Phase separation in biology; functional organization of a higher order

Diana Mitrea; Richard Kriwacki

doi:10.1186/s12964-015-0125-7

Phase separation in biology; functional organization of a higher order

Mitrea, Diana; Kriwacki, Richard 2016-01-05 00:00:00 Inside eukaryotic cells, macromolecules are partitioned into membrane-bounded compartments and, within these, some are further organized into non-membrane-bounded structures termed membrane-less organelles. The latter structures are comprised of heterogeneous mixtures of proteins and nucleic acids and assemble through a phase separation phenomenon similar to polymer condensation. Membrane-less organelles are dynamic structures maintained through multivalent interactions that mediate diverse biological processes, many involved in RNA metabolism. They rapidly exchange components with the cellular milieu and their properties are readily altered in response to environmental cues, often implicating membrane-less organelles in responses to stress signaling. In this review, we discuss: (1) the functional roles of membrane-less organelles, (2) unifying structural and mechanistic principles that underlie their assembly and disassembly, and (3) established and emerging methods used in structural investigations of membrane-less organelles. Keywords: Membrane-less organelles, Phase separation, Multivalency, Stress response, RNA metabolism Background specialized in protein sorting and trafficking through the Similar to the division of labor in human societies, the cell. Mitochondria supply the ATP energetic needs of a cellular “workforce”, macromolecules such as proteins, cell, and are enclosed in a double layer membrane, in DNA and RNA, is spatially organized in the cell based contrast to the single lipid bilayer that surrounds the on functional specialization. Subcellular organization of other membrane-bounded organelles. macromolecules underlies vital cellular processes such With the advent of electron microscopy that allowed as development, division and homeostasis, while disrup- visualization of nanometer scale structures [1] and ad- tion of this organization is often associated with disease. vances in fluorescent dyes and light microscopy, it be- A large proportion of the enzymatic and signaling came evident that there is further sub-division and local reactions in biology occurs in aqueous solution. Lipid organization within the nucleus and cytosol in the form bilayers, immiscible with the aqueous phase, enclose the of non-membrane bounded, macromolecular assemblies. water-soluble components of a cell. The plasma mem- Currently characterized membrane-less bodies or or- brane engulfs all the internal components of a cell. ganelles range in size from tens of nm to tens of μm and Membrane-bounded organelles provide the physical sep- were defined as highly dynamic macromolecular assem- aration required for specialized processes to occur in blies, whose components rapidly cycle between the functionally optimized compartments within a cell. organelle and surrounding milieu [2–7]. Nucleoli Thus, the nucleus contains the machinery dedicated for (reviewed in [8]), nuclear speckles (reviewed in [3, 9]), DNA and RNA synthesis, while the cytoplasm houses paraspeckles (reviewed in [2, 10]), and PML (reviewed in components that control protein synthesis and degrad- [11, 12]) and Cajal bodies (reviewed in [4]) are enclosed ation. The endoplasmic reticulum, Golgi apparatus and within the nuclear envelope and are specialized in vari- the lipid vesicles are membrane-bounded compartments ous aspects of gene regulation and RNA metabolism. Cytoplasmic messenger ribonucleoprotein (mRNP) gran- * Correspondence: [email protected] ules, such as P-bodies, germ granules, and stress gran- Department of Structural Biology, St. Jude Children’s Research Hospital, ules (reviewed in [13]) fulfill specific roles in mRNA Memphis, TN 38105, USA Department of Microbiology, Immunology and Biochemistry, University of metabolism and homeostasis. Analogous forms of RNA Tennessee Health Sciences Center, Memphis, TN 38163, USA © 2016 Mitrea and Kriwacki. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 2 of 20 granules have recently been identified in mitochondria component (DFC) and granular component (GC). Dur- with roles in mitochondrial ribosome biogenesis and ing mitosis, the GC dissolves, disrupting nucleolar RNA processing [14]. organization but components of the FC and DFC main- In this review we will present an overview of current tain interactions as diffusible sub-structures. knowledge regarding the structural biology of membrane- Nucleolar assembly (reviewed in [8]) is initiated by less organelles and the molecular mechanisms involved in RNA Polymerase I (RNA Pol I) transcription of clustered regulating their structure and function. ribosomal RNA (rRNA) genes (rDNA) bound to the transcription factor UBF. Ribosome biogenesis occurs Overview of membrane-less organelles vectorially, starting from the FCs, where rDNA is tran- Membrane-less organelles were described as dynamic scribed into rRNA. pre-rRNA molecules transit through structures which often display liquid-like physical prop- the DFC, where they are spliced and the small ribosomal erties [5, 6]. Although it is well established that they are subunit is assembled, then move into the GC where the implicated in important biological processes, their pre- large ribosomal subunit is assembled. Pre-ribosomal par- cise roles remain elusive, often being associated with ticles are then released into the nucleoplasm and subse- more than a single functional pathway. As will be de- quently exported into the cytoplasm where functional scribed in greater detail in the following sections, the ribosomes are assembled. proteinaceous composition of membrane-less organelles p53-dependent stress sensing mechanisms are inte- and their morphology are altered in response to changes grated into the nucleolus, thereby allowing the cell to in the cellular environment. This ability to respond to halt the energetically expensive process of ribosome bio- environmental cues may represent the mechanistic basis genesis under conditions that are unfavorable for growth for the involvement of the membrane-less organelles dis- and proliferation. For example, in response to oncogenic cussed herein in stress sensing [2, 4, 9, 11, 13, 15]. The stress (e.g., activation of Myc), Mdm2, the E3 ubiquitin lack of a lipid-rich barrier to enclose the constituents of ligase responsible for rapid turnover of p53, is immobi- ARF membrane-less organelles presents the advantage that lized in the nucleolus through interactions with p14 changes in the surrounding environment can readily in order to upregulate p53 and its downstream cell cycle alter their internal equilibrium. Release or sequestration arrest effectors [17]. of constituent proteins or RNAs from or within membrane-less organelles alters their concentrations in Paraspeckles the surrounding freely diffusing pool of macromolecules, Paraspeckles are nuclear bodies located in the interchro- thereby sending signals that impinge upon stress re- matin space, with roles in control of gene expression sponse pathways. One example is the accumulation into through nuclear retention of specific RNA molecules, the nucleolus, followed by release into the nucleoplasm marked by adenosine-inosine editing [2]. The proteins ARF of the tumor suppressor p14 in response to DNA that comprise paraspeckles are associated with RNA damage, which activates the p53 tumor suppressor path- Polymerase II (RNA Pol II) transcription and processing way [16]. The nuclear volume is partitioned into mul- of RNA. The DBHS family of splicing proteins, tiple membrane-less organelles, also called nuclear P54NRB/NONO, PSPC1, PSF/SFPQ [2, 10, 18, 19], and bodies. Cytoplasmic bodies further partition the cyto- the long non-coding RNAs (lcnRNA) NEAT1/Men ε/β solic components. Nuclear and cytoplasmic bodies are and Ctn are integral components of paraspeckles [2]. dynamic structures, with well-defined compositions, Paraspeckles are responsive to stress and exchange com- which have the ability to exchange components in re- ponents with the nucleolus in response to environmental sponse to alterations to their environment. In the follow- cues. For example, paraspeckle protein 1 (PSPC1) was ing section we will discuss the functional roles of first identified as a nucleolar protein; however, it was membrane-less organelles and the unique features that later shown that, under conditions of active RNA Pol II- define them. dependent transcription, it partitions into a different nuclear body, the paraspeckles, and only becomes re- Nuclear membrane-less bodies localized to the nucleolus when RNA Pol II activity is The nucleolus suppressed [10, 18]. Interestingly, this re-localization oc- The largest and best studied membrane-less organelle, curs at the peri-nucleolar caps, which are structures that the nucleolus, functions as the center for ribosome bio- appear to be physically associated with nucleoli, but are genesis in eukaryotic cells. The nucleolus exhibits com- not integrated into the nucleolar matrix [10]. This plex, compartmentalized organization in interphase and suggests that either the physical properties of PSPC1- disassembles in mitosis. Three distinct regions can be containing bodies and of the nucleolus are different, pre- observed by transmission electron microscopy (TEM) in cluding fusion, or their dynamic behavior is restricted in intact nucleoli: the fibrillar centers (FC), dense fibrillar response to the signals that inhibit RNA Pol II activity. Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 3 of 20 Nuclear speckles is dispensable with respect to the formation of PML Similar in appearance to paraspeckles and localized adja- bodies [29]. cent to nucleoplasmic interchromatin regions [3], nu- clear speckles, also referred to as snurposomes, are a Cytosolic membrane-less bodies distinct class of dynamic organelles [1]. The compos- Dynamic membrane-less organelles were also described ition of nuclear speckles, enriched in pre-mRNA spli- in the cytoplasm. They are generally referred to as cing factors, such as small nuclear ribonucleoproteins mRNP granules, are involved in mRNA metabolism and (snRNPs) and serine/arginine-rich (SR) proteins [20], homeostasis, and include structures such as P-bodies, and poly(A) RNA[21],aswell astheir spatialprox- stress granules and germ granules (reviewed in [13, 30]). imity to sites of active transcription, suggest they may Several different types of mRNP granules share protein playarole inregulatinggeneexpressionby supplying and mRNA components and it has been demonstrated or storing factors associated with the splicing of pre- that they have the ability to physically interact with one mRNAs [22]. another in vivo, undergoing docking and fusion events [13]. These observations suggest that not only are these Cajal bodies membrane-less organelles functionally related, but under Although not fully elucidated, the role of the Cajal bod- certain conditions they exhibit similar physico-chemical ies is linked to regulation of snRNPs and small nucleolar properties that allow for their structural miscibility. The ribonucleoprotein particles (snoRNPs) [4]. Time lapse major types of mRNP granules are discussed below. experiments monitoring fluorescently tagged coilin and survival of motor neurons (SMN) proteins, two well de- P-bodies scribed markers of Cajal bodies, showed that they are Processing or P-bodies are ubiquitous to all types of cells dynamic structures within the nucleus that undergo fu- and contain proteins involved in mRNA transport, sion and fission events [23]. Similar to other nuclear modification and translation (reviewed in [31]). Studies membrane-less organelles, Cajal bodies are responsive to in yeast demonstrated that deletion of any single protein stress conditions. The tumor suppressor p53 associates component was not sufficient to fully abrogate the as- with Cajal bodies under conditions of UV-irradiation sembly of P-bodies [32], but highlighted the importance and chemotoxic stress [24], while coilin re-localizes to of partner-specific interactions to the accumulation of a nucleolar caps, along with fibrillarin and components of number of proteins into the organelle [33, 34]. For ex- the RNA Pol I machinery [25]. Furthermore, similar to ample, recruitment of the Dcp1 decapping enzyme to the nucleolus, the structural integrity of Cajal bodies is the organelle is mediated by interactions with its co- cell cycle dependent; they are intact during interphase factor, Dcp2 [34], while Dcp2 directly interacts with the and dissolve during mitosis [26]. scaffold protein Edc3 [33, 34]. As with other membrane- less organelles, RNA plays a central role in the assembly PML bodies of P-bodies. Elevated levels of non-translating mRNA, Localized primarily in the nucleus, PML bodies are char- achieved by inhibition of translation initiation or stress, acterized by the presence of promyelocytic leukemia is correlated with an increase in the size and number of (PML) protein. A member of the TRIM family of pro- P-bodies [35]. Conversely, entrapment of mRNA into teins, PML contains a RING domain, two B-box do- polysomes by inhibiting the elongation step or enzymatic mains and a predicted coiled-coil domain, all of which degradation of mRNA correlated with dissolution of have been shown to be required for proper assembly of P-bodies [31, 35]. PML bodies. The exact role of these organelles is yet to be fully elucidated. Evidence that transcriptional regula- Stress granules tors such as p53, CBP and Daxx are transiently targeted Stress granules, as the name suggests, assemble in re- and retained in PML bodies suggests that they function sponse to stress signals to sequester transcriptionally as a storage compartment and thus regulate pathways silent mRNA molecules and transcription factors involved in tumor suppression, viral defense and apop- (reviewed in [30]). Translation initiation factors and tosis [12]. As with other membrane-less organelles, the components of the small ribosomal subunit are amongst number and structural integrity of PML bodies are influ- the proteins enriched within stress granules [13]. Re- enced by cell cycle phase and stress stimuli [27]. In sen- moval of the stress signals and re-initiation of mRNA escent cells, PML bodies become enlarged and associate translation caused stress granules to disassemble [36]. with the nucleolar caps [28]. Newly synthesized RNA Similarly to P-bodies, sequestration of non-translating accumulates at the periphery of PML bodies, support- mRNA molecules in polysomes inhibited formation of ing a role in RNA metabolism. However, unlike the stress granules [36], thus suggesting that mRNA is re- other membrane-less organelles described herein, RNA quired in their assembly. P-bodies and stress granules in Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 4 of 20 yeast exhibit extensive compositional overlap, but dis- the stress granule microenvironment, where high local tinct physical properties [37]. Furthermore, yeast strains protein concentrations are achieved [37, 42, 44, 45]. Fur- deficient in formation of P-bodies were also unable to ef- thermore, genetic mutations within the prion-like do- ficiently form stress granules. The formation of P-bodies mains of these proteins known to be associated with ALS in yeast was not affected in mutant strains that were accelerated formation of amyloid-like fibrils and inhibited deficient in stress granules assembly. Together, these stress granule clearance in vivo, thereby disrupting mRNA observations suggested that pre-assembly of mRNA/ homeostasis [41–44]. These findings suggest that the protein complexes in P-bodies is a pre-requisite for the highly dense environment of mRNP granules facilitates fi- formation of stress granules [32], highlighting a functional bril formation by the proteins noted above, especially connection between the two types of membrane-less when their aggregation propensity is enhanced by muta- organelles. tion. Further, these studies establish correlations between ALS-associated mutations in mRNP granule proteins, and Germ granules heightened fibril formation and altered mRNA metabol- The term, germ granules, encompasses a class of non- ism. Additional research is needed, however, to under- membrane bounded organelles found in the specialized stand how these changes to mRNP granule structure and germ cells that generate sexual cells upon meiosis in the function are related to neuropathogenesis. developing embryo and are referred to as P-granules, In the next section we will discuss the common germinal bodies or Nuage bodies, depending on the or- physico-chemical features of membrane-less organelles ganism of origin (reviewed in [38]). Significant advances and unifying mechanistic insights that describe their have been made in understanding both the biology and assembly into multicomponent dense phases. the biophysics of P-granules in the nematode, C. elegans. P-granules are enriched in mRNA, RNA helicases and Common features of membrane-less organelles RNA modifying enzymes and are involved in the post A hallmark of the membrane-less organelles described transcriptional regulation of mRNA in primordial germ above is that their composition and physical properties cells [38]. For example, nos-2 RNA is asymmetrically vary depending upon cellular factors such as cell cycle segregated during C. elegans larval development [39]. stage, growth stimuli and stress conditions. In addition, P-bodies physically dock, but do not fuse with germ they exhibit dynamic structural features. Brangwynne granules in C. elegans embryos. This physical association and colleagues demonstrated that the nucleolus [5] and between the two types of organelles allows P-bodies to P-granules [6] exhibit liquid-like behavior in vivo and segregate within the germline blastomere, a property that this fluid organization arises from phase separation borrowed from the germ granules. Furthermore, these of their molecular components. This concept is sup- P-bodies that are associated with germ granules fail ported by a growing body of evidence identifying pro- to undergo maturation into organelles that degrade teins, sometimes co-mixed with nucleic acids, that phase mRNA [40]. Collectively, these observations exemplify separate in vitro into dense liquid-like [46–49] or hydro- how distinct physico-chemical properties preserve or- gel [50, 51] structures (reviewed in [52]). The proteins ganelle integrity and suggest inter-organelle interac- and nucleic acids are concentrated ~ 10-100-fold in the tions as a novel mechanism for regulating function. dense phase [46, 48], where they can reach concentra- tions in the millimolar range [53]; the dilute phase is mRNP granules in neurodegenerative disease maintained at the critical phase separation concentra- Debilitating neurodegenerative