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The protein α-synuclein, which is well-known for its links to Parkinson's Disease, is associated with synaptic vesicles (SVs) in nerve terminals. Despite intensive studies, its precise physiological function remains elusive. Accumulating evidence indicates that liquid-liquid phase separation takes part in the assembly and/or maintenance of different synaptic compartments. The current review discusses recent data suggesting α-synuclein as a component of the SV liquid phase. We also consider possible implications of these data for disease., 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 Brodin, Milovanovic, Rizzoli and Shupliakov.)

Many recent references show that living cells can form some membrane-less organelles by liquid-liquid phase separation (LLPS) of biomolecules, like proteins and nucleic acids. LLPS has been confirmed to link with many important biological functions in living cells, and one of the most important functions of biomolecular condensates is in the field of RNA transcription. Many studies confirm that mammalian RNA polymerase II (Pol II) molecules containing the CTD with different phosphorylation level are purposed to shuttle between initiation condensates and elongation condensates of RNA transcription. Traditional ensemble assays often experience difficulties in quantitively and directly recording the transient recruitment of Pol II CTD. Novel single-molecule approach - DNA curtains can be used to directly visualize biomolecular condensates formation and also recruitment of RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) at the target sites in solution and in real time. This method can offer the potential for new insights into the mechanism of gene transcription. Here, we highlight the detailed protocol of DNA curtains method for studying LLPS., Competing Interests: Linyu Zuo, Jiawei Ding and Zhi Qi declare that they have no conflict of interest., (© The Author(s) 2022.)

Phase-separated condensates participate in various biological activities. Liquid–liquid phase separation (LLPS) can be driven by collective interactions between multivalent and intrinsically disordered proteins. The manner in which chromatin—with various morphologies and activities—is organized in a complex and small nucleus still remains to be fully determined. Recent findings support the claim that phase separation is involved in the regulation of chromatin organization and chromosome behavior. Moreover, phase separation also influences key events during mitosis and meiosis. This review elaborately dissects how phase separation regulates chromatin and chromosome organization and controls mitotic and meiotic chromosome behavior. [ABSTRACT FROM AUTHOR]

Protein recruitment to DNA break sites is an integral part of the DNA damage response (DDR). Elucidation of the hierarchy and temporal order with which DNA damage sensors as well as repair and signaling factors assemble around chromosome breaks has painted a complex picture of tightly regulated macromolecular interactions that build specialized compartments to facilitate repair and maintenance of genome integrity. While many of the underlying interactions, e.g. between repair factors and damage-induced histone marks, can be explained by lock-and-key or induced fit binding models assuming fixed stoichiometries, structurally less well defined interactions, such as the highly dynamic multivalent interactions implicated in phase separation, also participate in the formation of multi-protein assemblies in response to genotoxic stress. Although much remains to be learned about these types of cooperative and highly dynamic interactions and their functional roles, the rapidly growing interest in material properties of biomolecular condensates and in concepts from polymer chemistry and soft matter physics to understand biological processes at different scales holds great promises. Here, we discuss nuclear condensates in the context of genome integrity maintenance, highlighting the cooperative potential between clustered stoichiometric binding and phase separation. Rather than viewing them as opposing scenarios, their combined effects can balance structural specificity with favorable physicochemical properties relevant for the regulation and function of multilayered nuclear condensates. [ABSTRACT FROM AUTHOR]

Keywords: antibodies; intracellular delivery; liquid droplet; liquid-liquid phase separation (LLPS); peptides EN antibodies intracellular delivery liquid droplet liquid-liquid phase separation (LLPS) peptides 19493 19493 1 08/28/21 20210901 NES 210901 B Large amounts of antibodies b can be rapidly delivered into cells from coacervates (liquid droplets) formed by mixing a fluorescently labeled antibody and a delivery peptide, as reported by Shiroh Futaki et al. in their Research Article on page 19804. The cover illustration shows a representation of the intracellular delivery of antibodies by coacervate. The cover illustration shows a representation of the intracellular delivery of antibodies by coacervate. [Extracted from the article]

