Advertisement

Matter over mind: Liquid phase separation and neurodegeneration

  • Shana Elbaum-Garfinkle
    Correspondence
    To whom correspondence should be addressed: Structural Biology Initiative, CUNY Advanced Science Research Center, New York, NY 10031. Tel.:212-413-3245;
    Affiliations
    Structural Biology Initiative, CUNY Advanced Science Research Center, New York, New York 10031

    Ph.D. Program in Biochemistry, Graduate Center, CUNY, New York, New York 10031
    Search for articles by this author
Open AccessPublished:March 26, 2019DOI:https://doi.org/10.1074/jbc.REV118.001188
      Phase separation of biomolecules leading to the formation of assemblies with distinct material properties has recently emerged as a new paradigm underlying subcellular organization. The discovery that disordered proteins, long associated with aggregation in neurodegenerative disease, are also implicated in driving liquid phase separation has galvanized significant interest in exploring the relationship between misregulated phase transitions and disease. This review summarizes recent work linking liquid phase separation to neurodegeneration, highlighting a pathological role for altered phase behavior and material properties of proteins assembled via liquid phase separation. The techniques that recent and current work in this area have deployed are also discussed, as is the potential for these discoveries to promote new research directions for investigating the molecular etiologies of neurodegenerative diseases.

      Introduction

      Over a century of research has sought to identify the origins of protein aggregation associated with neurodegenerative disease (
      • Goedert M.
      • Spillantini M.G.
      A century of Alzheimer's disease.
      ). First reported in 1911 by Dr. Alois Alzheimer (
      • Alzheimer A.
      Concerning unsual medical cases in old age.
      ), protein aggregates have been associated with an array of neurodegenerative diseases, including Alzheimer's disease (AD),
      The abbreviations used are:
      AD
      Alzheimer's disease
      FTD
      frontotemporal dementia
      FRAP
      fluorescence recovery after photobleaching
      SG
      stress granule
      hnRNP
      heterogeneous nuclear ribonucleoprotein
      polyQ
      polyglutamine.
      Parkinson's disease, ALS, frontotemporal dementia (FTD), and traumatic brain injury (
      • Chiti F.
      • Dobson C.M.
      Protein misfolding, functional amyloid, and human disease.
      ,
      • Knowles T.P.
      • Vendruscolo M.
      • Dobson C.M.
      The amyloid state and its association with protein misfolding diseases.
      ). Determining the biogenesis of protein aggregation and the mechanisms by which protein misfolding or misregulation impacts pathology is crucial for the development of efficacious therapeutic solutions. The emerging paradigm of intracellular phase separation provides a new framework for studying the longstanding and widespread problem of misregulated protein self-assembly in neurodegenerative disease.

      Pathological aggregation

      Neurodegenerative disease is broadly defined as the degeneration and loss of neuronal function—either in the brain in cases of dementia, such as AD, or in motor neurons in neuromuscular disorders, such as ALS. Whereas neurodegenerative diseases encompass a diverse spectrum of clinical and pathological presentations, they share striking features, including the predominant risk factor of aging and the aggregation of disordered proteins in the nervous system (
      • Wyss-Coray T.
      Ageing, neurodegeneration and brain rejuvenation.
      ,
      • Aguzzi A.
      • O'Connor T.
      Protein aggregation diseases: pathogenicity and therapeutic perspectives.
      ). Disordered proteins, including tau and Aβ in AD, α-synuclein in Parkinson's disease, huntingtin protein in Huntington's disease, and FUS/TDP43 in ALS, pathologically self-assemble into insoluble fibers that further aggregate into the plaques, tangles, or inclusions characteristic of each disorder (
      • Chiti F.
      • Dobson C.M.
      Protein misfolding, functional amyloid, and human disease.
      ,
      • Knowles T.P.
      • Vendruscolo M.
      • Dobson C.M.
      The amyloid state and its association with protein misfolding diseases.
      ).
      The pathological hallmarks shared by this diverse disease class imply a common, systemic origin and mechanism of toxicity. Given these shared features, a major focus of study has been the misregulation of cellular pathways associated with aging that could affect protein health or homeostasis (i.e. proteostasis). Such processes include the misregulation of the proteostasis machinery, protein clearance, posttranslational modifications, and protein damage due to oxidative stress (
      • Chiti F.
      • Dobson C.M.
      Protein misfolding, functional amyloid, and human disease.
      ,
      • Knowles T.P.
      • Vendruscolo M.
      • Dobson C.M.
      The amyloid state and its association with protein misfolding diseases.
      ). Despite much progress, many important questions remain, including how soluble disordered proteins transform to insoluble structured fibers, how aggregation pathology propagates throughout the brain, and why certain neurons are particularly susceptible to aggregation. A fundamental advance in recent years has been the accruing evidence suggesting that cellular toxicity precedes the formation of fibrous aggregates (
      • Chiti F.
      • Dobson C.M.
      Protein misfolding, functional amyloid, and human disease.
      ,
      • Knowles T.P.
      • Vendruscolo M.
      • Dobson C.M.
      The amyloid state and its association with protein misfolding diseases.
      ,
      • Bucciantini M.
      • Giannoni E.
      • Chiti F.
      • Baroni F.
      • Formigli L.
      • Zurdo J.
      • Taddei N.
      • Ramponi G.
      • Dobson C.M.
      • Stefani M.
      Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.
      ). These findings suggest that fibrous protein deposits may not be a useful therapeutic target—they may simply “report” on or even protect against an otherwise misregulated/dysfunctional process. Significant effort has thus been made to identify events or protein states that promote toxicity before the formation of fibrous aggregates, such as aberrant protein conformational changes (
      • Elbaum-Garfinkle S.
      • Rhoades E.
      Identification of an aggregation-prone structure of tau.
      ,
      • Trexler A.J.
      • Rhoades E.
      Single molecule characterization of alpha-synuclein in aggregation-prone states.
      ), or the formation of toxic soluble oligomeric species (
      • Bucciantini M.
      • Giannoni E.
      • Chiti F.
      • Baroni F.
      • Formigli L.
      • Zurdo J.
      • Taddei N.
      • Ramponi G.
      • Dobson C.M.
      • Stefani M.
      Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.
      ,
      • Campioni S.
      • Mannini B.
      • Zampagni M.
      • Pensalfini A.
      • Parrini C.
      • Evangelisti E.
      • Relini A.
      • Stefani M.
      • Dobson C.M.
      • Cecchi C.
      • Chiti F.
      A causative link between the structure of aberrant protein oligomers and their toxicity.
      ,
      • Haass C.
      • Selkoe D.J.
      Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.
      • Winner B.
      • Jappelli R.
      • Maji S.K.
      • Desplats P.A.
      • Boyer L.
      • Aigner S.
      • Hetzer C.
      • Loher T.
      • Vilar M.
      • Campioni S.
      • Tzitzilonis C.
      • Soragni A.
      • Jessberger S.
      • Mira H.
      • Consiglio A.
      • et al.
      In vivo demonstration that α-synuclein oligomers are toxic.
      ). The identification of the events that result in protein aggregation would provide important clues to the mysterious origins of pathological aggregation.