diseases such as amyo- tion. Experimentally, the two physical states, liquid and trophic lateral sclerosis (ALS), multisystem proteinopathy hydrogel, are distinguished by their ability to flow when (MSP) and frontotemporal lobar degeneration (FTLD) are their surfaces are subjected to shear stress. The liquid- characterized by formation of pathological mRNP inclu- like features of membrane-less organelles and in vitro sions and disruption of normal mRNA metabolism phase separated protein and protein/RNA droplets, have (reviewed in [41]). These pathological inclusions are been demonstrated based upon measurements of their formed through aggregation of proteins found in en- viscoelastic properties [5, 6, 44, 47, 54, 55]. For example, dogenous mRNP granules. Interestingly, many of the pro- liquid-like P-bodies [37] and P-granules [6] adopted teins associated with pathological inclusions contain a spherical shapes in the cytoplasm that were governed by prion-like domain in their amino acid sequence, which surface tension, and coalesced and fused into larger promotes their assembly into amyloid-like fibrils. Several droplets that returned to spherical shapes. Additionally, proteins known to localize within stress granules, includ- P-granules became reversibly deformed when they en- ing FUS [42], hnRNPA1 [43–45] and hnRNPA2 [43], were countered a physical barrier (i.e. “dripped” on the sur- found in ALS-associated pathological inclusions. Interest- face of the nucleus) [6]. In contrast, hydrogels do not ingly, fibril formation by these proteins is promoted within exhibit flow under steady-state conditions [50, 51, 56]. Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 5 of 20 Microrheology analysis indicated that liquid-like mem- composition regulated in response to stress signals?In the brane-less organelles [5, 6] and protein and protein/RNA next section we address the molecular principles that droplets prepared in vitro are characterized by high vis- underlie phase separation and the structural organization cosity. Strikingly, the measured values for viscosity of membrane-less organelles. We also discuss current evi- varies widely, over a range of three orders of magni- dence that suggests how their dynamic structure and tude, from ~ 1 Pa · s for P-granules to ~ 10 Pa · s for compositions are regulated. nucleoli [5, 6, 47, 54, 55]. Although not necessarily a direct indicator of liquid-like behavior, macromolecules Structural and compositional features of proteins resident within membrane-less organelles ([7, 37, 44, 46]) and within membrane-less organelles liquid-like droplets [42, 44, 46, 53, 55] recover after photo- Results from knock-down and knock-out studies [32, 39, bleaching on a timescale of seconds to tens of seconds. 61–63] showed that the structural integrity of several This indicates rapid exchange of molecules within the membrane-less organelles depends upon heterogeneous liquid-like phase, or with the surrounding milieu, when interactions amongst multiple components. Knock-down the object is photobleached in part or in full, respectively. or genetic deletion of single proteins, such as NPM1 Membrane-less organelles exhibit compositions of [61] or nucleolin [62] in the nucleolus or PGL-1 and varied complexity. For example, P-granules are com- PGL-3 [63] in germ granules, altered organelle morph- prised of approximately 40 proteins [57] while mass ology but did not prevent other, unaltered organelle spectrometry has shown that human nucleoli contain a components from assembling into punctate structures. staggering ~4500 proteins [58]. Furthermore, the protein These observations are consistent with redundancy of composition of membrane-less organelles can vary de- the sequence features of proteins found within various pending upon cellular conditions. Notably, the nucleolar membrane-less organelles (Table 1). proteome is significantly altered under stress conditions and the alterations are specific to particular forms of stress Basic principles of phase separation by polymers; from [59, 60]. These observations raise two important ques- chemical polymers to proteins tions: (1) how is the specific molecular composition of Phase separation of organic polymers in solution has membrane-less organelles achieved and (2) how is their been extensively studied and can be described by Table 1 Protein and RNA composition of membrane-less organelles Organelle Biological role Protein Domains/Motifs RNA Nucleolus Ribosome biogenesis in nucleus Fibrillarin RGG box [133] rRNA [8] Nucleolin RRMs; RGG box [67] Paraspeckles Regulation of gene expression PSPC1 RRMs; Coil [2] ncRNA NEAT1 (Menε/β); Ctn [2, 19] in nucleus NONO/P54NRB RRMs; Coil [2] SFPQ/PSF RRMs; Coil [2] Nuclear speckles Regulation of gene expression via SRSF1 RRMs; RS [134] Poly(A) RNA; lncRNA MALAT1 [3, 134] storage of splicing factors Cajal bodies Regulation of snRNP maturation Coilin Coiled-coil [23] snRNA; snoRNA [4, 135] SMN Coiled-coil [23] PML bodies Regulation of transcription and PML Coiled-coil [12] None [11, 29] protein storage Germ granules Regulation of mRNA translation in GLH-1, GLH-2, FG [74] Developmentally regulated maternal the cytoplasm of germ cells GLH-4 mRNAs (nos-1, pos-1, mex-1, skn-1 , gld-2) [74, 136] PGL-1, PGL-3 RGG [63] DDX4 FG; RG [48] LAF-1 RGG box [47] P bodies mRNA processing and decay Pdc1 HLM; Coiled-coil [49] mRNA [31] Dcp2 HLM [49] Edc3 LSm; FDF [49] Stress granules Storage of translationally stalled FUS RRM; RGG box; [G/S]Y[G/S] [50, 137] Poly-(A) mRNA associated with PABP [30] mRNA and proteins of the hnRNPA1 RRM; RGG box; [G/S]Y[G/S] [50] translational machinery Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 6 of 20 simplified mathematical thermodynamic models. Flory- behavior of bimolecular and unimolecular protein sys- Huggins theory describes the free energy of mixing of a tems. However, the sequence complexity of protein poly- polymer with solvent, wherein polymers are treated as mers, in contrast with compositionally more simple simplified arrays of modules that represent their repeti- chemical polymers, provides the opportunity for add- tive segments. Liquid-liquid phase separation into a itional inter-molecular interactions that can “tune” the polymer-rich phase and a polymer-poor phase occurs phase separation phenomenon. These results provide a when a critical concentration or temperature threshold foundation for understanding the phase separation behav- is crossed, whereupon the polymer becomes a better ior of more complex systems in vitro in the future. Fur- solvent for itself than is the buffer it is dissolved in thermore, they provide a foundation for in depth study of (reviewed in [64]; Fig. 1). the behavior of membrane-less organelles in cells. Rosen and colleagues reported that multivalent, repeti- tive domains from two signaling proteins that regulate Protein elements associated with phase separation; low actin polymerization, NCK and N-WASP, phase separate complexity sequences and folded domains in vitro and that the phase separation threshold depends Proteins associated with membrane-less organelles often on the protein concentration and valency of each indi- exhibit multivalent features which are manifested struc- vidual interaction partner [46]. Employing a simplified turally in different ways. Folded domains are proteins protein representation akin to that used for organic segments which adopt discrete and stable secondary and polymers, the authors used an adaptation of the Flory- tertiary structures. Disordered regions, also referred to Huggins formalism to describe the phase transition be- as intrinsically disordered protein regions (IDRs), are havior of the binary NCK/N-WASP system. The model protein segments that do not adopt stable secondary and included four parameters: association/dissociation pa- tertiary structure and are conformationally heterogenous rameters, and diffusion and crowding coefficients. Quali- and dynamic. Some proteins within membrane-less or- tatively, this formalism, which assumed structural ganelles contain folded domains but may also contain uncoupling between individual binding domains, pre- IDRs, while others are entirely disordered (termed in- dicted the effect of varying valency on the concentration trinsically disordered proteins or IDPs). A subset of threshold for phase separation [46]. A similar adaptation disordered protein regions, termed low complexity re- of this model was used to describe the phase separation gions, exhibit compositional bias towards a small set behavior of the unimolecular RNA helicase, Ddx4 [48]. of amino acids. Interestingly, low complexity se- While the general phenomenology can be described quences and disorder [47, 48, 50, 56] are overrepre- using this simplified model, a recent report involving sented in proteins shown to phase separate in vitro. the binary NCK/N-WASP system demonstrated that These features provide a high degree of conform- charged residues within the disordered linker connect- ational flexibility which is required for binding events ing SH3 domain binding modules caused weak self- to remain uncoupled [46]. NMR analysis of proteins association of NCK and reduction of the critical con- within the liquid-like phase after phase separation did centration for phase separation [65] (Fig. 1). Thus, not provide evidence of folding-upon-binding, thereby Flory-Huggins theory describes the basic phase separation suggesting that the disordered low complexity regions Low