A common viral replication strategy is characterized by the assembly of intracellular compartments that concentrate factors needed for viral replication and simultaneously conceal the viral genome from host-defense mechanisms. Recently, various membrane-less virus-induced compartments and cellular organelles have been shown to represent biomolecular condensates (BMCs) that assemble through liquid-liquid phase separation (LLPS). In the present work, we analyze biophysical properties of intranuclear replication compartments (RCs) induced during human adenovirus (HAdV) infection. The viral ssDNA-binding protein (DBP) is a major component of RCs that contains intrinsically disordered and low complexity proline-rich regions, features shared with proteins that drive phase transitions. Using fluorescence recovery after photobleaching (FRAP) and time-lapse studies in living HAdV-infected cells, we show that DBP-positive RCs display properties of liquid BMCs, which can fuse and divide, and eventually form an intranuclear mesh with less fluid-like features. Moreover, the transient expression of DBP recapitulates the assembly and liquid-like properties of RCs in HAdV-infected cells. These results are of relevance as they indicate that DBP may be a scaffold protein for the assembly of HAdV-RCs and should contribute to future studies on the role of BMCs in virus-host cell interactions. [ABSTRACT FROM AUTHOR]

Antibodies, intracellular delivery, liquid droplet, liquid-liquid phase separation (LLPS), peptides Titelbild: Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides (Angew. Keywords: antibodies; intracellular delivery; liquid droplet; liquid-liquid phase separation (LLPS); peptides EN antibodies intracellular delivery liquid droplet liquid-liquid phase separation (LLPS) peptides 19645 19645 1 08/28/21 20210901 NES 210901 B Große Mengen von Antikörpern b können aus Koazervaten (Flüssigkeitströpfchen), die durch Mischen eines fluoreszenzmarkierten Antikörpers und eines Transportpeptids gebildet werden, schnell in Zellen eingebracht werden, wie Shiroh Futaki und Mitarbeiter im Forschungsartikel auf S. 19957 berichten. [Extracted from the article]

The human mitochondrial genome (mtDNA) regulates its transcription products in specialised and distinct ways as compared to nuclear transcription. Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. The RNA regulation within mitochondria is organised within specialised, membraneless, compartments of RNA-protein complexes, called the Mitochondrial RNA Granules (MRGs). MRGs were first identified to contain nascent mRNA, complexed with many proteins involved in RNA processing and maturation and ribosome assembly. Most recently, double-stranded RNA (dsRNA) species, a hybrid of the two complementary mRNA strands, were found to form granules in the matrix of mitochondria. These RNA granules are therefore components of the mitochondrial post-transcriptional pathway and as such play an essential role in mitochondrial gene expression. Mitochondrial dysfunctions in the form of, for example, RNA processing or RNA quality control defects, or inhibition of mitochondrial fission, can cause the loss or the aberrant accumulation of these RNA granules. These findings underline the important link between mitochondrial maintenance and the efficient expression of its genome. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 nucleocapsid (N) protein plays essential roles in many steps of the viral life cycle, thus representing a key drug target. N protein contains the folded N-/C-terminal domains (NTD/CTD) and three intrinsically disordered regions, while its functions including liquid-liquid phase separation (LLPS) depend on the capacity in binding various viral/host-cell RNA/DNA of diverse sequences. Previously NTD was established to bind various RNA/DNA while CTD to dimerize/oligomerize for forming high-order structures. By NMR, here for the first time we decrypt that CTD is not only capable of binding S2m, a specific probe derived from SARS-CoV-2 gRNA but with the affinity even higher than that of NTD. Very unexpectedly, ATP, the universal energy currency for all living cells with high cellular concentrations (2-16 mM), specifically binds CTD with Kd of 1.49 ± 0.28 mM. Strikingly, the ATP-binding residues of NTD/CTD are identical in the SARS-CoV-2 variants while ATP and S2m interplay in binding NTD/CTD, as well as in modulating LLPS critical for the viral life cycle. Results together not only define CTD as a novel binding domain for ATP and nucleic acid, but enforce our previous proposal that ATP has been evolutionarily exploited by SARS-CoV-2 to complete its life cycle in the host cell. Most importantly, the unique ATP-binding pockets on NTD/CTD may offer promising targets for design of specific anti-SARS-CoV-2 molecules to fight the pandemic. Fundamentally, ATP emerges to act at mM as a cellular factor to control the interface between the host cell and virus lacking the ability to generate ATP., (© 2021 The Protein Society.)

