: MeV is a pathogen that remains a global concern, with an estimated 9 million measles cases and 128,000 measles deaths in 2022 according to the World Health Organization. A large population of the world still has inadequate access to the effective vaccine against the exceptionally transmissible MeV. Measles disease is characterized by a high morbidity in children and in immunocompromised individuals. An important area of research for negative-sense RNA viruses, including MeV, is the characterization of the complex interactome between virus and host occurring at cytoplasmic IBs where viral replication occurs. Despite the progress made in understanding IB structures, little is known regarding the virus-host interactions within IBs and the role of these interactions in promoting viral replication and antagonizing host innate immunity. Herein we provide evidence suggesting a model by which MeV IBs utilize the host protein WDR5 to suppress the protein kinase R-integrated stress response pathway., Competing Interests: The authors declare no conflict of interest.

Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid. In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid-liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies. These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA double-strand break (DSB) is the most dangerous type of DNA damage, which may lead to cell death or oncogenic mutations. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are two typical DSB repair mechanisms. Recently, many studies have revealed that liquid-liquid phase separation (LLPS) plays a pivotal role in DSB repair and response. Through LLPS, the crucial biomolecules are quickly recruited to damaged sites with a high concentration to ensure DNA repair is conducted quickly and efficiently, which facilitates DSB repair factors activating downstream proteins or transmitting signals. In addition, the dysregulation of the DSB repair factor's phase separation has been reported to promote the development of a variety of diseases. This review not only provides a comprehensive overview of the emerging roles of LLPS in the repair of DSB but also sheds light on the regulatory patterns of phase separation in relation to the DNA damage response (DDR).

Background: The growth and metastasis of pancreatic cancer (PC) has been found to be closely associated with liquid-liquid phase separation (LLPS). This study sought to identify LLPS-related biomarkers in PC to construct a robust prognostic model., Methods: Transcriptomic data and clinical information related to PC were retrieved from publicly accessible databases. The PC-related data set was subjected to differential expression, Mendelian randomization (MR), univariate Cox, and least absolute selection and shrinkage operator analyses to identify biomarkers. Using the biomarkers, we subsequently constructed a risk model, identified the independent prognostic factors of PC, established a nomogram, and conducted an immune analysis., Results: The study identified four genes linked with an increased risk of PC; that is, PYGB, ACTR3, CCNA2 , and ITGB1 . Conversely, ATP8A1 , and RAP1GAP2 were found to provide protection against PC. These findings contributed significantly to the development of a highly precise risk model in which risk, age, and pathology N stage were categorized as independent factors in predicting the prognosis of PC patients. Using these factors, a nomogram was established to predict survival outcomes accurately. An immune analysis revealed varying levels of eosinophils, gamma delta T cells, and other immune cells between the distinct risk groups. The high-risk patients exhibited increased potential for immune escape, while the low-risk patients showed a higher response to immunotherapy., Conclusions: Six genes were identified as having potential causal relationships with PC. These genes were integrated into a prognostic risk model, thereby serving as unique prognostic signatures. Our findings provide novel insights into predicting the prognosis of PC patients., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-426/coif). The authors have no conflicts of interest to declare., (2024 Journal of Gastrointestinal Oncology. All rights reserved.)

Prion-like proteins (PrLPs) have emerged as beneficial molecules with implications in adaptive responses. These proteins possess a conserved prion-like domain (PrLD) which is an intrinsically disordered region capable of adopting different conformations upon perceiving external stimuli. Owing to changes in protein conformation, functional characteristics of proteins harboring PrLDs get altered thereby, providing a unique mode of protein-based regulation. Since PrLPs are ubiquitous in nature and involved in diverse functions, through this study, we aim to explore the role of such domains in yet another important physiological process viz. plant-microbe interactions to get insights into the mechanisms dictating cross-kingdom interactions. We have evaluated the presence and functions of PrLPs in 18 different plant-associated fungi of agricultural importance to unravel their role in plant-microbe interactions. Of the 241,997 proteins scanned, 3,820 (~ 1.6%) were identified as putative PrLPs with pathogenic fungi showing significantly higher PrLP density than their beneficial counterparts. Further, through GO enrichment analysis, we could predict several PrLPs from pathogenic fungi to be involved in virulence and formation of stress granules. Notably, PrLPs involved in (retro)transposition were observed exclusively in pathogenic fungi. We even analyzed publicly available data for the expression alterations of fungal PrLPs upon their interaction with their respective hosts which revealed perturbation in the levels of some PrLP-encoding genes during interactions with plants. Overall, our work sheds light into the probable role of prion-like candidates in plant-fungi interaction, particularly in context of pathogenesis, paving way for more focused studies for validating their role., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Autophagy - a highly regulated intracellular degradation process - is pivotal in maintaining cellular homeostasis. Liquid-liquid phase separation (LLPS) is a fundamental mechanism regulating the formation and function of membrane-less compartments. Recent research has unveiled connections between LLPS and autophagy, suggesting that phase separation events may orchestrate the spatiotemporal organization of autophagic machinery and cargo sequestration. The Unc-51-like kinase (ULK)/autophagy-related 1 (Atg1) family of proteins is best known for its regulatory role in initiating autophagy, but there is growing evidence that the functional spectrum of ULK/Atg1 extends beyond autophagy regulation. In this review, we explore the spatial and temporal regulation of the ULK/Atg1 family of kinases, focusing on their recruitment to LLPS-driven compartments, and highlighting their multifaceted functions beyond their traditional role., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ribosome biogenesis is initiated in the nucleolus, a multiphase biomolecular condensate formed by liquid-liquid phase separation. The nucleolus is a powerful disease biomarker and stress biosensor whose morphology reflects function. Here we have used digital holographic microscopy (DHM), a label-free quantitative phase contrast microscopy technique, to detect nucleoli in adherent and suspension human cells. We trained convolutional neural networks to detect and quantify nucleoli automatically on DHM images. Holograms containing cell optical thickness information allowed us to define a novel index which we used to distinguish nucleoli whose material state had been modulated optogenetically by blue-light-induced protein aggregation. Nucleoli whose function had been impacted by drug treatment or depletion of ribosomal proteins could also be distinguished. We explored the potential of the technology to detect other natural and pathological condensates, such as those formed upon overexpression of a mutant form of huntingtin, ataxin-3, or TDP-43, and also other cell assemblies (lipid droplets). We conclude that DHM is a powerful tool for quantitatively characterizing nucleoli and other cell assemblies, including their material state, without any staining., (© 2024. The Author(s).)