High PTMs, temperature, component component ionic strength, etc. concentration concentration Decreased threshold, assembly is enhanced Increased threshold, disassembly is promoted Disassembly Phase separation/ Critical assembly concentration for phase separation (M) Fig. 1 Macromolecular condensation mediates the formation of membrane-less organelles. Membrane-less organelles are dynamic structures formed via a polymer-condensation-like, concentration-dependent phase separation mechanism. The critical concentration threshold (grey line) for phase separation can be tuned within a range of concentrations (shaded green box) through physico-chemical alterations to the system (i.e., posttranslational modifications to domains and/or motifs that alter the affinity of their interactions, changes in temperature, altered ionic strength, etc.). These changes can drive phase separation and assembly of membrane-less organelles, or their disassembly Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 7 of 20 preserve their conformational flexibility within the to form membrane-less organelles? Given the large liquid-like phase [48, 53]. The detailed interpretation differences in critical concentration measured for the of these data is complicated, however, by the possibil- various systems, one possible answer is that compo- ity for organizational heterogeneity of the protein mol- nents with the lowest critical concentration phase ecules outside and possibly within liquid-like droplets, separate first, thus increasing the local concentration and the influence of inter-molecular interactions and above the critical concentration for phase separation apparent molecular size on resonance line widths and of other components which subsequently become in- intensities. corporated into the dense phase. Both folded domains Multivalent interactions are likely to contribute to the and disordered/low complexity regions have been re- dynamic, liquid-like properties of phase separated uni- ported to initiate phase separation in vitro and in cellulo. molecular assemblies [47, 48], as well as of more com- The folded domains are often implicated in specific plex assemblies [46, 49]. Amongst proteins associated protein-nucleic acid [67–69] and protein-protein [19, 70] with phase separation in membrane-less organelles, interactions and may provide an organizational scaffold multivalency is achieved through repetitive display of for the assembly of a membrane-less organelle. Low com- two types of protein modules: i) folded domains and plexity domains, on the other hand, provide a means for ii) low complexity disordered segments (summarized more dynamic interactions with a potentially broader in Tables 1 & 2; Fig. 2). In vitro studies had shown range of binding partners (Fig. 2). A compelling example that one of the two types of multivalency is necessary of such a synergistic cooperation between multivalent and sufficient for protein phase separation. The pro- folded domains and their respective connecting flexible tein concentrations associated with phase separation linkers was reported by Bajade et al., on the Nck/N- varied over several orders of magnitude for different WASP/nephrin system [65]. Nck constructs that are systems, ranging from sub-micromolar [44, 47] to divalent in SH3 motifs bind to PRM motifs in N-WASP hundreds of micromolar [44, 46, 48, 53]. Membrane- with micromolar to millimolar affinity and undergo phase less organelles are multicomponent systems and their separation. Through weak, largely electrostatically driven assembly, as demonstrated for the nucleolus, depends interactions, the disordered linker connecting the SH3 on the total concentration of their constituents [66]. domains in Nck promotes self-assembly, effectively Given the observations noted above that the accumu- lowering the critical concentration for phase separ- lation of components with nucleoli is temporally de- ation. Furthermore, addition of a disordered region of fined (reviewed in [8]) and occurs at pre-formed Nephrin containing multiple phospho-tyrosine resi- nucleolar organizing regions (NORs) raises an import- dues, which bind to a folded SH2 domain within Nck, ant question. Are some components more important enhances multivalent interactions and further lowers the others for initiating the phase separation process the critical concentration for phase separation. Thus, multivalent display of folded domains and low com- plexity sequences with disordered regions within pro- teins enables synergy between the various components Table 2 Examples of protein regions involved in phase of complex liquid-like droplets. Similar synergy be- separation and their functional roles tween multivalent components is likely to promote Domains Sequence/Structural Role features formation of membrane-less organelles in cells. FG FG/GFGG low complexity Association of P granules to repeats the NPC [74] Initiation events in the assembly of membrane-less RRM Folded domain RNA binding [19, 68] organelles Many of the proteins that participate in the formation of Coiled-coil Coiled-coil fold Homo/hetero-dimerization [12] membrane-less organelles exhibit segments with low RS RS low complexity repeats RNA binding; protein-protein interactions (Reviewed in complexity sequence features, often containing multiple [138, 139]) motifs enriched in the amino acids arginine, serine, gly- RGG RGG low complexity RNA binding (Reviewed in cine, glutamine, asparagine and/or aromatic residues repeats [140, 141]) (Tables 1 & 2). However, despite the low complexity of HLM Short helical leucine-rich LSm domains binding in their sequences, these proteins are often associated with motif P granules [49, 75] specific membrane-less organelles. What is the basis for SH3 Folded domain PRM motif finding [46] the incorporation of particular proteins and nucleic acid SH2 Folded domain Phosphorylated tyrosine molecules within particular membrane-less organelles? recognition [46] The emerging solution to this conundrum, at least in PRM Proline-rich short linear SH3 domain binding [46] some cases, is that specific protein-nucleic acid or motif protein-protein interactions initiate the assembly of Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 8 of 20 Catalytic Low domain complexity region Binding Low domain complexity region Active transcription, Multivalent RNA levels increase interactions Binding Catalytic domain domain Stress signals, altered Dimerization domain critical concentration threshold, reduced Dimerization Low domain complexity RNA level region Structural modularity and Pre-initiation Phase separation multivalency in soluble proteins intermediates Fig. 2 Molecular basis for membrane-less organelles assembly. The proteins enriched within the matrices of membrane-less organelles commonly exhibit multiple modules that create multivalency, including folded binding domains (red) and low complexity regions (purple). Valency is often amplified by domains that enable homo-, or hetero-oligomerization (orange). Interactions between proteins containing different combinations of these interaction modules provide a framework for building a heterogeneous, infinitely expandable network within membrane-less organelles. Formation of this type of network drives phase separation when the critical concentration threshold is reached. For many of the examples discussed herein, active RNA transcription is needed for membrane-less organelle assembly. We hypothesize that expression of RNA in excess of a critical concentration threshold is needed to nucleate interactions with specific, multi-modular proteins, and for nucleating formation of membrane-less organelles. Stress signals can alter the multivalent interactions that drive phase separation and lead to partial or complete disassembly of the organelle membrane-less organelles, which then create a micro- scattering (SAXS) to study the polymerization of DBHS environment that is conducive to phase separation of family of splicing factors, localized to and enriched in additional components (Fig. 2). This concept was de- paraspeckles [19, 70]. Extended coiled-coil interaction scribed for the nucleolus, which assembles around motifs within the polymerization domain of these pro- NORs, stable nucleolar precursors, comprised of clus- teins provided the structural scaffold for formation of tered arrays (i.e. multivalency) of the genes for rRNA, extended polymers of indefinite length. Weak, polar bound to the transcription factor UBF [71]. Notably, contacts stabilize the coiled-coil interactions and are UBF contains an array of six HMG box domains that ex- thought to be advantageous in maintaining the solubility hibit a broad range of binding affinities for DNA [69]. of unpaired extended helical structures [70]. The valency RNA Pol I is recruited to the NORs to transcribe pre- of the molecular assembly is enhanced by an additional rRNA, which initiates the assembly of the nucleolus. In dimerization domain which mediates homo- and hetero- the case of germ granules [63] and PML bodies [12], dimerization between DBHS family proteins, such as their formation is initiated by self-association of the PSPC1 and NONO [19] or SFPQ and NONO [70]. Fur- coiled-coil domains of the proteins PGL-1/3 and PML, thermore, multivalent interactions with RNA are medi- respectively. In these examples, structured domains me- ated by tandem RRM domains present in NONO, diate specific interactions to form assemblies that serve PSPC1 and SFPQ [19, 70]. These