      Phase separation and biomolecular condensates

      Phase separation has recently emerged as a new paradigm underlying the intracellular assembly of proteins and RNA with emergent collective material properties (thoroughly reviewed in Refs.
      • Boeynaems S.
      • Alberti S.
      • Fawzi N.L.
      • Mittag T.
      • Polymenidou M.
      • Rousseau F.
      • Schymkowitz J.
      • Shorter J.
      • Wolozin B.
      • Van Den Bosch L.
      • Tompa P.
      • Fuxreiter M.
      Protein phase separation: a new phase in cell biology.
      • Shin Y.
      • Brangwynne C.P.
      Liquid phase condensation in cell physiology and disease.
      ,
      • Banani S.F.
      • Lee H.O.
      • Hyman A.A.
      • Rosen M.K.
      Biomolecular condensates: organizers of cellular biochemistry.
      • Dolgin E.
      What lava lamps and vinaigrette can teach us about cell biology.
      ). Liquid–liquid phase separation, or the reversible process of a homogenous fluid de-mixing into two distinct liquid phases (Fig. 1A), has been particularly recognized as a new driving force for cellular self-assembly. The advent of this new “phase” in cell biology (
      • Dolgin E.
      What lava lamps and vinaigrette can teach us about cell biology.
      ) was pioneered with the demonstrated liquid-like properties of membraneless organelles, including the apparent wetting, fusion, and dynamic exchange of internal components of P granules, (germ granules native to Caenorhabditis elegans) (
      • Brangwynne C.P.
      • Eckmann C.R.
      • Courson D.S.
      • Rybarska A.
      • Hoege C.
      • Gharakhani J.
      • Jülicher F.
      • Hyman A.A.
      Germline P granules are liquid droplets that localize by controlled dissolution/condensation.
      ), stress granules (
      • Wippich F.
      • Bodenmiller B.
      • Trajkovska M.G.
      • Wanka S.
      • Aebersold R.
      • Pelkmans L.
      Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling.
      ), and the nucleolus (
      • Brangwynne C.P.
      • Mitchison T.J.
      • Hyman A.A.
      Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes.
      ). In addition to membraneless organelles, various cellular functions, including signaling, cytoskeletal organization, and transcriptional regulation, have been shown to be accompanied by phase separation into condensed states with a range of material properties and are increasingly referred to as biomolecular condensates (
      • Banani S.F.
      • Lee H.O.
      • Hyman A.A.
      • Rosen M.K.
      Biomolecular condensates: organizers of cellular biochemistry.
      ).
      Figure thumbnail gr1
      Figure 1.Disordered proteins drive liquid phase separation and neurodegenerative aggregation. A, in classic liquid–liquid phase separation, a coexistence curve, or binodal, delineates the phase-separating region. At a given interaction strength, any concentration above the saturation concentration (Csat) gives rise to phase separation into liquid droplets made up of a higher concentration (Cdrop). Inset, P granules in the early C. elegans embryo (adapted from Ref.
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      ). B, list of disordered proteins implicated in both liquid phase separation and pathological aggregation into fibers in neurodegenerative diseases.
      Whereas many of the molecular and mechanistic details underlying the assembly and dynamics of biomolecular condensates remain to be determined, the role of multivalent protein–protein and protein–RNA interactions has been a dominant theme. Multivalent interactions of varying strength between disordered proteins of low sequence complexity (
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      • Nott T.J.
      • Petsalaki E.
      • Farber P.
      • Jervis D.
      • Fussner E.
      • Plochowietz A.
      • Craggs T.D.
      • Bazett-Jones D.P.
      • Pawson T.
      • Forman-Kay J.D.
      • Baldwin A.J.
      Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.
      ,
      • Lin Y.
      • Protter D.S.
      • Rosen M.K.
      • Parker R.
      Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.
      ,
      • Kato M.
      • Han T.W.
      • Xie S.
      • Shi K.
      • Du X.
      • Wu L.C.
      • Mirzaei H.
      • Goldsmith E.J.
      • Longgood J.
      • Pei J.
      • Grishin N.V.
      • Frantz D.E.
      • Schneider J.W.
      • Chen S.
      • Li L.
      • et al.
      Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.
      • Molliex A.
      • Temirov J.
      • Lee J.
      • Coughlin M.
      • Kanagaraj A.P.
      • Kim H.J.
      • Mittag T.
      • Taylor J.P.
      Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.
      ), modular structured domains (
      • Li P.
      • Banjade S.
      • Cheng H.C.
      • Kim S.
      • Chen B.
      • Guo L.
      • Llaguno M.
      • Hollingsworth J.V.
      • King D.S.
      • Banani S.F.
      • Russo P.S.
      • Jiang Q.X.
      • Nixon B.T.
      • Rosen M.K.
      Phase transitions in the assembly of multivalent signalling proteins.
      ,
      • Su X.
      • Ditlev J.A.
      • Hui E.
      • Xing W.
      • Banjade S.
      • Okrut J.
      • King D.S.
      • Taunton J.
      • Rosen M.K.
      • Vale R.D.
      Phase separation of signaling molecules promotes T cell receptor signal transduction.
      • Banjade S.
      • Rosen M.K.
      Phase transitions of multivalent proteins can promote clustering of membrane receptors.
      ), and combinations of these two archetypes give rise to condensed phases with a spectrum of material properties from liquids to solids.
      The discovery that disordered proteins or domains are important drivers of liquid phase separation (
      • Uversky V.N.
      Intrinsically disordered proteins in overcrowded milieu: membrane-less organelles, phase separation, and intrinsic disorder.
      ) (Fig. 1) builds on a growing appreciation of the diverse roles that disordered proteins play in the cellular milieu (
      • Uversky V.N.
      Intrinsically disordered proteins in overcrowded milieu: membrane-less organelles, phase separation, and intrinsic disorder.
      ,
      • Forman-Kay J.D.
      • Mittag T.
      From sequence and forces to structure, function, and evolution of intrinsically disordered proteins.
      ). Disordered proteins that have been identified as drivers of the assembly of membraneless organelles include the P granule proteins LAF-1 (
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      ), PGL-3 (
      • Saha S.
      • Weber C.A.
      • Nousch M.
      • Adame-Arana O.
      • Hoege C.
      • Hein M.Y.
      • Osborne-Nishimura E.
      • Mahamid J.
      • Jahnel M.
      • Jawerth L.
      • Pozniakovski A.
      • Eckmann C.R.
      • Jülicher F.
      • Hyman A.A.
      Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism.
      ), and MEG proteins (
      • Smith J.
      • Calidas D.
      • Schmidt H.
      • Lu T.
      • Rasoloson D.
      • Seydoux G.
      Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3.
      ,
      • Wang J.T.
      • Smith J.
      • Chen B.C.
      • Schmidt H.
      • Rasoloson D.
      • Paix A.
      • Lambrus B.G.
      • Calidas D.
      • Betzig E.
      • Seydoux G.
      Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans.
      ); the nucleolar protein Fib1 (
      • Berry J.
      • Weber S.C.
      • Vaidya N.
      • Haataja M.
      • Brangwynne C.P.
      RNA transcription modulates phase transition-driven nuclear body assembly.
      ,
      • Feric M.
      • Vaidya N.
      • Harmon T.S.
      • Mitrea D.M.
      • Zhu L.
      • Richardson T.M.
      • Kriwacki R.W.
      • Pappu R.V.
      • Brangwynne C.P.
      Coexisting liquid phases underlie nucleolar subcompartments.
      ); and stress granule proteins FUS (
      • Burke K.A.
      • Janke A.M.
      • Rhine C.L.
      • Fawzi N.L.
      Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II.
      ,
      • Murakami T.
      • Qamar S.
      • Lin J.Q.
      • Schierle G.S.
      • Rees E.
      • Miyashita A.
      • Costa A.R.
      • Dodd R.B.
      • Chan F.T.
      • Michel C.H.
      • Kronenberg-Versteeg D.
      • Li Y.
      • Yang S.P.
      • Wakutani Y.
      • Meadows W.
      • et al.
      ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function.
      ), TDP-43 (
      • Conicella A.E.
      • Zerze G.H.
      • Mittal J.
      • Fawzi N.L.
      ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain.
      ), and hnRNPA1 (
      • Lin Y.
      • Protter D.S.
      • Rosen M.K.
      • Parker R.
      Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.
      ,
      • Molliex A.
      • Temirov J.
      • Lee J.
      • Coughlin M.
      • Kanagaraj A.P.
      • Kim H.J.
      • Mittag T.
      • Taylor J.P.
      Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.
      ). These stress granule proteins are further implicated in the pathology of ALS and FTD, where they are found in fibrous form. In fact, low complexity sequences of RNA-binding proteins that associate with condensates have generally been linked to neurodegenerative disease (
      • King O.D.
      • Gitler A.D.
      • Shorter J.
      The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease.
      ,
      • Ramaswami M.
      • Taylor J.P.
      • Parker R.
      Altered ribostasis: RNA-protein granules in degenerative disorders.
      ). Additionally, proteins long implicated in neurodegenerative aggregation, such as tau (
      • Ambadipudi S.
      • Biernat J.
      • Riedel D.
      • Mandelkow E.
      • Zweckstetter M.
      Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau.
      ,
      • Zhang X.
      • Lin Y.
      • Eschmann N.A.
      • Zhou H.
      • Rauch J.N.
      • Hernandez I.
      • Guzman E.
      • Kosik K.S.
      • Han S.
      RNA stores tau reversibly in complex coacervates.
      • Wegmann S.
      • Eftekharzadeh B.
      • Tepper K.
      • Zoltowska K.M.
      • Bennett R.E.
      • Dujardin S.
      • Laskowski P.R.
      • MacKenzie D.
      • Kamath T.
      • Commins C.
      • Vanderburg C.
      • Roe A.D.
      • Fan Z.
      • Molliex A.M.
      • Hernandez-Vega A.
      • et al.
      Tau protein liquid-liquid phase separation can initiate tau aggregation.
      ), FMRP (
      • Vernon R.M.
      • Chong P.A.
      • Tsang B.
      • Kim T.H.
      • Bah A.
      • Farber P.
      • Lin H.
      • Forman-Kay J.D.
      Pi-Pi contacts are an overlooked protein feature relevant to phase separation.
      ), and huntingtin protein (
      • Peskett T.R.
      • Rau F.
      • O'Driscoll J.
      • Patani R.
      • Lowe A.R.
      • Saibil H.R.
      A liquid to solid phase transition underlying pathological huntingtin exon1 aggregation.
      ), have recently been demonstrated to undergo liquid phase separation.
      The newfound role for disordered proteins in liquid phase separation in general and the evidence for both liquid and fiber formation for specific neuronal proteins introduces compelling new questions about the relationship between phase separation and neurodegeneration. What role, if any, does liquid phase separation play in neurodegeneration? Under what conditions will a disordered protein assume a liquid or fibrous state, and what is the relationship between these distinct material states? The previously unrealized potential for disordered proteins to drive formation into reversible dynamic liquid phases adds an important new dimension to research questions focused on unearthing the origins of pathological aggregation associated with disease. Here we review recent work on the capacity for neuronal proteins to phase separate and discuss the potential for these discoveries to impact new directions in neurodegeneration research. Included first is an outline of the approaches commonly used to probe condensate properties.

      Probing material properties

      Probing material properties of condensates has been and continues to be crucial in realizing the role of phase separation in pathology and its corresponding potential as a therapeutic target. Summarized here are current approaches to probe the material properties of liquids and the divergence from viscous liquid behavior.

      Shape/morphology

      Pure viscous liquid droplets take on spherical shapes in solution, resulting from the energetic need to relax interfacial tension at the liquid–liquid interface (
      • Hyman A.A.
      • Weber C.A.
      • Jülicher F.
      Liquid-liquid phase separation in biology.
      ). Observance of spherical morphologies of protein assemblies has been used frequently to identify the presence of liquid material states (
      • Shin Y.
      • Brangwynne C.P.
      Liquid phase condensation in cell physiology and disease.
      ,
      • Banani S.F.
      • Lee H.O.
      • Hyman A.A.
      • Rosen M.K.
      Biomolecular condensates: organizers of cellular biochemistry.
      ). Conversely, divergence from a liquid state has been dramatically depicted by the extreme morphological shape change of P granule proteins that can form square solid sheets (
      • Hubstenberger A.
      • Noble S.L.
      • Cameron C.
      • Evans T.C.
      Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development.
      ) or the maturation of FUS droplets into a “starburst” morphology (
      • Patel A.
      • Lee H.O.
      • Jawerth L.
      • Maharana S.
      • Jahnel M.
      • Hein M.Y.
      • Stoynov S.
      • Mahamid J.
      • Saha S.
      • Franzmann T.M.
      • Pozniakovski A.
      • Poser I.
      • Maghelli N.
      • Royer L.A.
      • Weigert M.
      • et al.
      A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.
      ) (Fig. 2).
      Figure thumbnail gr2
      Figure 2.Probing material properties. A, from left to right, LAF-1 droplets form spherical droplets on a pluronic F127-treated glass surface (
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      ); LAF-1 droplets deform upon wetting untreated glass surface; P granule protein CAR-1 forms square sheet structures upon silencing CGH1 expression (
      • Hubstenberger A.
      • Noble S.L.
      • Cameron C.
      • Evans T.C.
      Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development.
      ); three-dimensional rendering of FUS protein droplets after maturation (
      • Patel A.
      • Lee H.O.
      • Jawerth L.
      • Maharana S.
      • Jahnel M.
      • Hein M.Y.
      • Stoynov S.
      • Mahamid J.
      • Saha S.
      • Franzmann T.M.
      • Pozniakovski A.
      • Poser I.
      • Maghelli N.
      • Royer L.A.
      • Weigert M.
      • et al.
      A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.
      ). B, FRAP experiments of FUS-IDR droplets demonstrating decreasing recovery and dynamics as a function of time (
      • Lin Y.
      • Protter D.S.
      • Rosen M.K.
      • Parker R.
      Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.
      ). C, coalescence of LAF-1 droplet fusion is measured by the relaxation of the aspect ratio to 1 (
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      ). Inset, timescale of fusion scales linearly with size of droplets (
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      ). D, microrheology of condensates. Left, MSD versus lag time for LAF1 droplets. Diffusive exponent α reflects the viscoelasticity of a material (see “Viscoelasticity”) (
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      ). Right, RNA can tune LAF-1 droplet viscosity (
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      ). Error bars, S.D.
      However, shape alone is insufficient for determining the material character of condensates. For instance, liquids are not always spherical, as they can deform upon wetting different surfaces, such as membranes in the case of P granules in the adult C. elegans germ line (
      • Brangwynne C.P.
      • Eckmann C.R.
      • Courson D.S.
      • Rybarska A.
      • Hoege C.
      • Gharakhani J.
      • Jülicher F.
      • Hyman A.A.
      Germline P granules are liquid droplets that localize by controlled dissolution/condensation.
      ) or glass slides with varying surface treatments (
      • Feric M.
      • Vaidya N.
      • Harmon T.S.
      • Mitrea D.M.
      • Zhu L.
      • Richardson T.M.
      • Kriwacki R.W.
      • Pappu R.V.
      • Brangwynne C.P.
      Coexisting liquid phases underlie nucleolar subcompartments.
      ) (Fig. 2). Furthermore, when droplets approach the resolution limit, as is common for small condensates in vivo, accurately resolving droplet morphology becomes increasingly challenging. Additionally, if a system transitions from a liquid to solid state, as has been suggested for balbiani bodies (
      • Boke E.
      • Mitchison T.J.
      The balbiani body and the concept of physiological amyloids.
      ) and centrosomal SPD-5 condensates (
      • Woodruff J.B.
      • Ferreira Gomes B.
      • Widlund P.O.
      • Mahamid J.
      • Honigmann A.
      • Hyman A.A.
      The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.
      ), it may retain a spherical shape despite no longer being a pure viscous liquid.