α-Synuclein (α-Syn) is the major protein component of Lewy bodies, a key pathological feature of Parkinson's disease (PD). The manganese ion Mn 2+ has been identified as an environmental risk factor of PD. However, it remains unclear how Mn 2+ regulates α-Syn aggregation. Here, we discovered that Mn 2+ accelerates α-Syn amyloid aggregation through the regulation of protein phase separation. We found that Mn 2+ not only promotes α-Syn liquid-to-solid phase transition but also directly induces soluble α-Syn monomers to form solid-like condensates. Interestingly, the lipid membrane is integrated into condensates during Mn 2+ -induced α-Syn phase transition; however, the preformed Mn 2+ /α-syn condensates can only recruit lipids to the surface of condensates. In addition, this phase transition can largely facilitate α-Syn amyloid aggregation. Although the Mn 2+ -induced condensates do not fuse, our results demonstrated that they could recruit soluble α-Syn monomers into the existing condensates. Furthermore, we observed that a manganese chelator reverses Mn 2+ -induced α-Syn aggregation during the phase transition stage. However, after maturation, α-Syn aggregation becomes irreversible. These findings demonstrate that Mn 2+ facilitates α-Syn phase transition to accelerate the formation of α-Syn aggregates and provide new insights for targeting α-Syn phase separation in PD treatment., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Phase separation is one of the fundamental processes to compartmentalize biomolecules in living cells. RNA-protein complexes (RNPs) often scaffold biomolecular condensates formed through phase separation. We here present a statistical thermodynamics approach to investigate intracellular phase separation. We first present the statistical thermodynamic theory of the liquid-liquid phase separation (LLPS) of two molecules (such as proteins and solvent molecules) and of a polymer solution (such as RNPs and solvent molecules). Condensates produced by LLPS show coarsening and/or coalescence to minimize their total surface area. In addition to the LLPS, there are other types of self-assembly, such as microphase separation, micellization, emulsification, and vesiculation, with which the growth of the assembly stops with optimal size and shape. We also describe a scaling theory of micelles of block copolymers, where their structures are analogous to the core-shell structure of paraspeckle nuclear bodies scaffolded by RNPs of NEAT1_2 long noncoding RNAs (lncRNAs) and RNA-binding proteins (RBPs). These theories treat the self-assembly of polymers in the thermodynamic equilibrium, where their concentrations and compositions do not change with time. In contrast, RNPs are produced according to the transcription of RNAs and are degraded with time. We therefore take into account the dynamical aspect of the production of RNPs in an extension of the theory of the self-assembly of soft matter. Finally, we discuss the structure of paraspeckles as an example to demonstrate that an approach combining experiment and theory is powerful to investigate the mechanism of intracellular phase separation., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid–liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology. [ABSTRACT FROM AUTHOR]

Infections by negative strand RNA viruses (NSVs) induce the formation of viral inclusion bodies (IBs) in the host cell that segregate viral as well as cellular proteins to enable efficient viral replication. The induction of those membrane-less viral compartments leads inevitably to structural remodeling of the cellular architecture. Recent studies suggested that viral IBs have properties of biomolecular condensates (or liquid organelles), as have previously been shown for other membrane-less cellular compartments like stress granules or P-bodies. Biomolecular condensates are highly dynamic structures formed by liquid-liquid phase separation (LLPS). Key drivers for LLPS in cells are multivalent protein:protein and protein:RNA interactions leading to specialized areas in the cell that recruit molecules with similar properties, while other non-similar molecules are excluded. These typical features of cellular biomolecular condensates are also a common characteristic in the biogenesis of viral inclusion bodies. Viral IBs are predominantly induced by the expression of the viral nucleoprotein (N, NP) and phosphoprotein (P); both are characterized by a special protein architecture containing multiple disordered regions and RNA-binding domains that contribute to different protein functions. P keeps N soluble after expression to allow a concerted binding of N to the viral RNA. This results in the encapsidation of the viral genome by N, while P acts additionally as a cofactor for the viral polymerase, enabling viral transcription and replication. Here, we will review the formation and function of those viral inclusion bodies upon infection with NSVs with respect to their nature as biomolecular condensates. [ABSTRACT FROM AUTHOR]