studies exemplify how as scaffolds for further assembly of components of modular, multivalent proteins can mediate the formation membrane-less organelles. Some of the proteins that of heterogeneous, dynamic molecular assemblies, thereby promote assembly contain both structured domains and providing the structural basis for formation of a low complexity segments that mediate multivalent inter- membrane-less organelle (Fig. 2). actions. The formation of membrane-less organelles may thus involve hierarchical assembly of specific, higher Forces that mediate the interactions associated with protein affinity protein-nucleic acid complexes followed by the phase separation recruitment of additional components through weaker, As discussed above, proteins that undergo phase separ- multivalent interactions. ation commonly contain segments with low sequence The assembly behavior of proteins associated with complexity. Further, these regions are often enriched in paraspeckles provides another example of how initiation charged and aromatic amino acids, highlighting the im- events can mediate the recruitment of components portance of electrostatic and hydrophobic interactions in within a membrane-less organelle. Bond and co-workers the process of phase separation. For example, disordered used X-ray crystallography and small angle X-ray segments of the DEAD-box helicases Ddx4 [48] and Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 9 of 20 LAF-1 [47], as well as hnRNPA1 [44] that mediate phase variable contributions of the different types of intermo- separation are enriched in arginine residues within their lecular interactions that promote phase separation deter- low complexity RGG box and RRM domains. Due to mine selective accumulation of specific proteins within their overall positive charge, the formation of liquid-like specific types of membrane-less organelles. droplets by these proteins is highly sensitive to the ionic strength of the surrounding solution. Numerous other Mechanisms involved in achieving local organization and proteins associated with nuclear bodies and mRNP gran- compositional complexity in membrane-less organelles ules are enriched in arginine residues (e.g. RGG and SR The localization of specific macromolecules within par- domains; see Table 1). For example, the low complexity ticular membrane-less organelles is achieved through SR repeats common to the SR family of splicing factors specific interactions with the molecular network that ex- were identified as targeting signals for nuclear speckle tends from the nucleating region. As discussed above, a localization [72, 73]. These observations strongly suggest large proportion of the proteins known to associate with that electrostatic interactions play a key role in the phase membrane-less organelles exhibit multivalency through separation of a subset of proteins (Fig. 1). the display of repeated low complexity motifs (e.g., SR, Electrostatics are not, however, the only interactions RGG or FG motifs) and/or of multiple copies of folded that promote the formation of the protein-rich phase domains, such as RRM domains. Through combinatorial separated state. Low complexity regions that are rich in utilization of a finite number of intermolecular inter- aromatic residues (i.e. phenylalanine, tyrosine) are over- action modules, complex mixtures of proteins and nu- represented in proteins that reside within membrane- cleic acids can thus be recruited into the condensed less organelles [48, 74] and other phase separated phase. For example, the formation of P-granules is initi- matrixes, as is the case for the FUS protein in mRNP ated by self-association of the coiled-coil domains of granules [50, 53] and the FG-Nups in the nuclear pore PGL-1 and PGL-3 proteins, which further bind mRNA complex [51]. Interestingly, mutations of F to Y, but not via their low complexity RGG domains. Vasa-related F to S, within the FG repeat domain preserved in vitro helicases GLH-1, 2, 3 and 4 that contain FG repeats are hydrogel formation by the yeast nucleoporin Nsp1p [51], then incorporated to facilitate P-granule association with demonstrating the importance of aromatic residues in nuclei, through interactions with and expansion of the assembly phenomena associated with the nuclear pore nuclear pore complex hydrogel matrix [74]. The pres- complex. Furthermore, the critical concentration for for- ence of homo- and hetero-oligomerization domains fur- mation of in vitro FUS liquid droplets was lowered by ther enhances the degree of multivalency and promotes increasing the ionic strength of the solution, consistent integration within membrane-less organelles (Fig. 2). with the interpretation that salting out the hydrophobic The PML protein forms homo- and hetero-oligomers via interactions reduced the solubility threshold for the pro- its coiled-coil domain, but valency can be increased by tein in buffer [53]. Nott et al., noted that evolutionarily homo-dimerization through the RING domain. Muta- conserved clustering of similarly-charged amino acid tions in either the coiled-coil or RING domains led to residues and regular spacing between the RG and FG disruption of PML bodies [12]. Components of the motifs are required for the phase separation of a Ddx4 mRNA decapping machinery found in P-bodies, includ- construct [48]. These studies highlight the roles of ing Pdc1, Dcp2 and Edc3, assemble into liquid-like drop- cation-π [48] and π-π [50, 51] interactions in phase sep- lets in vitro. Two LSm domains in dimeric Edc3 interact aration phenomena. with Dcp2 and Pdc1, which both contain multivalent In the absence of a lipid membrane barrier, the move- HLM motifs. Edc3 binds to various HLM motifs with af- ment of molecules into and out of membrane-less or- finities within the low micromolar to millimolar range ganelles is diffusion limited [1], and their accumulation [49]. The valency of the HLM motifs in Pdc1 is in- is mainly dependent on retention based on interactions creased through oligomerization via a central coiled-coil with the organelle matrix. Interestingly, the diffusion domain [49, 75]. These examples illustrate how multiva- barrier for exogenous macromolecules such as dextrans, lent interaction modules and oligomerization domains is dictated by the physical properties of the membrane- can cooperate to initiate phase separation in the context less organelle matrix [1]. The DFC of the nucleolus is of different types of membrane-less organelles. Add- less permissive to accumulation of dextrans compared to itional domains within these proteins, which are not dir- the surrounding GC, consistent with the observations ectly involved in the mechanism of phase separation, that the DFC is denser than the GC [1]. Furthermore, can mediate the recruitment of additional components the dynamic features of components specifically retained into the liquid phase. For example, the helicase Ddx6/ within membrane-less organelles vary based on the Dhh1 and mRNA can be recruited to P-bodies via the nature of their interactions with other constituents of FDF domain of Edc3 and the RNA binding domain of the matrix [7, 23]. Together, these results suggest that the helicase, respectively [49]. We thus distinguish Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 10 of 20 between two basic types of components of membrane- induced by DRB, a small molecule that selectively in- less organelles: (i) multivalent macromolecules that dir- hibits RNA Pol II, caused dissolution of paraspeckes be- ectly participate in interactions involved in the process fore a significant decrease in the total Mem ε/β lncRNA of phase separation and underlie the structural features levels could be measured [77]. This finding suggests that of the liquid phase and (ii) other macromolecules that a currently unknown regulatory mechanism controls the are recruited via specific interactions with the phase sep- structural integrity of paraspeckles and that there is a arated assembly, which lack multivalent interaction sharp and sensitive threshold for sensing and responding elements, but perform specialized functions within the li- to cellular stress. This raises an important general ques- quid phase (i.e., enzymes that catalyze specific biochemical tion: how are changes in environmental conditions, for reactions). However, the capability for assembly/phase example in response to different types of stress, transmit- separation and biochemical functionality can be embodied ted to the membrane-less organelle matrix and mani- within a single protein, as is seen with Ddx4, which har- fested as changes in structure and function? This topic is bors a helicase domain and a multivalent, low complexity discussed in the next section. RGG domain that mediates phase separation [48]. Structural and dynamic regulation of phase separated RNA within membrane-less organelles structures While much attention has been given to understanding The lack of a lipid bilayer barrier between membrane- the roles of multivalent proteins in the formation of less organelles and their surroundings circumvents the membrane-less organelles, the primary functions of need for active transport of macromolecules across many of these organelles are different aspects of RNA membranes and enables rapid signal transduction. Stress metabolism and, consequently, RNA is also involved in signals influence the structural integrity of membrane- their assembly and structural integrity. The assembly of less organelles, providing