Adenosine triphosphate (ATP) acts as the universal energy currency that drives various biological processes, while nucleic acids function to store and transmit genetic information for all living organisms. Liquid-liquid phase separation (LLPS) represents the common principle for the formation of membrane-less organelles (MLOs) composed of proteins rich in intrinsically disordered regions (IDRs) and nucleic acids. Currently, while IDRs are well recognized to facilitate LLPS through dynamic and multivalent interactions, the precise mechanisms by which ATP and nucleic acids affect LLPS still remain elusive. This review summarizes recent NMR results on the LLPS of human FUS, TDP-43, and the viral nucleocapsid (N) protein of SARS-CoV-2, as modulated by ATP and nucleic acids, revealing the following: (1) ATP binds to folded domains overlapping with nucleic-acid-binding interfaces; (2) ATP and nucleic acids interplay to biphasically modulate LLPS by competitively binding to overlapping pockets of folded domains and Arg/Lys within IDRs; (3) ATP energy-independently induces protein folding with the highest efficiency known so far. As ATP likely emerged in the prebiotic monomeric world, while LLPS represents a pivotal mechanism to concentrate and compartmentalize rare molecules for forming primordial cells, ATP appears to control protein homeostasis and shape genome-proteome interfaces throughout the evolutionary trajectory, from prebiotic origins to modern cells., Competing Interests: The author declares no competing interests.

Inclusion bodies (IBs) of respiratory syncytial virus (RSV) are formed by liquid-liquid phase separation (LLPS) and contain internal structures termed "IB-associated granules" (IBAGs), where anti-termination factor M2-1 and viral mRNAs are concentrated. However, the mechanism of IBAG formation and the physiological function of IBAGs are unclear. Here, we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein (mRNP) condensates formed by secondary LLPS. Mechanistically, the RSV nucleoprotein (N) and M2-1 interact with and recruit PABP to IBs, promoting PABP to bind viral mRNAs transcribed in IBs by RNA-recognition motif and drive secondary phase separation. Furthermore, PABP-eIF4G1 interaction regulates viral mRNP condensate composition, thereby recruiting specific translation initiation factors (eIF4G1, eIF4E, eIF4A, eIF4B and eIF4H) into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment. Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase., Competing Interests: Conflict of interest Prof. Mingzhou Chen is an ​editorial board member ​for ​Virologica Sinica, and was not involved in the editorial review or the decision to publish this article. ​The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1 , show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin-binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome-Rpn1, Rpn10, and Rpn13-and the Ubl domains of Rad23 and Dsk2 are critical under different condensate-inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes., Competing Interests: Competing interests statement:The authors declare no competing interest.

Liquid‒liquid phase separation (LLPS) is a general phenomenon recently recognized to be critically involved in the regulation of a variety of cellular biological processes, such as transcriptional regulation, heterochromatin formation and signal transduction, through the compartmentalization of proteins or nucleic acids into droplet-like condensates. These processes are directly or indirectly related to tumor initiation and treatment. Posttranslational modifications (PTMs), which represent a rapid and reversible mechanism involved in the functional regulation of proteins, have emerged as key events in modulating LLPS under physiological or pathophysiological conditions, including tumorigenesis and antitumor therapy. In this review, we introduce the biological functions participated in cancer-associated LLPS, discuss the potential roles of LLPS during tumor onset or therapy, and emphasize the mechanistic characteristics of LLPS regulated by PTMs and its effects on tumor progression. We then provide a perspective on further studies on LLPS and its regulation by PTMs in cancer research. This review aims to broaden the understanding of the functions of LLPS and its regulation by PTMs under normal or aberrant cellular conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Liquid-liquid phase separation (LLPS) in biology describes a process by which proteins form membraneless condensates within a cellular compartment when conditions are met, including the concentration and posttranslational modifications of the protein components, the condition of the aqueous solution (pH, ionic strength, pressure, and temperature), and the existence of assisting factors (such as RNAs or other proteins). In these supramolecular liquid droplet-like inclusion bodies, molecules are held together through weak intermolecular and/or intramolecular interactions. With the aid of LLPS, cells can assemble functional sub-units within a given cellular compartment by enriching or excluding specific factors, modulating cellular function, and rapidly responding to environmental or physiological cues. Hence, LLPS is emerging as an important means to regulate biology and physiology. Yet, excessive inclusion body formation by, for instance, higher-than-normal concentrations or mutant forms of the protein components could result in the conversion from dynamic liquid condensates into more rigid gel- or solid-like aggregates, leading to the disruption of the organelle's function followed by the development of human disorders like neurodegenerative diseases. In summary, well-controlled formation and de-formation of LLPS is critical for normal biology and physiology from single cells to individual organisms, whereas abnormal LLPS is involved in the pathophysiology of human diseases. In turn, targeting these aggregates or their formation represents a promising approach in treating diseases driven by abnormal LLPS including those neurodegenerative diseases that lack effective therapies., (© 2024. The Author(s).)