      Molecular dynamics, exchange, and mobility

      Molecules in viscous liquids are free to diffuse rapidly within droplets and undergo constant exchange between the droplet and bulk solution for liquid systems in thermodynamic equilibrium. Fluorescence recovery after photobleaching (FRAP) allows for the quantification of molecular dynamics, exchange, and mobility within droplets, and is by far the most widely used technique to probe condensate properties both in vitro and in vivo. Quantifying the rate of fluorescence recovery within a droplet allows for the estimation of an effective diffusion coefficient of the molecule probed. A decrease in the recovery rate of a molecule could indicate changes in material properties as well as oligomerization events or altered binding (
      • Reits E.A.
      • Neefjes J.J.
      From fixed to FRAP: measuring protein mobility and activity in living cells.
      ). Incomplete fluorescence recovery, or a mobile fraction less than one, reports on any immobile molecules that are not free to exchange and could indicate a divergence from liquid behavior (
      • Lin Y.
      • Protter D.S.
      • Rosen M.K.
      • Parker R.
      Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.
      ,
      • Molliex A.
      • Temirov J.
      • Lee J.
      • Coughlin M.
      • Kanagaraj A.P.
      • Kim H.J.
      • Mittag T.
      • Taylor J.P.
      Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.
      ,
      • Murakami T.
      • Qamar S.
      • Lin J.Q.
      • Schierle G.S.
      • Rees E.
      • Miyashita A.
      • Costa A.R.
      • Dodd R.B.
      • Chan F.T.
      • Michel C.H.
      • Kronenberg-Versteeg D.
      • Li Y.
      • Yang S.P.
      • Wakutani Y.
      • Meadows W.
      • et al.
      ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function.
      ,
      • Wegmann S.
      • Eftekharzadeh B.
      • Tepper K.
      • Zoltowska K.M.
      • Bennett R.E.
      • Dujardin S.
      • Laskowski P.R.
      • MacKenzie D.
      • Kamath T.
      • Commins C.
      • Vanderburg C.
      • Roe A.D.
      • Fan Z.
      • Molliex A.M.
      • Hernandez-Vega A.
      • et al.
      Tau protein liquid-liquid phase separation can initiate tau aggregation.
      ,
      • Patel A.
      • Lee H.O.
      • Jawerth L.
      • Maharana S.
      • Jahnel M.
      • Hein M.Y.
      • Stoynov S.
      • Mahamid J.
      • Saha S.
      • Franzmann T.M.
      • Pozniakovski A.
      • Poser I.
      • Maghelli N.
      • Royer L.A.
      • Weigert M.
      • et al.
      A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.
      ,
      • Mackenzie I.R.
      • Nicholson A.M.
      • Sarkar M.
      • Messing J.
      • Purice M.D.
      • Pottier C.
      • Annu K.
      • Baker M.
      • Perkerson R.B.
      • Kurti A.
      • Matchett B.J.
      • Mittag T.
      • Temirov J.
      • Hsiung G.R.
      • Krieger C.
      • et al.
      TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics.
      ,
      • Zhang H.
      • Elbaum-Garfinkle S.
      • Langdon E.M.
      • Taylor N.
      • Occhipinti P.
      • Bridges A.A.
      • Brangwynne C.P.
      • Gladfelter A.S.
      RNA controls PolyQ protein phase transitions.
      ) (Fig. 2).
      It is important to note that fluorescence recovery rates are only a proxy for liquidity of a material and depend strongly on the probe being monitored. Viscosity is a collective material property of a network of molecules and may not always be directly coupled to molecular diffusion rates. This was recently demonstrated by the length scale dependence of diffusion within LAF1 viscous droplets (
      • Wei M.T.
      • Elbaum-Garfinkle S.
      • Holehouse A.S.
      • Chen C.C.
      • Feric M.
      • Arnold C.B.
      • Priestley R.D.
      • Pappu R.V.
      • Brangwynne C.P.
      Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles.
      ). This work showed that for particles larger than 14 nm in radius, diffusion scaled with particle size, in accordance with the Stokes–Einstein relationship for the given droplet viscosity. However, for molecules smaller than 14 nm, diffusion no longer correlated with particle size and was no longer coupled to the viscosity within the droplet.

      Coalescence

      Coalescence, or fusion dynamics of droplets, can provide additional information about their material properties. For liquid droplets diffusing in a medium of lower viscosity, the timescale of fusion scales with the droplet size and the ratio of viscosity over surface tension, also known as the inverse capillary velocity (
      • Eggers J.
      • Lister J.R.
      • Stone H.A.
      Coalescence of liquid drops.
      ). Importantly, single-exponential fusion kinetics as well as a linear scaling relationship between time and length are expected for viscous liquids (
      • Ceballos A.V.
      • McDonald C.J.
      • Elbaum-Garfinkle S.
      Methods and strategies to quantify phase separation of disordered proteins.
      ). Linear scaling of droplet coalescence has been demonstrated for P granules (
      • Brangwynne C.P.
      • Eckmann C.R.
      • Courson D.S.
      • Rybarska A.
      • Hoege C.
      • Gharakhani J.
      • Jülicher F.
      • Hyman A.A.
      Germline P granules are liquid droplets that localize by controlled dissolution/condensation.
      ), nucleoli (
      • Brangwynne C.P.
      • Mitchison T.J.
      • Hyman A.A.
      Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes.
      ), droplets of LAF1 (
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      ), and Whi3 (
      • Zhang H.
      • Elbaum-Garfinkle S.
      • Langdon E.M.
      • Taylor N.
      • Occhipinti P.
      • Bridges A.A.
      • Brangwynne C.P.
      • Gladfelter A.S.
      RNA controls PolyQ protein phase transitions.
      ). Conversely, arrested coalescence or incomplete fusion, as indicated by aspherical shapes or large aspect ratios, signifies divergence from pure liquid materials. As this method offers only an approximation of the ratio of viscosity to surface tension, additional measurements are necessary to define either viscosity or surface tension and their respective changes.

      Viscoelasticity

      Materials exist on a viscoelastic spectrum, from liquids with only a viscous component to solids with only an elastic component. A material that has both viscous and elastic components is considered to be viscoelastic. The viscoelasticity of materials can be accurately measured using methods of rheology, the study of how materials flow or deform in response to pressure or stress. In particle tracking microrheology, small micrometer-sized particles are embedded into materials, and their mean squared displacement (MSD) is calculated and plotted as a function of lag time. The diffusive exponent, α, derived from the slope of a log–log plot of MSD versus lag time, can be used to quantify the viscoelasticity of the material (
      • Mason T.G.
      • Weitz D.A.
      Linear viscoelasticity of colloidal hard sphere suspensions near the glass transition.
      ,
      • Wirtz D.
      Particle-tracking microrheology of living cells: principles and applications.
      ), where α ranges from 0 (for elastic solids) to 1 (for viscous fluids), with intermediate values indicating a viscoelastic material (Fig. 2). Microrheology has been used to directly measure the viscosity of droplets composed of the P granule protein LAF1 (
      • Elbaum-Garfinkle S.
      • Kim Y.
      • Szczepaniak K.
      • Chen C.C.
      • Eckmann C.R.
      • Myong S.
      • Brangwynne C.P.
      The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
      ) as well as the polyglutamine (polyQ) protein Whi3 (
      • Zhang H.
      • Elbaum-Garfinkle S.
      • Langdon E.M.
      • Taylor N.
      • Occhipinti P.
      • Bridges A.A.
      • Brangwynne C.P.
      • Gladfelter A.S.
      RNA controls PolyQ protein phase transitions.
      ) and nucleolar proteins (
      • Feric M.
      • Vaidya N.
      • Harmon T.S.
      • Mitrea D.M.
      • Zhu L.
      • Richardson T.M.
      • Kriwacki R.W.
      • Pappu R.V.
      • Brangwynne C.P.
      Coexisting liquid phases underlie nucleolar subcompartments.
      ).
      Microrheology is a powerful tool to directly quantify viscoelastic properties of droplets in vitro; however, it can be more challenging to perform accurate microrheological measurements within small condensates in vivo. Nanorheology, a modified fluorescence correlation spectroscopy technique that measures diffusion of smaller sub-micrometer particles, has recently been used to measure viscosity of protein droplets (
      • Wei M.T.
      • Elbaum-Garfinkle S.
      • Holehouse A.S.
      • Chen C.C.
      • Feric M.
      • Arnold C.B.
      • Priestley R.D.
      • Pappu R.V.
      • Brangwynne C.P.
      Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles.
      ); this and related imaging-based technologies have the potential to be powerful tools for measurements in vivo. Further development of rheological techniques and their application to biomolecular condensates will offer important insight into the role of changing material properties in neurodegenerative disease. Microrheology measurements could, for example, be used to quantify the liquid–solid transition of condensates, as has been analogously used in measuring the gel point of stereotypical gel systems, such as acrylamide (
      • Larsen T.H.
      • Furst E.M.
      Microrheology of the liquid-solid transition during gelation.
      ).