Membraneless organelles (MLOs) are micro-compartments that lack delimiting membranes, concentrating several macro-molecules with a high local concentration in eukaryotic cells. Recent studies have shown that MLOs have pivotal roles in multiple biological processes, including gene transcription, RNA metabolism, translation, protein modification, and signal transduction. These biological processes in cells have essential functions in many diseases, such as cancer, neurodegenerative diseases, and virus-related diseases. The liquid-liquid phase separation (LLPS) microenvironment within cells is thought to be the driving force for initiating the formation of micro-compartments with a liquid-like property, becoming an important organizing principle for MLOs to mediate organism responses. In this review, we comprehensively elucidated the formation of these MLOs and the relationship between biological functions and associated diseases. The mechanisms underlying the influence of protein concentration and valency on phase separation in cells are also discussed. MLOs undergoing the LLPS process have diverse functions, including stimulation of some adaptive and reversible responses to alter the transcriptional or translational processes, regulation of the concentrations of biomolecules in living cells, and maintenance of cell morphogenesis. Finally, we highlight that the development of this field could pave the way for developing novel therapeutic strategies for the treatment of LLPS-related diseases based on the understanding of phase separation in the coming years., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/tcr-21-1111). The authors have no conflicts of interest to declare., (2021 Translational Cancer Research. All rights reserved.)

The function of synapses depends on spatially and temporally controlled molecular interactions between synaptic components that can be described in terms of copy numbers, binding affinities, and diffusion properties. To understand the functional role of a given synaptic protein, it is therefore crucial to quantitatively characterise its biophysical behaviour in its native cellular environment. Single molecule localisation microscopy (SMLM) is ideally suited to obtain quantitative information about synaptic proteins on the nanometre scale. Molecule counting of recombinant proteins tagged with genetically encoded fluorophores offers a means to determine their absolute copy numbers at synapses due to the known stoichiometry of the labelling. As a consequence of its high spatial precision, SMLM also yields accurate quantitative measurements of molecule concentrations. In addition, live imaging of fluorescently tagged proteins at synapses can reveal diffusion dynamics and local binding properties of behaving proteins under normal conditions or during pathological processes. In this perspective, it is argued that the detailed structural information provided by super-resolution imaging can be harnessed to gain new quantitative information about the organisation and dynamics of synaptic components in cellula . To illustrate this point, I discuss the concentration-dependent aggregation of α-synuclein in the axon and the concomitant changes in the dynamic equilibrium of α-synuclein at synapses in quantitative terms., Competing Interests: The author declares 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 © 2021 Specht.)

Most protein-coding genes in eukaryotes possess at least two poly(A) sites, and alternative polyadenylation is considered a contributing factor to transcriptomic and proteomic diversity. Following transcription, a nascent RNA usually undergoes capping, splicing, cleavage, and polyadenylation, resulting in a mature messenger RNA (mRNA); however, increasing evidence suggests that transcription and RNA processing are coupled. Plants, which must produce rapid responses to environmental changes because of their limited mobility, exhibit such coupling. In this review, we summarize recent advances in our understanding of the coupling of transcription with RNA processing in plants, and we describe the possible spatial environment and important proteins involved. Moreover, we describe how liquid–liquid phase separation, mediated by the C-terminal domain of RNA polymerase II and RNA processing factors with intrinsically disordered regions, enables efficient co-transcriptional mRNA processing in plants. [ABSTRACT FROM AUTHOR]

The basic helix–loop–helix/Per-ARNT-SIM (bHLH-PAS) proteins are a family of transcription factors regulating expression of a wide range of genes involved in different functions, ranging from differentiation and development control by oxygen and toxins sensing to circadian clock setting. In addition to the well-preserved DNA-binding bHLH and PAS domains, bHLH-PAS proteins contain long intrinsically disordered C-terminal regions, responsible for regulation of their activity. Our aim was to analyze the potential connection between disordered regions of the bHLH-PAS transcription factors, post-transcriptional modifications and liquid-liquid phase separation, in the context of disease-associated missense mutations. Highly flexible disordered regions, enriched in short motives which are more ordered, are responsible for a wide spectrum of interactions with transcriptional co-regulators. Based on our in silico analysis and taking into account the fact that the functions of transcription factors can be modulated by posttranslational modifications and spontaneous phase separation, we assume that the locations of missense mutations inducing disease states are clearly related to sequences directly undergoing these processes or to sequences responsible for their regulation. [ABSTRACT FROM AUTHOR]