Global climate change has caused severe abiotic and biotic stresses, affecting plant growth and food security. The mechanical understanding of plant stress responses is critical for achieving sustainable agriculture. Intrinsically disordered proteins (IDPs) are a group of proteins without unique three-dimensional structures. The environmental sensitivity and structural flexibility of IDPs contribute to the growth and developmental plasticity for sessile plants to deal with environmental challenges. This article discusses the roles of various disordered proteins in plant stress tolerance and resistance, describes the current mechanistic insights into unstructured proteins such as the disorder-to-order transition for adopting secondary structures to interact with specific partners (i.e., cellular membranes, membrane proteins, metal ions, and DNA), and elucidates the roles of liquid-liquid phase separation driven by protein disorder in stress responses. By comparing IDP studies in animal systems, this article provides conceptual principles of plant protein disorder in stress adaptation, reveals the current research gaps, and advises on the future research direction. The highlighting of relevant unanswered questions in plant protein disorder research aims to encourage more studies on these emerging topics to understand the mechanisms of action behind their stress resistance phenotypes.

In recent years, the role of liquid-liquid phase separation (LLPS) and intrinsically disordered proteins (IDPs) in cellular molecular processes has received increasing attention from researchers. One such intrinsically disordered protein is TSPYL5, considered both as a marker and a potential therapeutic target for various oncological diseases. However, the role of TSPYL5 in intracellular processes remains unknown, and there is no clarity even in its intracellular localization. In this study, we characterized the intracellular localization and exchange dynamics with intracellular contents of TSPYL5 and its parts, utilizing TSPYL5 fusion proteins with EGFP. Our findings reveal that TSPYL5 can be localized in both the cytoplasm and nucleoplasm, including the nucleolus. The nuclear (nucleolar) localization of TSPYL5 is mediated by the nuclear/nucleolar localization sequences (NLS/NoLS) identified in the N-terminal intrinsically disordered region (4-27 aa), while its cytoplasmic localization is regulated by the ordered NAP-like domain (198-382 aa). Furthermore, our results underscore the significant role of the TSPYL5 N-terminal disordered region (1-198 aa) in the exchange dynamics with the nucleoplasm and its potential ability for phase separation. Bioinformatics analysis of the TSPYL5 interactome indicates its potential function as a histone and ribosomal protein chaperone. Taken together, these findings suggest a significant contribution of liquid-liquid phase separation to the processes involving TSPYL5, providing new insights into the role of this protein in the cell's molecular life.

The cellular environment is highly crowded, with up to 40% of the volume fraction of the cell occupied by various macromolecules. Most laboratory experiments take place in dilute buffer solutions; by adding various synthetic or organic macromolecules, researchers have begun to bridge the gap between in vitro and in vivo measurements. This is a review of the reported effects of macromolecular crowding on the compaction and extension of DNA, the effect of macromolecular crowding on DNA kinetics, and protein-DNA interactions. Theoretical models related to macromolecular crowding and DNA are briefly reviewed. Gaps in the literature, including the use of biologically relevant crowders, simultaneous use of multi-sized crowders, empirical connections between macromolecular crowding and liquid-liquid phase separation of nucleic materials are discussed.

This paper discusses the properties of proteins and their relations in the interactomes of the selected subsets of SARS-CoV-2 proteome-the membrane protein, nonstructural proteins, and, finally, full proteome. Protein disorder according to several measures, liquid-liquid phase separation probabilities, and protein node degrees in the interaction networks were singled out as the features of interest. Additionally, viral interactomes were combined with the interactome of human lung tissue so as to examine if the new connections in the resulting viral-host interactome are linked to protein disorder. Correlation analysis shows that there is no clear relationship between raw features of interest, whereas there is a positive correlation between the protein disorder and its neighborhood mean disorder. There are also indications that highly connected viral hubs tend to be on average more ordered than proteins with a small number of connections. This is in contrast to previous similar studies conducted on eukaryotic interactomes and possibly raises new questions in research on viral interactomes., (© 2023 Wiley Periodicals LLC.)

Long noncoding RNAs (lncRNAs), which are among the most well-characterized noncoding RNAs, have attracted much attention due to their regulatory functions and potential therapeutic options in many types of disease. Liquid-liquid phase separation (LLPS), the formation of droplet condensates, is involved in various cellular processes, but the molecular interactions of lncRNAs in LLPS are unclear. In this review, we describe the research development on LLPS, including descriptions of various methods established to identify LLPS, summarize the physiological and pathological functions of LLPS, identify the molecular interactions of lncRNAs in LLPS, and present the potential applications of leveraging LLPS in the clinic. The aim of this review is to update the knowledge on the association between LLPS and lncRNAs, which might provide a new direction for the treatment of LLPS-mediated disease., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The control of supramolecular complexes in living systems at the molecular level is an important goal in life-sciences. Spatiotemporal organization of molecular distribution & flow of such complexes are essential physicochemical processes in living cells and important for pharmaceutical processes. Membraneless organelles (MO) found in eukaryotic cells, formed by liquid-liquid phase-separation (LLPS) of intrinsically disordered proteins (IDPs) control and adjust intracellular organization. Artificially designed compartments based on LLPS open up a novel pathway to control chemical flux and partition in vitro and in vivo. We designed a library of chemically precisely defined block copolymer-like proteins based on elastin-like proteins (ELPs) with defined charge distribution and type, as well as polar and hydrophobic block domains. This enables the programmability of physicochemical properties and to control adjustable LLPS in vivo attaining control over intracellular partitioning and flux as role model for in vitro and in vivo applications. Tailor-made ELP-like block copolymer proteins exhibiting IDP-behavior enable LLPS formation in vitro and in vivo allowing the assembly of membrane-based and membraneless superstructures via protein phase-separation in E. coli. Subsequently, we demonstrate the responsiveness of protein phase-separated spaces (PPSSs) to environmental physicochemical triggers and their selective, charge-dependent and switchable interaction with DNA or extrinsic and intrinsic molecules enabling their selective shuttling across semipermeable phase boundaries including (cell)membranes. This paves the road for adjustable artificial PPSS-based storage and reaction spaces and the specific transport across phase boundaries for applications in pharmacy and synthetic biology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Membrane vesiculation is an energy-costing process. Previous studies paid much attention to proteins with curvature-inducing motifs. Recent publications reveal that the liquid-like protein assembly on membrane surfaces provides an efficient yet structure-independent mechanism for increasing the membrane curvature, which plays important roles in vesicle transport in many aspects. Intrinsically disordered regions (IDRs) within the proteins are highly potent drivers of membrane curvature by providing large hydrodynamic radii to generate steric pressure. Biomolecular condensates formed by phase separation can provide a reaction platform for sequential processes or generate a wetting surface to sequestrate cargos and trigger membrane remodeling. We review the latest progress in yeast and mammalian cells, focus on the mechanism of clathrin-mediated endocytosis (CME) and autophagy initiation, and compare with what we know in model plant Arabidopsis. The comparison may give important insights into the understanding of basic membrane trafficking mechanisms in plant cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