a mechanism for organelle- the nucleolus at the exit of mitosis is initiated by mediated stress responses. We next discuss various transcriptional activation of RNA Pol I [8, 76] and the factors that influence the structure and function of structural integrity of paraspeckles is dependent upon membrane-less organelles. transcriptional activity of RNA Pol II [2]. Proteins cap- able of undergoing phase separation often contain simi- Chemical and other environmental factors lar sets of folded and low complexity multivalent Changes in temperature [27, 48], ionic strength [47, 48], domains, giving rise to structural redundancy and the and chemotoxic and DNA damage [27, 59, 60, 78, 79] potential, under certain conditions, to promiscuously are environmental changes known to disrupt phase sepa- localize within more than one types of membrane-less rated cellular bodies and in vitro liquid droplets. The organelle. In contrast, the different types of organelles stiffness of nucleoli isolated from HeLa cells was de- generally contain specific types of RNA (summarized in creased or increased upon RNA Polymerase or prote- Table 1), suggesting that the RNA components are the asome inhibition, respectively, based on atomic force principal determinants of organelle identity. In support microscopy measurements [79]. Thus, stress signals of this hypothesis, disruption of RNA transcription affect the viscoelastic properties of nucleoli and conse- causes re-localization of the protein components of dif- quently modulate their functions. ferent nuclear and cytoplasmic bodies [25, 59]. For ex- Membrane-less organelles form, disassemble and func- ample, Mao et al., demonstrated that the lncRNA Mem tion in an intracellular environment crowded with mac- ε/β was required for the recruitment of specific protein romolecules. The high cumulative concentration of and RNA molecules to paraspeckles [77]. Additionally, macromolecules in the cell, which correlates with a high immobilization of PSP1, a modular, paraspeckle protein percentage of excluded volume (~20–30 % of the total shown to homo- and hetero-oligomerize [18], was able cell volume), affects the kinetics and thermodynamics of to recruit some paraspeckle protein components, but most biochemical processes [80]. In vitro, molecular was unable to recapitulate complete assembly of the or- crowding agents promote assembly of recombinant ganelle [77]. Recruitment of the full complement of pro- hnRNPA1 into protein dense liquid-like droplets at tein and RNA components of paraspeckles, coupled with lower critical concentrations than observed in buffer exclusion of macromolecules associated with nuclear alone [44, 45]. Thus, the increase in excluded volume speckles, was achieved only under conditions of active caused by macromolecular crowding increases the local transcription of the Mem ε/β lncRNA. While the obser- concentration of individual protein species, thereby de- vations summarized above clearly indicate the dominant creasing the effective concentration threshold for phase role of RNA in the molecular makeup of certain separation (Fig. 1). membrane-less organelles, other factors can also influ- Alterations in the morphology and viscoelastic proper- ence their structural integrity. For example, stress signals ties of mRNP granules, due to mutations in resident Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 11 of 20 proteins (e.g. hnRNPA1, FUS) are associated with debili- that ATP-hydrolyzing enzymes regulate the dynamics of tating neurodegenerative diseases [13, 42, 44, 45]. In vitro, macromolecules within membrane-less organelles. Simi- both FUS and hnRNPA1 phase separate into liquid-like larly, several other types of ATP-dependent enzymes, in- droplets [42, 44, 45, 53] or hydrogels [42, 44, 50], de- cluding kinases and DEAD-box helicases [47–49, 78], pending on protein concentration and experimental which are incorporated into these organelles, may be in- conditions. The low complexity regions in the two volved in maintaining their liquid-like physical proper- proteins, along with the RRM domains [44, 45, 53], ties. Helicases may modulate RNA structure as well as contribute to phase separation. Mutations within Q/N- protein-RNA interactions and, thereby, actively control rich low complexity regions, termed prion-like domains, the viscoelastic properties of membrane-less organelles. are associated with defects in mRNP granules and neuro- pathogenesis [42, 44]. These defects are attributed to a Role of posttranslational modifications in regulating kinetically slow step (tens of minutes to hours time scale) membrane-less organelle structure and dynamics that occurs in the dense liquid-like phase, referred to as The assembly of components within many of the phase “droplet aging” [42], wherein the liquid-like phase trans- separated systems we have discussed is electrostatically forms into a solid-like state. Phenomenological observa- driven. Therefore, posttranslational modifications that tions suggest that this physical transformation is a result alter the charge features of amino acids within the do- of a slow structural re-organization of the dense, liquid- mains and low complexity segments of proteins provide like phase. The reorganization leads to decreased dy- a means to modulate their multivalent interactions and namics within the phase separated state and culmi- phase separation behavior (Fig. 1). nates in a transition from a liquid-like state to a The importance of electrostatic interactions is illus- hydrogel or solid-like state. The transition between the trated by the phase separation behavior of LAF-1 [47], two physical states is accompanied by morphological hnRNPA1 [44, 45] and Ddx4 [48], whose ability to form changes, from nearly spherical droplets, shaped by surface liquid-like droplets is strongly influenced by the salt tension, to elongated, fibril-like structures [42, 44, 45]. A concentration of the surrounding buffer. The phase sep- similar transition was observed in vitro and in vivo aration concentration threshold for both scaled linearly droplets containing Whi3, a protein encoding a polyQ with ionic strength as the NaCl concentration was in- tract [55]. A potential underlying mechanism is that creased. In addition, methylation of arginine residues in under the conditions of the high local protein con- the RGG domain of Ddx4 increased the phase separation centration within the dense, liquid-like phase, new, threshold in vitro [48]. less dynamic interactions occur, perhaps between the Phosphorylation plays a crucial role in many signal low complexity prion-like domains. In time, these in- transduction pathways and also modulates the structural teractions may become dominant over the more dy- integrity and dynamics of membrane-less organelles. For namic, multivalent electrostatic interactions that give example, tyrosine phosphorylation of nephrin stimulates rise to the liquid-like state. We speculate that the balance the phase separation of the ternary system nephrin/ of the thermodynamic favorability of these two types of in- NCK/N-WASP [46]. Interestingly, a common feature of teractions may influence the physical nature of the phase certain well-characterized membrane-less organelles is separated state (i.e., liquid, hydrogel/solid) and determine that they incorporate kinases and phosphatases within the different propensities of wild-type and mutant proteins their matrixes [39, 78, 82]. Active phosphorylation/ to undergo the transition for the liquid-like to solid-like dephosphorylation cycles have been linked to regula- structural state. tion of organelle structural integrity. The activity of the nucleolar kinase CK2 controls the structural connect- Energy-dependent control of membrane-less organelle ivity between the GC and the DFC regions within the nu- dynamics cleolus [78] and increases the dynamics of NPM1 We have emphasized that the physical properties of exchange between the nucleolar and nucleoplasmic com- membrane-less organelles depend upon their protein partments [83]. Furthermore, phosphorylation of MEG-3 and RNA composition. In addition, however, the nucle- and MEG-4 proteins by MBK-2/DYRK kinase and de- PPTR-1/PPTR2 olus requires ATP in order to maintain its liquid-like phosphorylation by PP2A phosphatase regu- behavior, a physical state termed an “active liquid” [5]. It lates P-granule disassembly and assembly, respectively, is currently unclear what specific ATP-dependent pro- during mitosis in C. elegans in association with embryo- cesses are involved in maintaining this active liquid- genesis [39]. state. Furthermore, the activity of ATP-dependent chap- Assembly and disassembly of membrane-less organ- erones, such as Hsp70/Hsp40, which accumulate within elles provides a mechanism for controlling the concen- stress granules, is required for their disassembly upon tration and associated signaling behavior of freely recovery from stress [81]. These observations suggest diffusing molecules within the membrane-bounded Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 12 of 20 compartments of the cell. For example, the dynamic paraspeckles depends upon the concentrations of their properties of stress granules are coupled with mTORC1 constituent RNAs, which are controlled by the transcrip- signaling by immobilization of mTORC1 within the tional activity of RNA polymerases [2, 8], suggesting that granules, while phosphorylation-mediated dissolution of transcriptional control of RNA concentration