      Reversibility and/or presence of insoluble aggregates

      Another way to probe the properties of droplets is to probe the reversibility or presence of aggregates in solution over time or as a function of mutation/perturbation. The dissolution of phase-separated liquid droplets can be achieved by reversing the phase-separating condition (i.e. by changing the salt, temperature, or pH or “cycling” those parameters). Incomplete reversibility to a single soluble phase signals a divergence from a liquid state. This phenomenon has been referred to as “maturation” or even a “liquid to solid transition” (
      • Peskett T.R.
      • Rau F.
      • O'Driscoll J.
      • Patani R.
      • Lowe A.R.
      • Saibil H.R.
      A liquid to solid phase transition underlying pathological huntingtin exon1 aggregation.
      ,
      • Patel A.
      • Lee H.O.
      • Jawerth L.
      • Maharana S.
      • Jahnel M.
      • Hein M.Y.
      • Stoynov S.
      • Mahamid J.
      • Saha S.
      • Franzmann T.M.
      • Pozniakovski A.
      • Poser I.
      • Maghelli N.
      • Royer L.A.
      • Weigert M.
      • et al.
      A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.
      ). Additionally, droplet dissolution by 1,6-hexanediol is often used to probe droplet properties (
      • Molliex A.
      • Temirov J.
      • Lee J.
      • Coughlin M.
      • Kanagaraj A.P.
      • Kim H.J.
      • Mittag T.
      • Taylor J.P.
      Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.
      ,
      • Wegmann S.
      • Eftekharzadeh B.
      • Tepper K.
      • Zoltowska K.M.
      • Bennett R.E.
      • Dujardin S.
      • Laskowski P.R.
      • MacKenzie D.
      • Kamath T.
      • Commins C.
      • Vanderburg C.
      • Roe A.D.
      • Fan Z.
      • Molliex A.M.
      • Hernandez-Vega A.
      • et al.
      Tau protein liquid-liquid phase separation can initiate tau aggregation.
      ,
      • Peskett T.R.
      • Rau F.
      • O'Driscoll J.
      • Patani R.
      • Lowe A.R.
      • Saibil H.R.
      A liquid to solid phase transition underlying pathological huntingtin exon1 aggregation.
      ,
      • Lee K.H.
      • Zhang P.
      • Kim H.J.
      • Mitrea D.M.
      • Sarkar M.
      • Freibaum B.D.
      • Cika J.
      • Coughlin M.
      • Messing J.
      • Molliex A.
      • Maxwell B.A.
      • Kim N.C.
      • Temirov J.
      • Moore J.
      • Kolaitis R.M.
      • et al.
      C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles.
      ,
      • Kroschwald S.
      • Maharana S.
      • Mateju D.
      • Malinovska L.
      • Nüske E.
      • Poser I.
      • Richter D.
      • Alberti S.
      Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules.
      ). This aliphatic alcohol is thought to disrupt transient hydrophobic interactions between aromatic residues (
      • Ribbeck K.
      • Görlich D.
      The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.
      ) sometimes present within liquid phases, but fails to disrupt the more stable interactions present in other fibrous forms. However, it is not actually clear precisely which interactions 1,6-hexanediol disrupts, and responses to this compound do not directly report on the presence or absence of a liquid phase.
      The presence of insoluble aggregates within droplets can be further confirmed in a number of ways. A simple centrifugation assay after dissolution of the condensed liquid phase, can report on the formation of any insoluble material. EM can be used to analyze the aggregate structures in more detail and/or confirm the presence of nanoscale fibers. Additionally, small fluorescent molecular probes like thioflavin S or T, which report on the presence of cross-β-sheet structures, are often used to discern ordered fibers from amorphous aggregates (
      • Molliex A.
      • Temirov J.
      • Lee J.
      • Coughlin M.
      • Kanagaraj A.P.
      • Kim H.J.
      • Mittag T.
      • Taylor J.P.
      Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.
      ,
      • Wegmann S.
      • Eftekharzadeh B.
      • Tepper K.
      • Zoltowska K.M.
      • Bennett R.E.
      • Dujardin S.
      • Laskowski P.R.
      • MacKenzie D.
      • Kamath T.
      • Commins C.
      • Vanderburg C.
      • Roe A.D.
      • Fan Z.
      • Molliex A.M.
      • Hernandez-Vega A.
      • et al.
      Tau protein liquid-liquid phase separation can initiate tau aggregation.
      ,
      • Patel A.
      • Lee H.O.
      • Jawerth L.
      • Maharana S.
      • Jahnel M.
      • Hein M.Y.
      • Stoynov S.
      • Mahamid J.
      • Saha S.
      • Franzmann T.M.
      • Pozniakovski A.
      • Poser I.
      • Maghelli N.
      • Royer L.A.
      • Weigert M.
      • et al.
      A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.
      ,
      • Kroschwald S.
      • Maharana S.
      • Mateju D.
      • Malinovska L.
      • Nüske E.
      • Poser I.
      • Richter D.
      • Alberti S.
      Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules.
      ). It is important to note that these probes are quite prone to false negatives and positives (
      • Malmos K.G.
      • Blancas-Mejia L.M.
      • Weber B.
      • Buchner J.
      • Ramirez-Alvarado M.
      • Naiki H.
      • Otzen D.
      ThT 101:a primer on the use of thioflavin T to investigate amyloid formation.
      ), and careful controls along with complementary techniques should be applied.

      Phase separation of neuronal proteins

      Stress granule proteins

      Stress granule–related proteins have provided the first link between phase-separated compartments and neurological disease. The proteins FUS, TDP-43, hnRNPA1, and TIA1 and the C9orf72 translational dipeptide repeats GR/PR, are each implicated in the pathology of ALS and FTD with demonstrated roles in phase separation.

      FUS

      Fused in sarcoma (FUS) protein is an RNA binding protein that associates with liquid-like stress granules (SGs) and is further implicated in chromosomal translocations in cancer and aggregation in ALS/FTD (
      • Deng H.
      • Gao K.
      • Jankovic J.
      The role of FUS gene variants in neurodegenerative diseases.
      ). In 2015, Patel et al. (
      • Patel A.
      • Lee H.O.
      • Jawerth L.
      • Maharana S.
      • Jahnel M.
      • Hein M.Y.
      • Stoynov S.
      • Mahamid J.
      • Saha S.
      • Franzmann T.M.
      • Pozniakovski A.
      • Poser I.
      • Maghelli N.
      • Royer L.A.
      • Weigert M.
      • et al.
      A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.
      ) demonstrated that FUS-GFP can phase-separate into spherical droplets, capable of fusion and with fast and complete recovery by FRAP. They further showed that introduction of the ALS disease mutation G156E to their FUS-GFP construct resulted in dramatic changes to the material properties of the droplets over time, illustrated by significant loss of mobile fraction by FRAP and the appearance, upon shaking, of nonspherical structures (Fig. 2). Murakami et al. (
      • Murakami T.
      • Qamar S.
      • Lin J.Q.
      • Schierle G.S.
      • Rees E.
      • Miyashita A.
      • Costa A.R.
      • Dodd R.B.
      • Chan F.T.
      • Michel C.H.
      • Kronenberg-Versteeg D.
      • Li Y.
      • Yang S.P.
      • Wakutani Y.
      • Meadows W.
      • et al.
      ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function.
      ) further showed that reversibility of FUS aggregates in vitro and in vivo is compromised by ALS/FTD mutations, consequently leading to the sequestering of additional proteins within the aggregates. Together, these studies suggest that changing of material properties from reversible liquid states to less soluble states (i.e. “maturation” or a liquid-to-solid transition) could contribute to disease. Additionally, phosphorylation of FUS (
      • Monahan Z.
      • Ryan V.H.
      • Janke A.M.
      • Burke K.A.
      • Rhoads S.N.
      • Zerze G.H.
      • O'Meally R.
      • Dignon G.L.
      • Conicella A.E.
      • Zheng W.
      • Best R.B.
      • Cole R.N.
      • Mittal J.
      • Shewmaker F.
      • Fawzi N.L.
      Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity.
      ) has been shown to disrupt both FUS phase separation and aggregation, suggesting a potential treatment pathway for FUS assembly regulation.

      TDP-43

      Aggregates of TAR-DNA–binding protein of 43 kDa (TDP-43) are impressively found in ∼97% of all cases of ALS and 45% of FTD cases (
      • Ling H.
      • Kara E.
      • Bandopadhyay R.
      • Hardy J.
      • Holton J.
      • Xiromerisiou G.
      • Lees A.
      • Houlden H.
      • Revesz T.
      TDP-43 pathology in a patient carrying G2019S LRRK2 mutation and a novel p.Q124E MAPT.
      ,
      • Arnold E.S.
      • Ling S.C.
      • Huelga S.C.
      • Lagier-Tourenne C.
      • Polymenidou M.
      • Ditsworth D.
      • Kordasiewicz H.B.
      • McAlonis-Downes M.
      • Platoshyn O.
      • Parone P.A.
      • Da Cruz S.
      • Clutario K.M.
      • Swing D.
      • Tessarollo L.
      • Marsala M.
      • et al.
      ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43.
      ), but the connection between TDP-43 aggregation and toxicity remains unclear. The C-terminal domain of TDP-43 has been shown to phase-separate into droplets with rapid dynamics by FRAP (
      • Conicella A.E.
      • Zerze G.H.
      • Mittal J.
      • Fawzi N.L.
      ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain.
      ). Probing several disease mutants, the authors found that most mutants inhibited phase separation, demonstrated by a shift to the right in the phase diagram, aside from A321V, which exhibits a shift to the left. Interestingly, all disease mutants showed enhanced “conversion” to aggregates over time, as seen by a loss of reversibility upon temperature cycling.

      hnRNPA1

      Heterogeneous nuclear ribonucleoproteins (hnRNPs) form cytoplasmic inclusions that are pathologically deposited in ALS/FTD patients. Molliex et al. (
      • Molliex A.
      • Temirov J.
      • Lee J.
      • Coughlin M.
      • Kanagaraj A.P.
      • Kim H.J.
      • Mittag T.
      • Taylor J.P.
      Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.
      ) demonstrated that hnRNPA1 can form liquid droplets in a concentration-dependent manner. They show that fibrillization of the disease-causing mutation D262V is promoted by phase separation using a combination of temperature cycling and ThT staining (
      • Molliex A.
      • Temirov J.
      • Lee J.
      • Coughlin M.
      • Kanagaraj A.P.
      • Kim H.J.
      • Mittag T.
      • Taylor J.P.
      Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.
      ). Similarly, Lin et al. (
      • Lin Y.
      • Protter D.S.
      • Rosen M.K.
      • Parker R.
      Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.
      ) demonstrated the accumulation of aggregates in hnRNPA1 droplets over time that are further exacerbated by the amyloid-enhancing D262V mutation.

      TIA1

      T cell–restricted intracellular antigen-1 (TIA1) is an SG component with a demonstrated role in SG assembly (
      • Kedersha N.
      • Cho M.R.
      • Li W.
      • Yacono P.W.
      • Chen S.
      • Gilks N.
      • Golan D.E.
      • Anderson P.
      Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules.
      ,
      • Gilks N.
      • Kedersha N.
      • Ayodele M.
      • Shen L.
      • Stoecklin G.
      • Dember L.M.
      • Anderson P.
      Stress granule assembly is mediated by prion-like aggregation of TIA-1.
      ). The disordered domain of TIA1 has been shown to undergo liquid phase separation in the presence of RNA (
      • Lin Y.
      • Protter D.S.
      • Rosen M.K.
      • Parker R.
      Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.
      ). Using the full-length TIA1, Mackenzie et al. (
      • Mackenzie I.R.
      • Nicholson A.M.
      • Sarkar M.
      • Messing J.
      • Purice M.D.
      • Pottier C.
      • Annu K.
      • Baker M.
      • Perkerson R.B.
      • Kurti A.
      • Matchett B.J.
      • Mittag T.
      • Temirov J.
      • Hsiung G.R.
      • Krieger C.
      • et al.
      TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics.
      ) showed that disease mutations in TIA1 (P362L, A381T, and E384K) promote phase separation, demonstrated as a shift to the left in the phase diagram; decrease mobile fraction in TIA1 droplets by FRAP; and decrease droplet reversibility. In vivo, they found that TIA1 mutations alter stress granule dynamics, in that they inhibit the rate and completion of disassembly. Interestingly, stress-induced colocalization of TDP-43 into granules accelerates aggregation of TIA1 within SGs.