"Membraneless organelles," also referred to as biomolecular condensates, perform a variety of cellular functions and their dysregulation is implicated in cancer and neurodegeneration. In the last two decades, liquid-liquid phase separation (LLPS) of intrinsically disordered and multidomain proteins has emerged as a plausible mechanism underlying the formation of various biomolecular condensates. Further, the occurrence of liquid-to-solid transitions within liquid-like condensates may give rise to amyloid structures, implying a biophysical link between phase separation and protein aggregation. Despite significant advances, uncovering the microscopic details of liquid-to-solid phase transitions using experiments remains a considerable challenge and presents an exciting opportunity for the development of computational models which provide valuable, complementary insights into the underlying phenomenon. In this review, we first highlight recent biophysical studies which provide new insights into the molecular mechanisms underlying liquid-to-solid (fibril) phase transitions of folded, disordered and multi-domain proteins. Next, we summarize the range of computational models used to study protein aggregation and phase separation. Finally, we discuss recent computational approaches which attempt to capture the underlying physics of liquid-to-solid transitions along with their merits and shortcomings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

hPFN1 mutations including C71G cause ALS by gain of toxicity but the mechanism still remains unknown. Stress granules (SGs) are formed by phase separation of the prion-like domain (PLD) of RNA-binding proteins including FUS, whose inclusion was also associated with ALS. C71G-hPFN1 triggers seed-dependent co-aggregation with FUS/TDP-43 to manifest the prion-like propagandation but its biophysical basis remains unexplored. Here by DIC imaging we first showed that three hPFN1 mutants have differential capacity in disrupting the dynamics of liquid droplets formed by phase separation of FUS prion-like domain (PLD). C71G-hPFN1 co-exists with the folded and unfolded states, thus allowing to simultaneously characterize conformations, hydrodynamics and dynamics of the interactions of both states with the phase separated FUS PLD by NMR. The results reveal that the folded state is not significantly affected while by contrast, the unfolded state has extensive interactions with FUS PLD. As a consequence, the dynamics of FUS liquid droplets become significantly reduced. Such interactions might act to recruit C71G-hPFN1 into the droplets, thus leading to the increase of the local concentrations and subsequent co-aggregation of C71G-hPFN1 with FUS. Our study sheds the first light on the biophysical basis by which hPFN1 mutants gain toxicity to cause ALS. As other aggregation-prone proteins have no fundamental difference from hPFN1 mutants, aggregation-prone proteins might share a common capacity in disrupting phase separation responsible for organizing various membrane-less organelles. As such, the mechanism for C71G-hPFN1 might also be utilized by other aggregation-prone proteins for gain of toxicity to trigger diseases and aging., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Ribonucleic acid (RNA) biology has emerged as one of the most important areas in modern biology and biomedicine. RNA and RNA-binding proteins (RBPs) are involved in forming biomolecular condensates, which are crucial for RNA metabolism. To quantitively decipher the molecular mechanisms of RNP granules, researchers have turned to single-molecule biophysical techniques, such as single-molecule Förster resonance energy transfer (smFRET), in vivo single-molecule imaging technique with single particle tracking (SPT), DNA Curtains, optical tweezers, and atomic force microscopy (AFM). These methods are used to investigate the molecular biophysical properties within RNP granules, as well as the molecular interactions between RNA and RBPs and RBPs themselves, which are challenging to study using traditional experimental methods of the liquid-liquid phase separation (LLPS) field, such as fluorescence recovery after photobleaching (FRAP). In this work, we summarize the applications of single-molecule biophysical techniques in RNP granule studies and highlight how these methods can be used to reveal the molecular mechanisms of RNP granules.

PML nuclear body (NB) malfunction often leads to acute leukemia outbreaks and other severe diseases. PML NB rescue is the molecular basis of arsenic success in acute promyelocytic leukemia (APL) treatment. However, it is unclear how PML NBs are assembled. Here, we observed the presence of liquid-liquid phase separation (LLPS) in NB formation by fluorescence recovery after photobleaching (FRAP) experiment. Compared with the wild-type (WT) NBs, PML A216V derived from arsenic-resistant leukemia patients markedly crippled LLPS, but not altered the overall structure and PML RBCC oligomerization. In parallel, we also reported several Leu to Pro mutations that were critical to PML coiled-coil domain. FRAP characterization and comparison between L268P and A216V revealed markedly different LLPS activities in these mutant NBs. Transmission electron microscopy (TEM) inspections of LLPS-crippled and uncrippled NBs showed aggregation- and ring-like PML packing in A216V and WT/L268P NBs, respectively. More importantly, the correct LLPS-driven NB formation was the prerequisite for partner recruitment, post-translational modifications (PTMs), and PML-driven cellular regulations, such as ROS stress control, mitochondria production, and PML-p53-mediated senescence and apoptosis. Altogether, our results helped to define a critical LLPS step in PML NB biogenesis., (© 2023 Federation of American Societies for Experimental Biology.)