A distinguishing morphological feature of all herpesviruses is the multiprotein tegument layer located between the nucleocapsid and lipid envelope of the virion. Tegument proteins play multiple roles in viral replication, including viral assembly, but we do not yet understand their individual functions or how the tegument is assembled and organized. UL11, the smallest tegument protein, is important for several distinct processes in replication, including efficient virion morphogenesis and cell-cell spread. However, the mechanistic understanding of its role in these and other processes is limited in part by the scant knowledge of its biochemical and structural properties. Here, we report that UL11 from herpes simplex virus 1 (HSV-1) is an intrinsically disordered, conformationally dynamic protein that undergoes liquid-liquid phase separation (LLPS) in vitro Intrinsic disorder may underlie the ability of UL11 to exert multiple functions and bind multiple partners. Sequence analysis suggests that not only all UL11 homologs but also all HSV-1 tegument proteins contain intrinsically disordered regions of different lengths. The presence of intrinsic disorder, and potentially, the ability to form LLPS, may thus be a common feature of the tegument proteins. We hypothesize that tegument assembly may involve the formation of a biomolecular condensate, driven by the heterogeneous mixture of intrinsically disordered tegument proteins. IMPORTANCE Herpesvirus virions contain a unique tegument layer sandwiched between the capsid and lipid envelope and composed of multiple copies of about two dozen viral proteins. However, little is known about the structure of the tegument or how it is assembled. Here, we show that a conserved tegument protein UL11 from herpes simplex virus 1, a prototypical alphaherpesvirus, is an intrinsically disordered protein that undergoes liquid-liquid phase separation in vitro Through sequence analysis, we find intrinsically disordered regions of different lengths in all HSV-1 tegument proteins. We hypothesize that intrinsic disorder is a common characteristic of tegument proteins and propose a new model of tegument as a biomolecular condensate., (Copyright © 2020 Metrick et al.)

Recent studies have indicated that tau, a protein involved in Alzheimer's disease and other neurodegenerative disorders, has a propensity to undergo liquid-liquid phase separation (LLPS). However, the mechanism of this process remains unknown. Here, we demonstrate that tau LLPS is largely driven by intermolecular electrostatic interactions between the negatively charged N-terminal and positively charged middle/C-terminal regions, whereas hydrophobic interactions play a surprisingly small role. Furthermore, our results reveal that, in contrast to previous suggestions, phosphorylation is not required for tau LLPS. These findings provide a foundation for understanding the mechanism by which phosphorylation and other posttranslational modifications could modulate tau LLPS in the context of specific physiological functions as well as pathological interactions., (© 2019 Boyko et al.)

Ribonucleoprotein (RNP) granules are membrane-less organelles consisting of RNA-binding proteins (RBPs) and RNA. RNA granules form through liquid-liquid phase separation (LLPS), whereby weak promiscuous interactions among RBPs and/or RNAs create a dense network of interacting macromolecules and drive the phase separation. Post-translational modifications (PTMs) of RBPs have emerged as important regulators of LLPS and RNP granule dynamics, as they can directly weaken or enhance the multivalent interactions between phase-separating macromolecules or can recruit or exclude certain macromolecules into or from condensates. Here, we review recent insights into how PTMs regulate phase separation and RNP granule dynamics, in particular arginine (Arg)-methylation and phosphorylation. We discuss how these PTMs regulate the phase behavior of prototypical RBPs and how, as "friend or foe," they might influence the assembly, disassembly, or material properties of cellular RNP granules, such as stress granules or amyloid-like condensates. We particularly highlight how PTMs control the phase separation and aggregation behavior of disease-linked RBPs. We also review how disruptions of PTMs might be involved in aberrant phase transitions and the formation of amyloid-like protein aggregates as observed in neurodegenerative diseases., (© 2019 Hofweber and Dormann.)