      C9orf72

      Expansion of hexanucleotide repeat GGGCC in the intron of chromosome 9 ORF 72 (C9ORF72) is the most common cause of FTD and ALS, serving as evidence for the connection between the pathologies of the two neurological diseases (
      • DeJesus-Hernandez M.
      • Mackenzie I.R.
      • Boeve B.F.
      • Boxer A.L.
      • Baker M.
      • Rutherford N.J.
      • Nicholson A.M.
      • Finch N.A.
      • Flynn H.
      • Adamson J.
      • Kouri N.
      • Wojtas A.
      • Sengdy P.
      • Hsiung G.Y.R.
      • Karydas A.
      • et al.
      Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.
      ,
      • Renton A.E.
      • Majounie E.
      • Waite A.
      • Simón-Sánchez J.
      • Rollinson S.
      • Gibbs J.R.
      • Schymick J.C.
      • Laaksovirta H.
      • van Swieten J.C.
      • Myllykangas L.
      • Kalimo H.
      • Paetau A.
      • Abramzon Y.
      • Remes A.M.
      • Kaganovich A.
      • et al.
      A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.
      ). One potential consequence of this expansion lies in the misregulated translation of this sequence, which produces dipeptide repeats GR and PR (
      • Zu T.
      • Liu Y.
      • Bañez-Coronel M.
      • Reid T.
      • Pletnikova O.
      • Lewis J.
      • Miller T.M.
      • Harms M.B.
      • Falchook A.E.
      • Subramony S.H.
      • Ostrow L.W.
      • Rothstein J.D.
      • Troncoso J.C.
      • Ranum L.P.W.
      RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia.
      ). In 2016, Lee et al. (
      • Lee K.H.
      • Zhang P.
      • Kim H.J.
      • Mitrea D.M.
      • Sarkar M.
      • Freibaum B.D.
      • Cika J.
      • Coughlin M.
      • Messing J.
      • Molliex A.
      • Maxwell B.A.
      • Kim N.C.
      • Temirov J.
      • Moore J.
      • Kolaitis R.M.
      • et al.
      C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles.
      ) found that expression and colocalization of GR and PR dipeptides with nucleoli and SGs can impair dynamics and mobile fraction in living cells by FRAP. They further showed that GR/PR peptides colocalize with hnRNPA1 and TIA1 droplets in vitro and promote phase separation by decreasing the saturation concentration. Boeynaems et al. (
      • Boeynaems S.
      • Bogaert E.
      • Kovacs D.
      • Konijnenberg A.
      • Timmerman E.
      • Volkov A.
      • Guharoy M.
      • De Decker M.
      • Jaspers T.
      • Ryan V.H.
      • Janke A.M.
      • Baatsen P.
      • Vercruysse T.
      • Kolaitis R.M.
      • Daelemans D.
      • Taylor J.P.
      • Kedersha N.
      • Anderson P.
      • Impens F.
      • Sobott F.
      • Schymkowitz J.
      • Rousseau F.
      • Fawzi N.L.
      • Robberecht W.
      • Van Damme P.
      • Tompa P.
      • Van Den Bosch L.
      Phase separation of C9orf72 dipeptide repeats perturbs stress granule dynamics.
      ) further showed that PR peptides can alone phase-separate into dynamic liquids capable of fusion and recovery by FRAP.

      Other neurodegenerative proteins

      More recently, additional neuronal proteins not previously known to be associated with liquid compartments have also been demonstrated to undergo liquid phase separation. These include tau (
      • Ambadipudi S.
      • Biernat J.
      • Riedel D.
      • Mandelkow E.
      • Zweckstetter M.
      Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau.
      ,
      • Zhang X.
      • Lin Y.
      • Eschmann N.A.
      • Zhou H.
      • Rauch J.N.
      • Hernandez I.
      • Guzman E.
      • Kosik K.S.
      • Han S.
      RNA stores tau reversibly in complex coacervates.
      • Wegmann S.
      • Eftekharzadeh B.
      • Tepper K.
      • Zoltowska K.M.
      • Bennett R.E.
      • Dujardin S.
      • Laskowski P.R.
      • MacKenzie D.
      • Kamath T.
      • Commins C.
      • Vanderburg C.
      • Roe A.D.
      • Fan Z.
      • Molliex A.M.
      • Hernandez-Vega A.
      • et al.
      Tau protein liquid-liquid phase separation can initiate tau aggregation.
      ,
      • Zhou L.
      • McInnes J.
      • Wierda K.
      • Holt M.
      • Herrmann A.G.
      • Jackson R.J.
      • Wang Y.C.
      • Swerts J.
      • Beyens J.
      • Miskiewicz K.
      • Vilain S.
      • Dewachter I.
      • Moechars D.
      • De Strooper B.
      • Spires-Jones T.L.
      • et al.
      Tau association with synaptic vesicles causes presynaptic dysfunction.
      ,
      • Hernández-Vega A.
      • Braun M.
      • Scharrel L.
      • Jahnel M.
      • Wegmann S.
      • Hyman B.T.
      • Alberti S.
      • Diez S.
      • Hyman A.A.
      Local nucleation of microtubule bundles through tubulin concentration into a condensed tau phase.
      ), FMRP (
      • Vernon R.M.
      • Chong P.A.
      • Tsang B.
      • Kim T.H.
      • Bah A.
      • Farber P.
      • Lin H.
      • Forman-Kay J.D.
      Pi-Pi contacts are an overlooked protein feature relevant to phase separation.
      ,
      • Boeynaems S.
      • Bogaert E.
      • Kovacs D.
      • Konijnenberg A.
      • Timmerman E.
      • Volkov A.
      • Guharoy M.
      • De Decker M.
      • Jaspers T.
      • Ryan V.H.
      • Janke A.M.
      • Baatsen P.
      • Vercruysse T.
      • Kolaitis R.M.
      • Daelemans D.
      • Taylor J.P.
      • Kedersha N.
      • Anderson P.
      • Impens F.
      • Sobott F.
      • Schymkowitz J.
      • Rousseau F.
      • Fawzi N.L.
      • Robberecht W.
      • Van Damme P.
      • Tompa P.
      • Van Den Bosch L.
      Phase separation of C9orf72 dipeptide repeats perturbs stress granule dynamics.
      ), and huntingtin protein (
      • Peskett T.R.
      • Rau F.
      • O'Driscoll J.
      • Patani R.
      • Lowe A.R.
      • Saibil H.R.
      A liquid to solid phase transition underlying pathological huntingtin exon1 aggregation.
      ).

      Tau

      Tau is a neuronal protein that pathologically aggregates into fibers and tangles associated with a long list of tauopathies, from Alzheimer's disease to traumatic brain injury (
      • Arendt T.
      • Stieler J.T.
      • Holzer M.
      Tau and tauopathies.
      ). Recent work has found that in the presence of macromolecular crowder, phosphorylation, and/or RNA, tau can phase-separate into liquid droplets. Wegmann et al. (
      • Wegmann S.
      • Eftekharzadeh B.
      • Tepper K.
      • Zoltowska K.M.
      • Bennett R.E.
      • Dujardin S.
      • Laskowski P.R.
      • MacKenzie D.
      • Kamath T.
      • Commins C.
      • Vanderburg C.
      • Roe A.D.
      • Fan Z.
      • Molliex A.M.
      • Hernandez-Vega A.
      • et al.
      Tau protein liquid-liquid phase separation can initiate tau aggregation.
      ) have shown that full-length tau (tau441-GFP) forms droplets in cultured neurons that exhibit rapid recovery after photobleaching. In vitro, they show that phosphorylated tau, naturally phosphorylated from expression in insect cells, forms droplets that become less dynamic over time. They further show that increased phosphorylation levels of tau, as well as the addition of co-factors known to increase tau aggregation, such as heparin and RNA, induce or enhance droplet formation.
      Similarly, the microtubule-binding region fragment of tau, K18, has been shown to form droplets that are enhanced by increasing the temperature to 37 °C (
      • Ambadipudi S.
      • Biernat J.
      • Riedel D.
      • Mandelkow E.
      • Zweckstetter M.
      Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau.
      ). Phosphorylation also enhances K18 phase separation by lowering the saturation concentration and the apparent rate of droplet formation. Both studies demonstrated the formation of ThS (
      • Wegmann S.
      • Eftekharzadeh B.
      • Tepper K.
      • Zoltowska K.M.
      • Bennett R.E.
      • Dujardin S.
      • Laskowski P.R.
      • MacKenzie D.
      • Kamath T.
      • Commins C.
      • Vanderburg C.
      • Roe A.D.
      • Fan Z.
      • Molliex A.M.
      • Hernandez-Vega A.
      • et al.
      Tau protein liquid-liquid phase separation can initiate tau aggregation.
      ) and ThT (
      • Ambadipudi S.
      • Biernat J.
      • Riedel D.
      • Mandelkow E.
      • Zweckstetter M.
      Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau.
      ) positive aggregates in their respective droplet systems. Another fragment of tau, d187, has been shown to form droplets in an RNA-dependent manner (
      • Zhang X.
      • Lin Y.
      • Eschmann N.A.
      • Zhou H.
      • Rauch J.N.
      • Hernandez I.
      • Guzman E.
      • Kosik K.S.
      • Han S.
      RNA stores tau reversibly in complex coacervates.
      ). Using the insect cell–expressed phosphorylated tau441-GFP in the presence of 10% dextran, Hernández-Vega et al. (
      • Hernández-Vega A.
      • Braun M.
      • Scharrel L.
      • Jahnel M.
      • Wegmann S.
      • Hyman B.T.
      • Alberti S.
      • Diez S.
      • Hyman A.A.
      Local nucleation of microtubule bundles through tubulin concentration into a condensed tau phase.
      ) further showed that tau phase separation can enhance the organization of tubulin and MT polymerization, suggesting a potential functional role for tau phase separation.

      Huntingtin

      Recent work by Peskett et al. (
      • Peskett T.R.
      • Rau F.
      • O'Driscoll J.
      • Patani R.
      • Lowe A.R.
      • Saibil H.R.
      A liquid to solid phase transition underlying pathological huntingtin exon1 aggregation.
      ) has shown that HTT exon 1 with extended polyQ repeats can form assemblies with distinct morphologies in mammalian cells and yeast. They show that polyQ expansions can form two types of assemblies with distinct intensity features they refer to as “dim” and “bright” as well as distinct physical properties. The dim assemblies appear to be more consistent with a liquid state, in that they recover almost completely and within seconds by FRAP, can undergo fusion, and are reversible by 1,6-hexanediol, whereas the “bright” assemblies exhibit significantly slower recovery times with very low mobile fractions, are 1,6-hexanediol–insensitive, often have asymmetrical structures or spikes that protrude from their center of mass, and appear by electron tomography to contain a web of fibrous nanostructures.
      In vitro, they were able to show that polyQ25 could form droplets in the presence of 10% dextran that could fuse, recover rapidly by FRAP, and were sensitive to 1,6-hexanediol. Interestingly, they note that these droplets rapidly matured, showing fibrous protrusions and partial resistance to 1,6-hexanediol dissolution in just 30 min of incubation.