may be a these organelles liberates mTORC1, activating down- general mechanism to tune the physical properties of stream signaling [82]. As another example, Wippich et al. membrane-less organelles (Fig. 1). [82], demonstrated that the kinase DYRK3 condenses in Many membrane-less organelles are involved in cellu- cytoplasmic granules via its low complexity N-terminal lar responses to various types of stress and the sensitivity domain, in a concentration dependent manner, and local- of their structural integrity to protein and RNA concen- izes to stress granules under osmotic and oxidative stress. trations provides a mechanism for rapidly responding to Inactive DYRK3 condensed into stress granules, together stress signals that affect these levels. For example, inhib- with components of the mTORC1 pathway. Activation of ition of Pol I-, II- and III-dependent RNA transcription DYRK3 and downstream phosphorylation of PRAS40, an by Actinomycin D was associated with re-organization mTORC1 inhibitor, results in dissolution of stress gran- of constituents of both nuclear and cytoplasmic ules and disruption of the inhibitory PRAS40/mTORC1 membrane-less organelles [59]. After Actinomycin D interaction. treatment, NPM1, a major component of the GC of the Further evidence for the role of posttranslational mod- nucleolus, becomes delocalized to the nucleoplasm and ifications in regulation of the features of membrane-less cytoplasm due to inhibition of RNA Pol I-dependent organelles is provided by the observation that the amino transcription of rRNA. Under these conditions, cytoplas- acids arginine, serine and tyrosine are overrepresented mic NPM1 was found to interact with components of in the low complexity sequences of proteins within stress granules, such as mRNA, and the proteins them. These amino acids can be posttranslationally hnRNPU and hnRNPA1 [84]. modified, arginines by methylation and serines and tyro- Also under conditions of Actinomycin D treatment, sines by phosphorylation, providing general mechanisms protein and RNA components associated with para- for modulating protein condensation thresholds and speckles, and PML and Cajal bodies, re-localize to nucle- consequently the signaling pathways downstream of olar caps. Interestingly, while proteins from the GC are components sequestered within the phase separated ejected from the nucleolus, proteins from the DFC, such fraction. as fibrillarin, re-localize to nucleolar caps [25]. These ob- servations suggest that environmental changes can alter Component concentration as a factor in membrane-less the equilibria that maintain the integrity of membrane- organelle assembly/disassembly less organelles, thereby altering the concentrations of Another important factor in phase separation-dependent their components in the freely diffusing pools of mac- formation of membrane-less organelles is the local romolecules within the nucleoplasm and cytoplasm concentration of components (Fig. 1). For example, and allowing their redistribution within various other regulation of P-granules during the oocyte-to-embryo organelles. transition, when they transit from the perinuclear region to the cytoplasm, is regulated by a concentration gradi- Emerging methods for the study of phase separated ent, which causes dissolution of the perinuclear droplets structures and re-condensation in the cytoplasm. A similar mech- Detailed analysis of the structural features of membrane- anism is employed during the asymmetric segregation of less organelles and their underlying macromolecular as- P-granules into the germline founder cell [6]. Recently, semblies presents challenges not encountered in other Brangwynne and colleagues demonstrated that the levels areas of structural biology. Interactions relevant to the of RNA in LAF-1 droplets, a minimalistic in vitro model phase separation phenomenon occur over multiple of P-granules, tunes the viscosity and molecular dynam- length scales, from sub-nanometer to tens of microme- ics within the liquid-like phase [47]. The viscoelastic ters, thereby making any single analytical technique properties of liquid-like droplets containing Whi3 are insufficient for the study of phase separated macromol- also modulated by RNA concentration. While Whi3 is ecular assemblies. For example, while liquid-like droplets able to phase separate in a unimolecular fashion under exceed the size limitations associated with analysis by certain conditions, the presence of RNA is required for NMR spectroscopy, the structural and dynamic features the process to occur at physiological salt concentrations. of flexible components within them have been character- Furthermore, an increase in the RNA concentration ized [53]. However, the dynamic features of these correlates with an increase in droplet viscosity and a systems are incompatible with X-ray crystallography. Al- decrease in Whi3 dynamics of recovery after photo- though the macromolecular assemblies formed are read- bleaching [55]. In addition, assembly of nucleoli and ily observable by conventional microscopy techniques, Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 13 of 20 the interactions responsible for assembly occur on dynamics that characterize IDPs are often an advantage length scales that are below the resolution limit of detec- for NMR studies because they cause resonance narrow- tion. Additionally, these systems are highly heteroge- ing and enhance detection. However, some IDPs experi- neous and therefore, integrative solutions that combine ence motions on time scales that cause resonance complementary methods are needed in order to under- broadening and can hamper NMR studies. Despite these stand their structural features. limitations, NMR has already been demonstrated to pro- vide unique insights into the conformational and dynamic Atomic-resolution structure determination methods features of phase separation-prone IDPs both before and Several studies utilizing classical structural methods, in- after phase separation; several exemplary studies are dis- cluding solution NMR [46, 48, 49, 67–69] and X-ray cussed below under “Integrative approaches to understand crystallography [19, 70], have provided detailed insights the molecular basis of phase separation”. into the molecular interactions that mediate the network structure that drives phase separation of modular pro- Methods to study molecular interactions associated with teins within membrane-less organelles. However, due to phase separation technological limitations, these studies were performed Classical methods for characterization of biomolecular with truncated forms of the proteins and nucleic acids interactions, such as ITC [49] and SPR [68, 69], have corresponding to individual interaction modules. These been employed to characterize the wide range of binding traditional methods will be useful in the future for deter- affinities associated with the different types of interac- mining the structural basis of interactions between tions that occur within liquid-like droplets and/or folded domains within multi-domain phase separation- membrane-less organelles. NMR can also be used to prone proteins and their interaction partners, including characterize macromolecular interactions and is particu- peptides corresponding to short linear motifs and seg- larly well suited in studies of weak interactions that ments of RNA. However, because many phase separation- present challenges for other methods. For example, prone proteins exhibit low complexity and disordered chemical shift perturbations observed during titrations sequence features, these methods for determining discrete of an unlabeled binding partner into an isotope-labeled protein structure are likely to receive limited application protein can be quantitatively analyzed to report residue- in this emerging field. specific and global K values for interactions associated with phase separation [NPM1 integrates within the NMR spectroscopy; a versatile tool in studies of phase nucleolus via multi-modal interactions with proteins separation-prone proteins displaying R-rich linear motifs and rRNA: Mitrea DM, NMR spectroscopy offers unique capabilities in studies et al., under review]. However, the multivalent features of disordered proteins, by providing insights into confor- of phase separation-prone proteins can give rise to com- mations and dynamics of individual amino acids plex, multi-step interaction mechanisms, which compli- throughout the polypeptide chain. Measurements of cate the analysis of data from the methods discussed chemical shift values for nuclei of backbone atoms re- above. Therefore, experiments are often performed with port on secondary structure propensities and dynamics truncated macromolecules of reduced multivalency and can be probed on ps to ns, and μs to ms timescales using therefore do not address interactions under the conditions a variety of relaxation methods [85]. Furthermore, long- of phase separation. Despite these limitations, these bio- range structure within disordered proteins can be stud- physical methods provide important insights into the ied using paramagnetic relaxation enhancement (PRE) binding features of the individual elements within multi- methods and through the measurement of residual di- valent macromolecules that undergo phase separation. polar couplings [86]. The former method, however, re- quires that proteins be engineered to include single Scattering methods to probe structural features before and cysteine