During the dissolution of amorphous solid dispersion (ASD) formulations, the gel layer that forms at the ASD/water interface strongly dictates the release of the active pharmaceutical ingredient (API) and, hence, the dissolution performance. Several studies have demonstrated that the switch of the gel layer from eroding to non-eroding behavior is API-specific and drug-load (DL)-dependent. This study systematically classifies the ASD release mechanisms and relates them to the phenomenon of the loss of release (LoR). The latter is thermodynamically explained and predicted via a modeled ternary phase diagram of API, polymer, and water, and is then used to describe the ASD/water interfacial layers (below and above the glass transition). To this end, the ternary phase behavior of the APIs, naproxen, and venetoclax with the polymer poly(vinylpyrrolidone-co-vinyl acetate) (PVPVA64) and water was modeled using the perturbed-chain statistical associating fluid theory (PC-SAFT). The glass transition was modeled using the Gordon-Taylor equation. The DL-dependent LoR was found to be caused by API crystallization or liquid-liquid phase separation (LLPS) at the ASD/water interface. If crystallization occurs, it was found that API and polymer release was impeded above a threshold DL at which the APIs crystallized directly at the ASD interface. If LLPS occurs, an API-rich phase and a polymer-rich phase are formed. Above a threshold DL, the less mobile and hydrophobic API-rich phase accumulates at the interface which prevents API release. LLPS is further influenced by the composition and glass transition temperature of the evolving phases and was investigated at 37 °C and 50 °C regarding impact of temperature of. The modeling results and LoR predictions were experimentally validated by means of dissolution experiments, microscopy, Raman spectroscopy, and size exclusion chromatography. The experimental results were found to be in very good agreement with the predicted release mechanisms deduced from the phase diagrams. Thus, this thermodynamic modeling approach represents a powerful mechanistic tool that can be applied to classify and quantitatively predict the DL-dependent LoR release mechanism of PVPVA64-based ASDs in water.

The use of infectious bursal disease virus (IBDV) reverse genetics to engineer tagged reporter viruses has revealed that the virus factories (VFs) of the Birnaviridae family are biomolecular condensates that show properties consistent with liquid-liquid phase separation (LLPS). Although the VFs are not bound by membranes, it is currently thought that viral protein 3 (VP3) initially nucleates the formation of the VF on the cytoplasmic leaflet of early endosomal membranes, and likely drives LLPS. In addition to VP3, IBDV VFs contain VP1 (the viral polymerase) and the dsRNA genome, and they are the sites of de novo viral RNA synthesis. Cellular proteins are also recruited to the VFs, which are likely to provide an optimal environment for viral replication; the VFs grow due to the synthesis of the viral components, the recruitment of other proteins, and the coalescence of multiple VFs in the cytoplasm. Here, we review what is currently known about the formation, properties, composition, and processes of these structures. Many open questions remain regarding the biophysical nature of the VFs, as well as the roles they play in replication, translation, virion assembly, viral genome partitioning, and in modulating cellular processes.

Liquid-liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders., (© 2022 European Peptide Society and John Wiley & Sons Ltd.)

Compartmentalization of macromolecules into discrete non-lipid-bound bodies by liquid-liquid phase separation (LLPS) is a well-characterized regulatory mechanism frequently associated with the cellular stress response in eukaryotes. In contrast, the formation and importance of similar complexes is just becoming evident in bacteria. Here, we identify LLPS as the mechanism by which the DEAD-box RNA helicase, cyanobacterial RNA helicase redox (CrhR), compartmentalizes into dynamic membraneless organelles in a temporal and spatial manner in response to abiotic stress in the cyanobacterium Synechocystis sp. strain PCC 6803. Stress conditions induced CrhR to form a single crescent localized exterior to the thylakoid membrane, indicating that this region is a crucial domain in the cyanobacterial stress response. These crescents rapidly dissipate upon alleviation of the stress conditions. Furthermore, CrhR aggregation was mediated by LLPS in an RNA-dependent reaction. We propose that dynamic CrhR condensation performs crucial roles in RNA metabolism, enabling rapid adaptation of the photosynthetic apparatus to environmental stresses. These results expand our understanding of the role that functional compartmentalization of RNA helicases and thus RNA processing in membraneless organelles by LLPS-mediated protein condensation performs in the bacterial response to environmental stress. IMPORTANCE Oxygen-evolving photosynthetic cyanobacteria evolved ~3 billion years ago, performing fundamental roles in the biogeochemical evolution of the early Earth and continue to perform fundamental roles in nutrient cycling and primary productivity today. The phylum consists of diverse species that flourish in heterogeneous environments. A prime driver for survival is the ability to alter photosynthetic performance in response to the shifting environmental conditions these organisms continuously encounter. This study demonstrated that diverse abiotic stresses elicit dramatic changes in localization and structural organization of the RNA helicase CrhR associated with the photosynthetic thylakoid membrane. These dynamic changes, mediated by a liquid-liquid phase separation (LLPS)-mediated mechanism, reveal a novel mechanism by which cyanobacteria can compartmentalize the activity of ribonucleoprotein complexes in membraneless organelles. The results have significant consequences for understanding bacterial adaptation and survival in response to changing environmental conditions.

The Lewy bodies and Lewy neurites are key pathological hallmarks of Parkinson's disease (PD). Single-point mutations associated with familial PD cause α-synuclein (α-Syn) aggregation, leading to the formation of Lewy bodies and Lewy neurites. Recent studies suggest α-Syn nucleates through liquid-liquid phase separation (LLPS) to form amyloid aggregates in a condensate pathway. How PD-associated mutations affect α-Syn LLPS and its correlation with amyloid aggregation remains incompletely understood. Here, we examined the effects of five mutations identified in PD, A30P, E46K, H50Q, A53T, and A53E, on the phase separation of α-Syn. All other α-Syn mutants behave LLPS similarly to wild-type (WT) α-Syn, except that the E46K mutation substantially promotes the formation of α-Syn condensates. The mutant α-Syn droplets fuse to WT α-Syn droplets and recruit α-Syn monomers into their droplets. Our studies showed that α-Syn A30P, E46K, H50Q, and A53T mutations accelerated the formation of amyloid aggregates in the condensates. In contrast, the α-Syn A53E mutant retarded the aggregation during the liquid-to-solid phase transition. Finally, we observed that WT and mutant α-Syn formed condensates in the cells, whereas the E46K mutation apparently promoted the formation of condensates. These findings reveal that familial PD-associated mutations have divergent effects on α-Syn LLPS and amyloid aggregation in the phase-separated condensates, providing new insights into the pathogenesis of PD-associated α-Syn mutations.