RNA granules consist of membrane-less RNA-protein assemblies and contain dynamic liquid-like shells and stable solid-like cores, which are thought to function in numerous processes in mRNA sorting and translational regulation. However, how these distinct substructures are formed, whether they are assembled by different scaffolds, and whether different RNA granule scaffolds induce these different substructures remains unknown. Here, using fluorescence microscopy-based morphological and molecular-dynamics analyses, we demonstrate that RNA granule scaffold proteins (scaffolds) can be largely classified into two groups, liquid and solid types, which induce the formation of liquid-like and solid-like granules, respectively, when expressed separately in cultured cells. We found that when co-expressed, the liquid-type and solid-type scaffolds combine and form liquid- and solid-like substructures in the same granules, respectively. The combination of the different types of scaffolds reduced the immobile fractions of the solid-type scaffolds and their dose-dependent ability to decrease nascent polypeptides in granules, but had little effect on the dynamics of the liquid-type scaffolds or their dose-dependent ability to increase nascent polypeptides in granules. These results suggest that solid- and liquid-type scaffolds form different substructures in RNA granules and differentially affect each other. Our findings provide detailed insight into the assembly mechanism and distinct dynamics and functions of core and shell substructures in RNA granules., (© 2019 Shiina.)

Background: Accumulated evidence suggests a critical role of protein-mediated liquid-liquid phase separation (LLPS) in many key biological processes through organization of membrane-less compartments. However, the pathological role of protein-mediated LLPS remains elusive and it is unknown whether protein-mediated LLPS contributes to carcinogenesis., Methods: HPDE6-C7, BxPC-3, PANC-1, AsPC-1 and CFPAC-1 cells were treated with the LLPS inhibitor, 1,6-hexanediol. Cell proliferation was determined by CCK8 assay. An atopic BxPC-3 xenograft mouse model was established and treated with 1,6-hexanediol. Gene expression profiling was achieved by RNA-sequencing., Results: 1,6-hexanediol significantly inhibited cell proliferation and induced cell death in all tested pancreatic cancer cells (BxPC-3, PANC-1, AsPC-1 and CFPAC-1) but had a minimal effect on control cells (HPDE6-C7) at low doses. In an atopic BxPC-3 xenograft model, 1,6-hexanediol significantly limited pancreatic cancer growth. In whole genomic transcriptional analysis, 1,6-hexanediol significantly downregulated the expression of a set of genes that were enriched in cytokine-cytokine receptor interactions, WNT signaling pathway, ECM-receptor interaction, MAPK signaling pathway and focal adhesion. Furthermore, 1,6-hexanediol treatment specifically reduced the expression of MYC but not IL-6 and KLF5., Conclusions: Together, we provided evidence to demonstrate an important role of protein-mediated LLPS in pancreatic cancer. Cancer cells might be more addictive to LLPS and thus disruption of such a process might be a novel way to treat cancer., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr.2019.01.06). The authors have no conflicts of interest to declare., (2019 Translational Cancer Research. All rights reserved.)

Liquid-liquid phase separation of proteins and nucleic acids into membraneless organelles (MLOs) spatially organizes cellular components and reactions. The RNA-binding protein heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) carries mRNA targets in MLOs called transport granules in neurons and oligodendrocytes. At sites of local translation, hnRNPA2 is phosphorylated by the tyrosine protein kinase Fyn, releasing the mRNA for translation. Fyn recognizes targets through its SH3 domain (Fyn-SH3). However, hnRNPA2 lacks canonical SH3-binding sequences, raising the question of how Fyn-SH3 binds hnRNPA2 in phase-separated transport granules. Here, we characterize the structural details of the interaction of the hnRNPA2 low-complexity domain (LC) with Fyn-SH3 and the effect of Fyn-SH3 on hnRNPA2 phase separation. We combined in vitro microscopy and solution NMR spectroscopy to evaluate assembly of hnRNPA2 and Fyn-SH3 into in vitro phase-separated granules and probe the structural details of their interaction. We observed that Fyn-SH3 induces hnRNPA2 LC phase separation and that Fyn-SH3 is incorporated into in vitro hnRNPA2 LC granules. Moreover, we identified hnRNPA2 LC interaction sites on the surface of Fyn-SH3. Our data offer a structural view of how hnRNPA2 LC may interact with Fyn. To our knowledge, our study provides the first example of a single globular domain inducing phase separation of a disordered MLO scaffold protein., (© 2018 Amaya et al.)