      Novel therapeutic targets

      These recent discoveries position liquid phase separation as a compelling novel component in the neurodegeneration pathway and consequently a new avenue for therapeutic strategies. However, the unanswered questions already surrounding the area of pathological aggregation are equally present here. For instance, whether or not protein fibers promote, protect, or are simply an artifact of toxicity should frame interpretations of data that aim to parse the connections between liquids and fibers. Additionally, whether or not liquid phases contribute to loss of function, gain of dysfunction, or both remains to be determined. Furthermore, various cases of reversible and/or functional amyloid fibers have been reported (
      • Audas T.E.
      • Audas D.E.
      • Jacob M.D.
      • Ho J.J.D.
      • Khacho M.
      • Wang M.
      • Perera J.K.
      • Gardiner C.
      • Bennett C.A.
      • Head T.
      • Kryvenko O.N.
      • Jorda M.
      • Daunert S.
      • Malhotra A.
      • Trinkle-Mulcahy L.
      • et al.
      Adaptation to stressors by systemic protein amyloidogenesis.
      ,
      • Berchowitz L.E.
      • Kabachinski G.
      • Walker M.R.
      • Carlile T.M.
      • Gilbert W.V.
      • Schwartz T.U.
      • Amon A.
      Regulated formation of an amyloid-like translational repressor governs gametogenesis.
      ), further challenging assumptions about the pathological status of protein fibers. In light of these questions, it can be useful to break down the current evidence for the role of liquid phase separation in neurodegeneration into three effective modes of action: those that influence (a) propensity for phase separation, (b) material properties of phase-separated droplets, and (c) nucleation of fibers (Fig. 3). This framework further delineates three potential pathways for therapeutic intervention.
      Figure thumbnail gr3
      Figure 3.Regulating and targeting liquid phase separation. Three modes of action currently link phase separation to pathology with the potential to serve as pathways for intervention. A, the phase boundary, a metric for the propensity to phase-separate, is defined by the saturation concentration and the relative interaction strengths between molecules. B, material properties of condensed phases include viscoelasticity, reversibility of exchange, and the dynamics and mobility of molecules within and across droplets. C, fiber formation may be nucleated within droplets, potentially giving rise to pathology. These three modes of action, which are sensitive to disease-associated conditions, highlight distinct avenues for regulation and therapeutic targeting of liquid phase separation in disease.

      Propensity for phase separation

      Phase separation is extremely sensitive to perturbations, including protein concentration, cellular/environmental conditions, and the relative abundance of cofactors or binding partners. The demonstrated capacity for neuronal proteins to phase-separate raises new possibilities for assembly (mis)regulation in vivo. A shift in a phase boundary of a protein or group of biomolecules that enhances (left shift) or inhibits (right shift) phase separation could have significant consequences for the function/dysfunction of neuronal proteins. ALS disease mutations have been shown to both inhibit (TDP43 (
      • Conicella A.E.
      • Zerze G.H.
      • Mittal J.
      • Fawzi N.L.
      ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain.
      )) and enhance (TDP4-3 and A231V (
      • Conicella A.E.
      • Zerze G.H.
      • Mittal J.
      • Fawzi N.L.
      ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain.
      ) and TIA1 (
      • Mackenzie I.R.
      • Nicholson A.M.
      • Sarkar M.
      • Messing J.
      • Purice M.D.
      • Pottier C.
      • Annu K.
      • Baker M.
      • Perkerson R.B.
      • Kurti A.
      • Matchett B.J.
      • Mittag T.
      • Temirov J.
      • Hsiung G.R.
      • Krieger C.
      • et al.
      TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics.
      )) phase separation; post-translational modifications, such as phosphorylation, have been shown to inhibit phase separation in the case of FUS (
      • Monahan Z.
      • Ryan V.H.
      • Janke A.M.
      • Burke K.A.
      • Rhoads S.N.
      • Zerze G.H.
      • O'Meally R.
      • Dignon G.L.
      • Conicella A.E.
      • Zheng W.
      • Best R.B.
      • Cole R.N.
      • Mittal J.
      • Shewmaker F.
      • Fawzi N.L.
      Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity.
      ) but enhance phase separation in the case of tau (
      • Wegmann S.
      • Eftekharzadeh B.
      • Tepper K.
      • Zoltowska K.M.
      • Bennett R.E.
      • Dujardin S.
      • Laskowski P.R.
      • MacKenzie D.
      • Kamath T.
      • Commins C.
      • Vanderburg C.
      • Roe A.D.
      • Fan Z.
      • Molliex A.M.
      • Hernandez-Vega A.
      • et al.
      Tau protein liquid-liquid phase separation can initiate tau aggregation.
      ). Interestingly, in both cases, the effect is consistent with inhibiting or enhancing aggregation. Independent of the potential relationship between liquids and fibers or the fate of the phase-separated state, the liquid phase diagram and its tunable phase boundary could serve as a useful assay in screening effective drug candidates.

      Material properties

      The material properties of protein phases, such as viscoelasticity and internal diffusivity, are also sensitive to perturbations and highly tunable by condition and cofactors. We are just beginning to discern the role of these material states in regulating the components within. In the case of SGs, there is increasing evidence for the impact of changing dynamics on pathology. Overall, disease mutations in FUS (
      • Murakami T.
      • Qamar S.
      • Lin J.Q.
      • Schierle G.S.
      • Rees E.
      • Miyashita A.
      • Costa A.R.
      • Dodd R.B.
      • Chan F.T.
      • Michel C.H.
      • Kronenberg-Versteeg D.
      • Li Y.
      • Yang S.P.
      • Wakutani Y.
      • Meadows W.
      • et al.
      ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function.
      ,
      • Patel A.
      • Lee H.O.
      • Jawerth L.
      • Maharana S.
      • Jahnel M.
      • Hein M.Y.
      • Stoynov S.
      • Mahamid J.
      • Saha S.
      • Franzmann T.M.
      • Pozniakovski A.
      • Poser I.
      • Maghelli N.
      • Royer L.A.
      • Weigert M.
      • et al.
      A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.
      ), TDP-43 (
      • Conicella A.E.
      • Zerze G.H.
      • Mittal J.
      • Fawzi N.L.
      ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain.
      ,
      • Mackenzie I.R.
      • Nicholson A.M.
      • Sarkar M.
      • Messing J.
      • Purice M.D.
      • Pottier C.
      • Annu K.
      • Baker M.
      • Perkerson R.B.
      • Kurti A.
      • Matchett B.J.
      • Mittag T.
      • Temirov J.
      • Hsiung G.R.
      • Krieger C.
      • et al.
      TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics.
      ), hnRNPA1 (
      • Lin Y.
      • Protter D.S.
      • Rosen M.K.
      • Parker R.
      Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.
      ,
      • Molliex A.
      • Temirov J.
      • Lee J.
      • Coughlin M.
      • Kanagaraj A.P.
      • Kim H.J.
      • Mittag T.
      • Taylor J.P.
      Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.
      ), and TIA1 (
      • Mackenzie I.R.
      • Nicholson A.M.
      • Sarkar M.
      • Messing J.
      • Purice M.D.
      • Pottier C.
      • Annu K.
      • Baker M.
      • Perkerson R.B.
      • Kurti A.
      • Matchett B.J.
      • Mittag T.
      • Temirov J.
      • Hsiung G.R.
      • Krieger C.
      • et al.
      TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics.
      ) give rise to changes in material properties, including a decrease in fluidity, mobile fraction, coalescence, and internal diffusivity. Additionally, longer polyQ protein variants that are associated with disease appear to form assemblies with distinct morphologies and material properties (
      • Peskett T.R.
      • Rau F.
      • O'Driscoll J.
      • Patani R.
      • Lowe A.R.
      • Saibil H.R.
      A liquid to solid phase transition underlying pathological huntingtin exon1 aggregation.
      ). Changes in granule material properties can have direct consequences for both loss and gain of function. Decreasing dynamics would impact interactions and reactions associated with granule function, whereas loss of reversibility may impact the native availability of a particular protein in the cytoplasmic or nucleoplasmic bulk. Furthermore, as in the case of FUS, such changes can lead to the aberrant sequestration of additional proteins within the assembly with the potential for innumerable functional consequences. Together, this suggests that devising methods to modulate the material properties of granules could present a useful therapeutic strategy.

      Fiber formation

      It is an intriguing possibility that enhanced propensity to liquid phase-separate, increased duration or interactions in a liquid state, and/or modulated liquid phase properties can contribute to the formation of fibers. Evidence for this now exists for SG proteins as well as tau and huntingtin. Phase separation could enhance the formation of fibers by providing an unstable oversaturated highly concentrated environment. This begs the question of how liquid states, if that unstable, are normally maintained, suggesting the potential involvement of stabilizing cofactors or chaperones. Fiber formation could in turn contribute to material property changes with aberrant functional consequences, such as unwanted sequestration as described above. Strategies to preserve liquid properties or otherwise inhibit conditions that result in the nucleation of fibers, could prove to be effective. Whereas the precise relationship between fluids and fibers still needs to be determined, the growing evidence linking phase separation to the classic pathological hallmark of neurodegeneration holds significant potential for impactful discoveries.

      Conclusion

      The previously unrealized potential for intracellular proteins to assemble via phase separation into soft condensed material states provides a new lens through which to probe the origins of pathological aggregation associated with neurodegeneration. The evidence in support of a relationship between droplet properties, protein aggregation, and neurodegenerative pathology is rapidly growing; however, significant future work is necessary to determine the molecular and regulatory mechanisms underlying these processes. The new paradigm of phase separation coupled with the advancement of techniques to probe this new phenomenon in and out of cells has the potential to drive new therapeutic directions for treating many devastating neurodegenerative diseases.

      Acknowledgments

      I thank Clifford P. Brangwynne, Elizabeth Rhoades, and Stephanie Weber for providing useful feedback on early manuscript drafts and Rachel Fisher, Charles J. McDonald, and Alfredo Vidal Ceballos for proofreading the manuscript.