Glucocorticoids (GCs) are hormones involved in circadian adaptation and stress response, and it is also noteworthy that these steroidal molecules present potent anti-inflammatory action through GC receptors (GR). Upon ligand-mediated activation, GR translocates to the nucleus, and regulates gene expression related to metabolism, acute-phase response and innate immune response. GR field of research has evolved considerably in the last decades, providing varied mechanisms that contributed to the understanding of transcriptional regulation and also impacted drug design for treating inflammatory diseases. Liquid-liquid phase separation (LLPS) in cellular processes represents a recent topic in biology that conceptualizes membraneless organelles and microenvironments that promote, or inhibit, chemical reactions and interactions of protein or nucleic acids. The formation of these molecular condensates has been implicated in gene expression control, and recent evidence shows that GR and other steroid receptors can nucleate phase separation (PS). Here we briefly review the varied mechanisms of transcriptional control by GR, which are largely studied in the context of inflammation, and further present how PS can be involved in the control of gene expression. Lastly, we consider how the reported advances on LLPS during transcription control, specially for steroid hormone receptors, could impact the different modalities of GR action on gene expression, adding a new plausible molecular event in glucocorticoid signal transduction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pinheiro, Preato, Petrucci, dos Santos and Glezer.)

A rapidly evolving understanding of phase separation in the biological and physical sciences has led to the redefining of virus-engineered replication compartments in many viruses with RNA genomes. Condensation of viral, host and genomic and subgenomic RNAs can take place to evade the innate immunity response and to help viral replication. Divergent viruses prompt liquid-liquid phase separation (LLPS) to invade the host cell. During HIV replication there are several steps involving LLPS. In this review, we characterize the ability of individual viral and host partners that assemble into biomolecular condensates (BMCs). Of note, bioinformatic analyses predict models of phase separation in line with several published observations. Importantly, viral BMCs contribute to function in key steps retroviral replication. For example, reverse transcription takes place within nuclear BMCs, called HIV-MLOs while during late replication steps, retroviral nucleocapsid acts as a driver or scaffold to recruit client viral components to aid the assembly of progeny virions. Overall, LLPS during viral infections represents a newly described biological event now appreciated in the virology field, that can also be considered as an alternative pharmacological target to current drug therapies especially when viruses become resistant to antiviral treatment., (© 2023. The Author(s).)

To date, it has been shown that the phenomenon of liquid-liquid phase separation (LLPS) underlies many seemingly completely different cellular processes. This provided a new idea of the spatiotemporal organization of the cell. The new paradigm makes it possible to provide answers to many long-standing, but still unresolved questions facing the researcher. In particular, spatiotemporal regulation of the assembly/disassembly of the cytoskeleton, including the formation of actin filaments, becomes clearer. To date, it has been shown that coacervates of actin-binding proteins that arise during the phase separation of the liquid-liquid type can integrate G-actin and thereby increase its concentration to initiate polymerization. It has also been shown that the activity intensification of actin-binding proteins that control actin polymerization, such as N-WASP and Arp2/3, can be caused by their integration into liquid droplet coacervates formed by signaling proteins on the inner side of the cell membrane.

The misfolding and mutation of Cu/Zn superoxide dismutase (SOD1) is commonly associated with amyotrophic lateral sclerosis (ALS). SOD1 can accumulate within stress granules (SGs), a type of membraneless organelle, which is believed to form via liquid-liquid phase separation (LLPS). Using wild-type, metal-deficient, and different ALS disease mutants of SOD1 and computer simulations, we report here that the absence of Zn leads to structural disorder within two loop regions of SOD1, triggering SOD1 LLPS and amyloid formation. The addition of exogenous Zn to either metal-free SOD1 or to the severe ALS mutation I113T leads to the stabilization of the loops and impairs SOD1 LLPS and aggregation. Moreover, partial Zn-mediated inhibition of LLPS was observed for another severe ALS mutant, G85R, which shows perturbed Zn-binding. By contrast, the ALS mutant G37R, which shows reduced Cu-binding, does not undergo LLPS. In addition, SOD1 condensates induced by Zn-depletion exhibit greater cellular toxicity than aggregates formed by prolonged incubation under aggregating conditions. Overall, our work establishes a role for Zn-dependent modulation of SOD1 conformation and LLPS properties that may contribute to amyloid formation., (© 2022 The Authors.)

Androgen receptor (AR) and its splice variants (AR-SVs) promote prostate cancer (PCa) growth by orchestrating transcriptional reprogramming. Mechanisms by which the low complexity and intrinsically disordered primary transactivation domain (AF-1) of AR and AR-SVs regulate transcriptional programming in PCa remains poorly defined. Using omics, live and fixed fluorescent microscopy of cells, and purified AF-1 and AR-V7 recombinant proteins we show here that AF-1 and the AR-V7 splice variant form molecular condensates by liquid-liquid phase separation (LLPS) that exhibit disorder characteristics such as rapid intracellular mobility, coactivator interaction, and euchromatin induction. The LLPS and other disorder characteristics were reversed by a class of small-molecule-selective AR-irreversible covalent antagonists (SARICA) represented herein by UT-143 that covalently and selectively bind to C406 and C327 in the AF-1 region. Interfering with LLPS formation with UT-143 or mutagenesis resulted in chromatin condensation and dissociation of AR-V7 interactome, all culminating in a transcriptionally incompetent complex. Biochemical studies suggest that C327 and C406 in the AF-1 region are critical for condensate formation, AR-V7 function, and UT-143's irreversible AR inhibition. Therapeutically, UT-143 possesses drug-like pharmacokinetics and metabolism properties and inhibits PCa cell proliferation and tumor growth. Our work provides critical information suggesting that clinically important AR-V7 forms transcriptionally competent molecular condensates and covalently engaging C327 and C406 in AF-1, dissolves the condensates, and inhibits its function. The work also identifies a library of AF-1-binding AR and AR-SV-selective covalent inhibitors for the treatment of PCa.

In the last years, RNA-binding proteins (RBPs) have been highlighted for their capacity to undergo liquid-liquid phase separation (LLPS). Aberrant phase transitions of RBPs from a liquid to a solid state are believed to underlie the formation of pathological RBP aggregates in several neurodegenerative diseases. Both in the physiological and the disease state, RBPs are often decorated with diverse posttranslational modifications (PTMs) that can influence the phase separation behavior, the physiological function, and the pathological behavior of the RBP. Here we describe two simple methods, sedimentation assays in vitro and in cells, that allow the analysis of RBP solubility as a measure of RBP phase separation in the absence or presence of a certain PTM., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Phase-separated biomolecular condensates play important roles in virtually all cellular processes, and their dysregulation is associated with many pathological processes including cancer. Here we concisely review some basic methodologies and strategies to analyze the phase-separated biomolecular condensates in cancer, including physical characterization of phase separation for the protein of interest, functional demonstration of this property in cancer regulation, as well as mechanistic studies on how phase separation regulates the protein's function in cancer., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The mediation of liquid-liquid phase separation (LLPS) for fused in sarcoma (FUS) protein is generally attributed to the low-complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full-length (FL) FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. FL FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn 2+ , and Ca 2+ and at the isoelectric pH. The FUS RNA recognition motif (RRM) and zinc-finger (ZnF) domains are found to undergo cold denaturation above 0°C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS-promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Carboxysomes are large, cytosolic bodies present in all cyanobacteria and many proteobacteria that function as the sites of photosynthetic CO 2 fixation by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The carboxysome lumen is enriched with Rubisco and carbonic anhydrase (CA). The polyhedral proteinaceous shell allows the passage of HCO 3 - ions into the carboxysome, where they are converted to CO 2 by CA. Thus, the carboxysome functions as a CO 2 -concentrating mechanism (CCM), enhancing the efficiency of Rubisco in CO 2 fixation. In β-cyanobacteria, carboxysome biogenesis first involves the aggregation of Rubisco by CcmM, a scaffolding protein that exists in two isoforms. Both isoforms contain a minimum of three Rubisco small subunit-like (SSUL) domains, connected by flexible linkers. Multivalent interaction between these linked SSUL domains with Rubisco results in phase separation and condensate formation. Here, we use Rubisco and the short isoform of CcmM (M35) of the β-cyanobacterium Synechococcus elongatus PCC7942 to describe the methods used for in vitro analysis of the mechanism of condensate formation driven by the SSUL domains. The methods include turbidity assays, bright-field and fluorescence microscopy, as well as transmission electron microscopy (TEM) in both negative staining and cryo-conditions., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Uncontrolled assembly/disassembly of physiologically formed liquid condensates is linked to irreversible aggregation. Hence, the quest for understanding protein-misfolding disease mechanism might lie in the studies of protein:nucleic acid coacervation. Several proteins with intrinsically disordered regions as well as nucleic acids undergo phase separation in the cellular context, and this process is key to physiological signaling and is related to pathologies. Phase separation is reproducible in vitro by mixing the target recombinant protein with specific nucleic acids at various stoichiometric ratios and then examined by microscopy and nanotracking methods presented herein. We describe protocols to qualitatively assess hallmarks of protein-rich condensates, characterize their structure using intrinsic and extrinsic dyes, quantify them, and analyze their morphology over time. Analysis by nanoparticle tracking provides information on the concentration and diameter of high-order protein oligomers formed in the presence of nucleic acid. Using the model protein (globular domain of recombinant murine PrP) and DNA aptamers (high-affinity oligonucleotides with 25 nucleotides in length), we provide examples of a systematic screening of liquid-liquid phase separation in vitro., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Liquid-liquid phase separation (LLPS) often induces the formation of biomolecule condensates at the cellular level. The importance of this phenomenon has been demonstrated in many important biological functions, such as in transcription. However, the biophysical nature of LLPS containing transcriptional machinery has not yet been carefully examined. Here, we give an overview of a novel high-throughput single-molecule technique, termed as DNA Curtains. It was established recently to dissect the DNA compaction process in real time. The experimental procedures are further discussed in detail in the context of the biomolecular condensates of a transcription repressor., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Biomolecular condensates are nonmembrane cellular compartments whose formation in many cases involves phase separation (PS). Despite much research interest in this mechanism of macromolecular self-organization, the concept of PS as applied to a live cell faces certain challenges. In this review, we discuss a basic model of PS and the role of site-specific interactions and percolation in cellular PS-related events. Using a multivalent poly(ADP-ribose) molecule as an example, which has high PS-driving potential due to its structural features, we consider how site-specific interactions and network formation are involved in the formation of phase-separated cellular condensates.

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1-2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40-50 mOsm). Both reassembled into new structures within 1-2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90-175 mOsm) first rapidly disassembled within 1-3 min, and then, in most cells, spontaneously reassembled 7-15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress.

Liquid-liquid phase separation (LLPS) compartmentalizes and concentrates biomacromolecules into liquid-like condensates, which underlies membraneless organelles (MLOs) formation in eukaryotic cells. With increasing evidence of the LLPS concept and methods, this phenomenon as a novel principle accounts for explaining the precise spatial and temporal regulation of cellular functions. Moreover, the phenomenon that LLPS tends to concentrate proteins is often accompanied by several abnormal signals for human diseases. It is reported that multiple metabolic diseases are strongly associated with the deposition of insoluble proteinaceous aggregating termed amyloids. At present, recent studies have observed the roles of LLPS in several metabolic diseases, including type 2 diabetes mellitus (T2DM), Alzheimer's disease (AD), and metabolic bone diseases (MBDs). This review aims to expound on the current concept and methods of LLPS and summarize its vital roles in T2DM, AD, and MBDs, uncover novel mechanisms of these metabolic diseases, and thus provide powerful potential therapeutic strategies and targets for ameliorating these metabolic diseases.