      References

        • Goedert M.
        • Spillantini M.G.
        A century of Alzheimer's disease.
        Science. 2006; 314 (17082447): 777-781
        • Alzheimer A.
        Concerning unsual medical cases in old age.
        Z. Gesamte Neurol. Psy. 1911; 4: 356-385
        • Chiti F.
        • Dobson C.M.
        Protein misfolding, functional amyloid, and human disease.
        Annu. Rev. Biochem. 2006; 75 (16756495): 333-366
        • Knowles T.P.
        • Vendruscolo M.
        • Dobson C.M.
        The amyloid state and its association with protein misfolding diseases.
        Nat. Rev. Mol. Cell Biol. 2014; 15 (24854788): 384-396
        • Wyss-Coray T.
        Ageing, neurodegeneration and brain rejuvenation.
        Nature. 2016; 539 (27830812): 180-186
        • Aguzzi A.
        • O'Connor T.
        Protein aggregation diseases: pathogenicity and therapeutic perspectives.
        Nat. Rev. Drug. Discov. 2010; 9 (20190788): 237-248
        • Bucciantini M.
        • Giannoni E.
        • Chiti F.
        • Baroni F.
        • Formigli L.
        • Zurdo J.
        • Taddei N.
        • Ramponi G.
        • Dobson C.M.
        • Stefani M.
        Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.
        Nature. 2002; 416 (11932737): 507-511
        • Elbaum-Garfinkle S.
        • Rhoades E.
        Identification of an aggregation-prone structure of tau.
        J. Am. Chem. Soc. 2012; 134 (22998648): 16607-16613
        • Trexler A.J.
        • Rhoades E.
        Single molecule characterization of alpha-synuclein in aggregation-prone states.
        Biophys. J. 2010; 99 (21044603): 3048-3055
        • Campioni S.
        • Mannini B.
        • Zampagni M.
        • Pensalfini A.
        • Parrini C.
        • Evangelisti E.
        • Relini A.
        • Stefani M.
        • Dobson C.M.
        • Cecchi C.
        • Chiti F.
        A causative link between the structure of aberrant protein oligomers and their toxicity.
        Nat. Chem. Biol. 2010; 6 (20081829): 140-147
        • Haass C.
        • Selkoe D.J.
        Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.
        Nat. Rev. Mol. Cell Biol. 2007; 8 (17245412): 101-112
        • Winner B.
        • Jappelli R.
        • Maji S.K.
        • Desplats P.A.
        • Boyer L.
        • Aigner S.
        • Hetzer C.
        • Loher T.
        • Vilar M.
        • Campioni S.
        • Tzitzilonis C.
        • Soragni A.
        • Jessberger S.
        • Mira H.
        • Consiglio A.
        • et al.
        In vivo demonstration that α-synuclein oligomers are toxic.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21325059): 4194-4199
        • Boeynaems S.
        • Alberti S.
        • Fawzi N.L.
        • Mittag T.
        • Polymenidou M.
        • Rousseau F.
        • Schymkowitz J.
        • Shorter J.
        • Wolozin B.
        • Van Den Bosch L.
        • Tompa P.
        • Fuxreiter M.
        Protein phase separation: a new phase in cell biology.
        Trends Cell Biol. 2018; 28 (29602697): 420-435
        • Shin Y.
        • Brangwynne C.P.
        Liquid phase condensation in cell physiology and disease.
        Science. 2017; 357 (28935776): eaaf4382
        • Banani S.F.
        • Lee H.O.
        • Hyman A.A.
        • Rosen M.K.
        Biomolecular condensates: organizers of cellular biochemistry.
        Nat. Rev. Mol. Cell Biol. 2017; 18 (28225081): 285-298
        • Dolgin E.
        What lava lamps and vinaigrette can teach us about cell biology.
        Nature. 2018; 555 (29542707): 300-302
        • Brangwynne C.P.
        • Eckmann C.R.
        • Courson D.S.
        • Rybarska A.
        • Hoege C.
        • Gharakhani J.
        • Jülicher F.
        • Hyman A.A.
        Germline P granules are liquid droplets that localize by controlled dissolution/condensation.
        Science. 2009; 324 (19460965): 1729-1732
        • Wippich F.
        • Bodenmiller B.
        • Trajkovska M.G.
        • Wanka S.
        • Aebersold R.
        • Pelkmans L.
        Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling.
        Cell. 2013; 152 (23415227): 791-805
        • Brangwynne C.P.
        • Mitchison T.J.
        • Hyman A.A.
        Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21368180): 4334-4339
        • Elbaum-Garfinkle S.
        • Kim Y.
        • Szczepaniak K.
        • Chen C.C.
        • Eckmann C.R.
        • Myong S.
        • Brangwynne C.P.
        The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
        Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26015579): 7189-7194
        • Nott T.J.
        • Petsalaki E.
        • Farber P.
        • Jervis D.
        • Fussner E.
        • Plochowietz A.
        • Craggs T.D.
        • Bazett-Jones D.P.
        • Pawson T.
        • Forman-Kay J.D.
        • Baldwin A.J.
        Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.
        Mol. Cell. 2015; 57 (25747659): 936-947
        • Lin Y.
        • Protter D.S.
        • Rosen M.K.
        • Parker R.
        Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.
        Mol. Cell. 2015; 60 (26412307): 208-219
        • Kato M.
        • Han T.W.
        • Xie S.
        • Shi K.
        • Du X.
        • Wu L.C.
        • Mirzaei H.
        • Goldsmith E.J.
        • Longgood J.
        • Pei J.
        • Grishin N.V.
        • Frantz D.E.
        • Schneider J.W.
        • Chen S.
        • Li L.
        • et al.
        Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.
        Cell. 2012; 149 (22579281): 753-767
        • Molliex A.
        • Temirov J.
        • Lee J.
        • Coughlin M.
        • Kanagaraj A.P.
        • Kim H.J.
        • Mittag T.
        • Taylor J.P.
        Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.
        Cell. 2015; 163 (26406374): 123-133
        • Li P.
        • Banjade S.
        • Cheng H.C.
        • Kim S.
        • Chen B.
        • Guo L.
        • Llaguno M.
        • Hollingsworth J.V.
        • King D.S.
        • Banani S.F.
        • Russo P.S.
        • Jiang Q.X.
        • Nixon B.T.
        • Rosen M.K.
        Phase transitions in the assembly of multivalent signalling proteins.
        Nature. 2012; 483 (22398450): 336-340
        • Su X.
        • Ditlev J.A.
        • Hui E.
        • Xing W.
        • Banjade S.
        • Okrut J.
        • King D.S.
        • Taunton J.
        • Rosen M.K.
        • Vale R.D.
        Phase separation of signaling molecules promotes T cell receptor signal transduction.
        Science. 2016; 352 (27056844): 595-599
        • Banjade S.
        • Rosen M.K.
        Phase transitions of multivalent proteins can promote clustering of membrane receptors.
        Elife. 2014; 3 (25321392)
        • Uversky V.N.
        Intrinsically disordered proteins in overcrowded milieu: membrane-less organelles, phase separation, and intrinsic disorder.
        Curr. Opin Struct. Biol. 2017; 44 (27838525): 18-30
        • Forman-Kay J.D.
        • Mittag T.
        From sequence and forces to structure, function, and evolution of intrinsically disordered proteins.
        Structure. 2013; 21 (24010708): 1492-1499
        • Saha S.
        • Weber C.A.
        • Nousch M.
        • Adame-Arana O.
        • Hoege C.
        • Hein M.Y.
        • Osborne-Nishimura E.
        • Mahamid J.
        • Jahnel M.
        • Jawerth L.
        • Pozniakovski A.
        • Eckmann C.R.
        • Jülicher F.
        • Hyman A.A.
        Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism.
        Cell. 2016; 166 (27594427): 1572-1584.e16
        • Smith J.
        • Calidas D.
        • Schmidt H.
        • Lu T.
        • Rasoloson D.
        • Seydoux G.
        Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3.
        Elife. 2016; 5 (27914198): e21337
        • Wang J.T.
        • Smith J.
        • Chen B.C.
        • Schmidt H.
        • Rasoloson D.
        • Paix A.
        • Lambrus B.G.
        • Calidas D.
        • Betzig E.
        • Seydoux G.
        Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans.
        Elife. 2014; 3 (25535836): e04591
        • Berry J.
        • Weber S.C.
        • Vaidya N.
        • Haataja M.
        • Brangwynne C.P.
        RNA transcription modulates phase transition-driven nuclear body assembly.
        Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26351690): E5237-E5245
        • Feric M.
        • Vaidya N.
        • Harmon T.S.
        • Mitrea D.M.
        • Zhu L.
        • Richardson T.M.
        • Kriwacki R.W.
        • Pappu R.V.
        • Brangwynne C.P.
        Coexisting liquid phases underlie nucleolar subcompartments.
        Cell. 2016; 165 (27212236): 1686-1697
        • Burke K.A.
        • Janke A.M.
        • Rhine C.L.
        • Fawzi N.L.
        Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II.
        Mol. Cell. 2015; 60 (26455390): 231-241
        • Murakami T.
        • Qamar S.
        • Lin J.Q.
        • Schierle G.S.
        • Rees E.
        • Miyashita A.
        • Costa A.R.
        • Dodd R.B.
        • Chan F.T.
        • Michel C.H.
        • Kronenberg-Versteeg D.
        • Li Y.
        • Yang S.P.
        • Wakutani Y.
        • Meadows W.
        • et al.
        ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function.
        Neuron. 2015; 88 (26526393): 678-690
        • Conicella A.E.
        • Zerze G.H.
        • Mittal J.
        • Fawzi N.L.
        ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain.
        Structure. 2016; 24 (27545621): 1537-1549
        • King O.D.
        • Gitler A.D.
        • Shorter J.
        The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease.
        Brain Res. 2012; 1462 (22445064): 61-80
        • Ramaswami M.
        • Taylor J.P.
        • Parker R.
        Altered ribostasis: RNA-protein granules in degenerative disorders.
        Cell. 2013; 154 (23953108): 727-736
        • Ambadipudi S.
        • Biernat J.
        • Riedel D.
        • Mandelkow E.
        • Zweckstetter M.
        Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau.
        Nat. Commun. 2017; 8 (28819146): 275
        • Zhang X.
        • Lin Y.
        • Eschmann N.A.
        • Zhou H.
        • Rauch J.N.
        • Hernandez I.
        • Guzman E.
        • Kosik K.S.
        • Han S.
        RNA stores tau reversibly in complex coacervates.
        PLoS Biol. 2017; 15 (28683104): e2002183
        • Wegmann S.
        • Eftekharzadeh B.
        • Tepper K.
        • Zoltowska K.M.
        • Bennett R.E.
        • Dujardin S.
        • Laskowski P.R.
        • MacKenzie D.
        • Kamath T.
        • Commins C.
        • Vanderburg C.
        • Roe A.D.
        • Fan Z.
        • Molliex A.M.
        • Hernandez-Vega A.
        • et al.
        Tau protein liquid-liquid phase separation can initiate tau aggregation.