In recent decades, a growing number of biomolecular condensates have been identified in eukaryotic cells. These structures form through phase separation and have been linked to a diverse array of cellular processes. While a checklist of established membrane-bound organelles is present across the eukaryotic domain, less is known about the conservation of membrane-less subcellular structures. Many of these structures can be seen throughout eukaryotes, while others are only thought to be present in metazoans or a limited subset of species. In particular, the nucleus is a hub of biomolecular condensates. Some of these subnuclear domains have been found in a broad range of organisms, which is a characteristic often attributed to essential functionality. However, this does not always appear to be the case. For example, the nucleolus is critical for ribosomal biogenesis and is present throughout the eukaryotic domain, while the Cajal bodies are believed to be similarly conserved, yet these structures are dispensable for organismal survival. Likewise, depletion of the Drosophila melanogaster omega speckles reduces viability, despite the apparent absence of this domain in higher eukaryotes. By reviewing primary research that has analyzed the presence of specific condensates (nucleoli, Cajal bodies, amyloid bodies, nucleolar aggresomes, nuclear speckles, nuclear paraspeckles, nuclear stress bodies, PML bodies, omega speckles, NUN bodies, mei2 dots) in a cross-section of organisms (e.g., human, mouse, D. melanogaster , Caenorhabditis elegans , yeast), we adopt a human-centric view to explore the emergence, retention, and absence of a subset of nuclear biomolecular condensates. This overview is particularly important as numerous biomolecular condensates have been linked to human disease, and their presence in additional species could unlock new and well characterized model systems for health research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lacroix and Audas.)

A non-canonical DNA/RNA structure, G-quadruplex (G4), is a unique structure formed by two or more guanine quartets, which associate through Hoogsteen hydrogen bonding leading to form a square planar arrangement. A set of RNA-binding proteins specifically recognize G4 structures and play certain unique physiological roles. These G4-binding proteins form ribonucleoprotein (RNP) through a physicochemical phenomenon called liquid-liquid phase separation (LLPS). G4-containing RNP granules are identified in both prokaryotes and eukaryotes, but extensive studies have been performed in eukaryotes. We have been involved in analyses of the roles of G4-containing RNAs recognized by two G4-RNA-binding proteins, TDP-43 and FUS, which both are the amyotrophic lateral sclerosis (ALS) causative gene products. These RNA-binding proteins play the essential roles in both G4 recognition and LLPS, but they also carry the risk of agglutination. The biological significance of G4-binding proteins is controlled through unique 3D structure of G4, of which the risk of conformational stability is influenced by environmental conditions such as monovalent metals and guanine oxidation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ishiguro and Ishihama.)

Protein fibrillation and human neurodegenerative diseases, with a profound underlying connection suggested between them, have been the subject of intense investigations in the medical, biophysical and bio-engineering sciences. For gaining the molecular mechanistic insights into such connection, i.e., the cause and effect, atomic-resolution molecular structure information especially on the initial oligomeric states is of paramount importance, not only that on the mature amyloid fibrils. α-Synuclein (αSyn) and its amyloid fibril has a direct relevance to the Parkinson's disease and other synucleinopathies, but what triggers the fibrillation is still not entirely clear. We here describe the liquid-liquid phase separation (LLPS) of αSyn and investigate its conformational evolution from its monomeric state into oligomer state within the early-stage of the phase-separated droplets, mainly using solution and magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopies, aided with optical and fluorescent microscopies and CD spectroscopy. Based on the analysis of the intricately broadened shapes of the MAS NMR peaks observed for isotopically 13 C-labeled His-50 of αSyn, we show that the distribution of the αSyn conformation is skewed from the initial completely random state to a loose β-rich ensembles at/around His-50 as early as day-3 (d3) within the droplet. This intra-droplet loose β-rich assembly showed a very slow progression until d8, and eventually maturated into ThT-positive, long and unbranched amyloid fibrils after 8 weeks. The obtained information on the evolution of the distribution of the conformation ensemble is unique, and difficult to obtain with X-ray crystallography and cryo-electron microscopy (cryoEM). In particular, the sensitivity-enhanced MAS NMR based on the low-temperature dynamic nuclear polarization (DNP) technique was proven to be a key tool in characterizing the conformational ensemble with dilute protein samples such as the liquid-phase droplets., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

SARS-CoV-2 is the coronavirus causing the ongoing pandemic with > 460 millions of infections and > 6 millions of deaths. SARS-CoV-2 nucleocapsid (N) is the only structural protein which plays essential roles in almost all key steps of the viral life cycle with its diverse functions depending on liquid-liquid phase separation (LLPS) driven by interacting with various nucleic acids. The 419-residue N protein is highly conserved in all variants including delta and omicron, and composed of both folded N-/C-terminal domains (NTD/CTD) as well as three long intrinsically disordered regions (IDRs). Recent results have suggested that its CTD and IDRs are also cryptic nucleic acid-binding domains. In this context, any small molecules capable of interfering in its interaction with nucleic acids are anticipated to modulate its LLPS and associated functions. Indeed, ATP, the energy currency existing at very high concentrations (2-12 mM) in all living cells but absent in viruses, modulates LLPS of N protein, and consequently appears to be evolutionarily hijacked by SARS-CoV-2 to promote its life cycle. Hydroxychloroquine (HCQ) has been also shown to specifically bind NTD and CTD to inhibit their interactions with nucleic acids, as well as to disrupt LLPS. Particularly, the unique structure of the HCQ-CTD complex offers a promising strategy for further design of anti-SARS-CoV-2 drugs with better affinity and specificity. The finding may indicate that LLPS is indeed druggable by small molecules, thus opening up a promising direction for drug discovery/design by targeting LLPS in general., Competing Interests: Conflict of interestThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2022.)