        EMBO J. 2018; 37 (29472250): e98049
        • Vernon R.M.
        • Chong P.A.
        • Tsang B.
        • Kim T.H.
        • Bah A.
        • Farber P.
        • Lin H.
        • Forman-Kay J.D.
        Pi-Pi contacts are an overlooked protein feature relevant to phase separation.
        Elife. 2018; 7 (29424691): e31486
        • Peskett T.R.
        • Rau F.
        • O'Driscoll J.
        • Patani R.
        • Lowe A.R.
        • Saibil H.R.
        A liquid to solid phase transition underlying pathological huntingtin exon1 aggregation.
        Mol. Cell. 2018; 70 (29754822): 588-601.e6
        • Hyman A.A.
        • Weber C.A.
        • Jülicher F.
        Liquid-liquid phase separation in biology.
        Annu. Rev. Cell Dev. Biol. 2014; 30 (25288112): 39-58
        • Hubstenberger A.
        • Noble S.L.
        • Cameron C.
        • Evans T.C.
        Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development.
        Dev. Cell. 2013; 27 (24176641): 161-173
        • Patel A.
        • Lee H.O.
        • Jawerth L.
        • Maharana S.
        • Jahnel M.
        • Hein M.Y.
        • Stoynov S.
        • Mahamid J.
        • Saha S.
        • Franzmann T.M.
        • Pozniakovski A.
        • Poser I.
        • Maghelli N.
        • Royer L.A.
        • Weigert M.
        • et al.
        A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.
        Cell. 2015; 162 (26317470): 1066-1077
        • Boke E.
        • Mitchison T.J.
        The balbiani body and the concept of physiological amyloids.
        Cell Cycle. 2017; 16 (27736303): 153-154
        • Woodruff J.B.
        • Ferreira Gomes B.
        • Widlund P.O.
        • Mahamid J.
        • Honigmann A.
        • Hyman A.A.
        The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.
        Cell. 2017; 169 (28575670): 1066-1077.e10
        • Reits E.A.
        • Neefjes J.J.
        From fixed to FRAP: measuring protein mobility and activity in living cells.
        Nat. Cell Biol. 2001; 3 (11389456): E145-E147
        • Mackenzie I.R.
        • Nicholson A.M.
        • Sarkar M.
        • Messing J.
        • Purice M.D.
        • Pottier C.
        • Annu K.
        • Baker M.
        • Perkerson R.B.
        • Kurti A.
        • Matchett B.J.
        • Mittag T.
        • Temirov J.
        • Hsiung G.R.
        • Krieger C.
        • et al.
        TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics.
        Neuron. 2017; 95 (e809 28817800): 808-816
        • Zhang H.
        • Elbaum-Garfinkle S.
        • Langdon E.M.
        • Taylor N.
        • Occhipinti P.
        • Bridges A.A.
        • Brangwynne C.P.
        • Gladfelter A.S.
        RNA controls PolyQ protein phase transitions.
        Mol. Cell. 2015; 60 (26474065): 220-230
        • Wei M.T.
        • Elbaum-Garfinkle S.
        • Holehouse A.S.
        • Chen C.C.
        • Feric M.
        • Arnold C.B.
        • Priestley R.D.
        • Pappu R.V.
        • Brangwynne C.P.
        Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles.
        Nat. Chem. 2017; 9 (29064502): 1118-1125
        • Eggers J.
        • Lister J.R.
        • Stone H.A.
        Coalescence of liquid drops.
        J. Fluid Mech. 1999; 401: 293-310
        • Ceballos A.V.
        • McDonald C.J.
        • Elbaum-Garfinkle S.
        Methods and strategies to quantify phase separation of disordered proteins.
        Methods Enzymol. 2018; 611 (30471691): 31-50
        • Mason T.G.
        • Weitz D.A.
        Linear viscoelasticity of colloidal hard sphere suspensions near the glass transition.
        Phys. Rev. Lett. 1995; 75 (10059400): 2770-2773
        • Wirtz D.
        Particle-tracking microrheology of living cells: principles and applications.
        Annu. Rev. Biophys. 2009; 38 (19416071): 301-326
        • Larsen T.H.
        • Furst E.M.
        Microrheology of the liquid-solid transition during gelation.
        Phys. Rev. Lett. 2008; 100 (18518051): 146001
        • Lee K.H.
        • Zhang P.
        • Kim H.J.
        • Mitrea D.M.
        • Sarkar M.
        • Freibaum B.D.
        • Cika J.
        • Coughlin M.
        • Messing J.
        • Molliex A.
        • Maxwell B.A.
        • Kim N.C.
        • Temirov J.
        • Moore J.
        • Kolaitis R.M.
        • et al.
        C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles.
        Cell. 2016; 167 (27768896): 774-788.e17
        • Kroschwald S.
        • Maharana S.
        • Mateju D.
        • Malinovska L.
        • Nüske E.
        • Poser I.
        • Richter D.
        • Alberti S.
        Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules.
        Elife. 2015; 4 (26238190): e06807
        • Ribbeck K.
        • Görlich D.
        The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.
        EMBO J. 2002; 21 (12032079): 2664-2671
        • Malmos K.G.
        • Blancas-Mejia L.M.
        • Weber B.
        • Buchner J.
        • Ramirez-Alvarado M.
        • Naiki H.
        • Otzen D.
        ThT 101:a primer on the use of thioflavin T to investigate amyloid formation.
        Amyloid. 2017; 24 (28393556): 1-16
        • Deng H.
        • Gao K.
        • Jankovic J.
        The role of FUS gene variants in neurodegenerative diseases.
        Nat. Rev. Neurol. 2014; 10 (24840975): 337-348
        • Monahan Z.
        • Ryan V.H.
        • Janke A.M.
        • Burke K.A.
        • Rhoads S.N.
        • Zerze G.H.
        • O'Meally R.
        • Dignon G.L.
        • Conicella A.E.
        • Zheng W.
        • Best R.B.
        • Cole R.N.
        • Mittal J.
        • Shewmaker F.
        • Fawzi N.L.
        Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity.
        EMBO J. 2017; 36 (28790177): 2951-2967
        • Ling H.
        • Kara E.
        • Bandopadhyay R.
        • Hardy J.
        • Holton J.
        • Xiromerisiou G.
        • Lees A.
        • Houlden H.
        • Revesz T.
        TDP-43 pathology in a patient carrying G2019S LRRK2 mutation and a novel p.Q124E MAPT.
        Neurobiol. Aging. 2013; 34 (23664753): 2889.e5-2889.e9
        • Arnold E.S.
        • Ling S.C.
        • Huelga S.C.
        • Lagier-Tourenne C.
        • Polymenidou M.
        • Ditsworth D.
        • Kordasiewicz H.B.
        • McAlonis-Downes M.
        • Platoshyn O.
        • Parone P.A.
        • Da Cruz S.
        • Clutario K.M.
        • Swing D.
        • Tessarollo L.
        • Marsala M.
        • et al.
        ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43.
        Proc. Natl Acad. Sci. U.S.A. 2013; 110 (23382207): E736-E745
        • Kedersha N.
        • Cho M.R.
        • Li W.
        • Yacono P.W.
        • Chen S.
        • Gilks N.
        • Golan D.E.
        • Anderson P.
        Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules.
        J. Cell Biol. 2000; 151 (11121440): 1257-1268
        • Gilks N.
        • Kedersha N.
        • Ayodele M.
        • Shen L.
        • Stoecklin G.
        • Dember L.M.
        • Anderson P.
        Stress granule assembly is mediated by prion-like aggregation of TIA-1.
        Mol. Biol. Cell. 2004; 15 (15371533): 5383-5398
        • DeJesus-Hernandez M.
        • Mackenzie I.R.
        • Boeve B.F.
        • Boxer A.L.
        • Baker M.
        • Rutherford N.J.
        • Nicholson A.M.
        • Finch N.A.
        • Flynn H.
        • Adamson J.
        • Kouri N.
        • Wojtas A.
        • Sengdy P.
        • Hsiung G.Y.R.
        • Karydas A.
        • et al.
        Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.
        Neuron. 2011; 72 (21944778): 245-256
        • Renton A.E.
        • Majounie E.
        • Waite A.
        • Simón-Sánchez J.
        • Rollinson S.
        • Gibbs J.R.
        • Schymick J.C.
        • Laaksovirta H.
        • van Swieten J.C.
        • Myllykangas L.
        • Kalimo H.
        • Paetau A.
        • Abramzon Y.
        • Remes A.M.
        • Kaganovich A.
        • et al.
        A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.
        Neuron. 2011; 72 (21944779): 257-268
        • Zu T.
        • Liu Y.
        • Bañez-Coronel M.
        • Reid T.
        • Pletnikova O.
        • Lewis J.
        • Miller T.M.
        • Harms M.B.
        • Falchook A.E.
        • Subramony S.H.
        • Ostrow L.W.
        • Rothstein J.D.
        • Troncoso J.C.
        • Ranum L.P.W.
        RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (24248382): E4968-E4977
        • Boeynaems S.
        • Bogaert E.
        • Kovacs D.
        • Konijnenberg A.
        • Timmerman E.
        • Volkov A.
        • Guharoy M.
        • De Decker M.
        • Jaspers T.
        • Ryan V.H.
        • Janke A.M.
        • Baatsen P.
        • Vercruysse T.
        • Kolaitis R.M.
        • Daelemans D.
        • Taylor J.P.
        • Kedersha N.
        • Anderson P.
        • Impens F.
        • Sobott F.
        • Schymkowitz J.
        • Rousseau F.
        • Fawzi N.L.
        • Robberecht W.
        • Van Damme P.
        • Tompa P.
        • Van Den Bosch L.
        Phase separation of C9orf72 dipeptide repeats perturbs stress granule dynamics.
        Mol. Cell. 2017; 65 (28306503): 1044-1055.e5
        • Zhou L.
        • McInnes J.
        • Wierda K.
        • Holt M.
        • Herrmann A.G.
        • Jackson R.J.
        • Wang Y.C.
        • Swerts J.
        • Beyens J.
        • Miskiewicz K.
        • Vilain S.
        • Dewachter I.
        • Moechars D.
        • De Strooper B.
        • Spires-Jones T.L.
        • et al.
        Tau association with synaptic vesicles causes presynaptic dysfunction.
        Nat. Commun. 2017; 8 (28492240): 15295
        • Hernández-Vega A.
        • Braun M.
        • Scharrel L.
        • Jahnel M.
        • Wegmann S.
        • Hyman B.T.
        • Alberti S.
        • Diez S.
        • Hyman A.A.
        Local nucleation of microtubule bundles through tubulin concentration into a condensed tau phase.
        Cell Rep. 2017; 20 (28877466): 2304-2312
        • Arendt T.
        • Stieler J.T.
        • Holzer M.
        Tau and tauopathies.
        Brain Res. Bull. 2016; 126 (27615390): 238-292
        • Audas T.E.
        • Audas D.E.
        • Jacob M.D.
        • Ho J.J.D.
        • Khacho M.
        • Wang M.
        • Perera J.K.
        • Gardiner C.
        • Bennett C.A.
        • Head T.
        • Kryvenko O.N.
        • Jorda M.
        • Daunert S.
        • Malhotra A.
        • Trinkle-Mulcahy L.
        • et al.
        Adaptation to stressors by systemic protein amyloidogenesis.
        Dev. Cell. 2016; 39 (27720612): 155-168
        • Berchowitz L.E.
        • Kabachinski G.
        • Walker M.R.
        • Carlile T.M.
        • Gilbert W.V.
        • Schwartz T.U.
        • Amon A.
        Regulated formation of an amyloid-like translational repressor governs gametogenesis.
        Cell. 2015; 163 (26411291): 406-418