residues for labeling with a paramagnetic probe. after phase separation A limitation of these NMR approaches is that rapid con- Dynamic light scattering and small angle X-ray scatter- formational fluctuations of disordered polypeptides ing (SAXS) [19, 46] have been employed to gain insight causes ensemble averaging of NMR parameters. A sec- into the overall size and shape of the macromolecular ond limitation is that the structural and dynamic infor- assemblies. In particular, SAXS has been used to mation gained reports on the features of individual sites characterize the shapes (e.g., radius of gyration) of en- within a protein on a very limited length scale (Å or tens sembles of disordered proteins [88]. However, scattering of Å in the case of PRE measurements). An exception is methods can also detect long-range order within so- the use of pulsed field gradient methods to study protein called soft materials and uniquely provide insights into diffusion [87] but this has not yet been used in studies the structural makeup of these materials. Small-angle of proteins within liquid-like droplets. The extensive neutron scattering (SANS) has previously been employed Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 14 of 20 in the structural analysis of polymer blends [89–91] and High resolution and single-molecule microscopy polymeric soft nanomaterials [92] and has great potential Electron microscopy can extend into the length scale in studies of membrane-less organelles to provide infor- gap between the two sets of techniques described above mation about the spatial organization of macromolecules and has been extensively utilized to study cellular ultra- within the condensed state. One recent study used SANS structure [1]. A significant limitation of this technique is to characterize the regular spacing of molecules within the low certainty with which specific molecules can be droplets comprised of the nucleolar protein, nucleophos- identified based upon the greyscale contrast of images min (NPM1), and a peptide derived from the ribosomal [96]. The emerging field of correlated light and electron protein, rpL5, on length scales from 5.5 to 11.9 nm microscopy (CLEM; reviewed in [96]) presents the op- [NPM1 integrates within the nucleolus via multi-modal portunity of directly connecting dynamic information interactions with proteins displaying R-rich linear motifs obtained via live fluorescence microscopy methods with and rRNA: Mitrea DM, et al., under review]. SANS has ultrastructural detail acquired by electron microscopy. the advantage of allowing detection of scattering from Significant advances were made in the last decade in specific components within heterogenous, phase sepa- super resolution microscopy methods (reviewed in [97]) rated states through selective protonation and/or deu- and were successfully applied to decipher chromosomal teration and solvent contrast matching [93]. architecture [98]. Lattice sheet microscopy coupled with Furthermore, time-resolved SANS has been used in structured illumination microscopy, a method that the past in studies of mutant huntingtin exon 1 phase returns 3D images with resolution ~ 200 nm x 200 nm separation into amyloid fibers to determine the mech- in the x/z plane that exceeds the diffraction limit, was anism of macromolecular assembly and the geometry applied to study the ultrastructural organization of germ of monomer packing within the fibrils [94]. We envision granules in C. elegans [39]. The internal structure ob- that SAXS and SANS may be able to reveal the spacing of served in several membrane-less organelles suggests that partially ordered macromolecules within the liquid-like the condensed macromolecules are not homogenously structure of droplets prepared in vitro and possibly within distributed, but further partition into phase separated membrane-less organelles if technical issues associated fractions with distinct physical properties. These methods with sample preparation can be addressed. We envision provide opportunities to reveal the heterogenous ultra- that these scattering methods will be powerful tools in the structure of membrane-less organelles in the future. characterization of biological structures that arise from Single-molecule fluorescence microscopy holds great phase separation in the future. potential in the analysis of proteins within liquid-like droplets in vitro and membrane-less organelles in cells. For example, single-molecule fluorescence correlation Light microscopy spectroscopy (FCS) [99] and Förster resonance energy Light microscopy methods (reviewed in [95]) have been ex- transfer (smFRET) [100] have been used to study the tensively utilized to visualize the subcellular localization of structural and dynamic features of aggregation-prone fluorescently tagged molecules. Live imaging coupled with intrinsically disordered proteins in vitro (reviewed in fluorescence recovery after photobleaching (FRAP) or [101]). In addition, single-molecule FRET and other fluorescence loss in photobleaching (FLIP) methods probe methods have been applied to a wide range of disordered the dynamics of macromolecules within membrane-less or- proteins with varied charged residue compositions ganelles inside living cells [7, 46, 48, 77] and phase sepa- and distributions (reviewed in [102]). We envision rated states reconstituted in vitro [46–48, 50]. that these methods will be applied in the future to The information obtained from structural biology disordered proteins within liquid-like droplets to re- −10 −9 methods is on length scales of 10 –10 m, while the veal their structural and dynamics features. Further- classical light microscopy techniques provide informa- more, smFRET and fluorescence lifetime imaging have −7 −3 tion on much greater length scales, from 10 to 10 m. revealed the conformational features of a disordered This situation creates a gap corresponding to two orders protein within HeLa cells [103], providing opportunities of magnitude on the length scale in our understanding in the future for studies of phase-separation-prone pro- of the structural and dynamic features of micron-sized teins within membrane-less organelles in their natural cel- membrane-less organelles. Macromolecular interactions lular setting. that occur on the length scale of this gap are responsible for the structural organization that gives rise to phase Additional physical characterization methods separation and the liquid-like and/or gel-like properties Density [1], viscosity [5, 6, 47] and stiffness [79] are a few of membrane-less organelles and related structures. We of the physical properties that have been measured for next discuss structural methods that can peer into this bona fide membrane-less organelles or in vitro reconsti- length scale gap. tuted liquid droplets. Interferometer microscopy was Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 15 of 20 utilized to measure the density of nuclear membrane- ensembles—the so-called “sample-and-select” methods less organelles in isolated Xenopus laevis germinal ves- [88, 119–121]. Complementary computational methods icles, oocyte nuclei [1]. This method provided important have been developed for generating IDP ensembles based insights into the physical properties of refractory sub- on SAXS data [122]. The development of physically accur- cellular bodies in a quasi-natural environment. A few con- ate molecular ensembles with atomistic detail for IDPs is siderations when interpreting these data, however, are that important because, with the exception of single-molecule the results are based on the simplified assumptions that fluorescence methods, the experimental methods used to the organelles are spherical in shape and are exclusively characterize IDPs are subject to ensemble averaging. composed of homogenously mixed water, proteins and Therefore, computationally generated ensemble models of low molecular weight solutes [1]. IDPs enable examination of the features of large numbers Atomic force microscopy provides the advantage of of individual molecules. However, these approaches are performing surface scans of membrane-less organelles only beginning to be applied to proteins that undergo which produce topological maps with resolution in the phase separation. nanometer range. Also, this method provides a means to A key challenge in computational studies of phase measure other key biophysical properties, such as struc- separation-prone proteins is to gain insight into the tural stiffness, as done for nucleoli [79]. inter-molecular interactions that are the basis for self- Microrheology methods, traditionally used in the association and phase separation. Regarding this goal, characterization of viscoelastic properties of polymers and the field is in its infancy. However, methodologies complex fluids [104], were applied to the characterization applied to understand protein aggregation and fibril for- of membrane-less organelles [5, 6, 42, 105] and in vitro mation can be leveraged to understand the types of in- formed protein and protein-RNA liquid droplets [47, 55]. teractions that drive protein phase separation and In particular, the tracer bead technology provided import- possibly, in the future, protein-nucleic acid phase separ- ant insights into the effect of RNA onto the viscoelastic ation. In the protein aggregation field, course-grained properties of in vitro liquid droplets [47, 55]. computational methods have been applied to understand the aggregation of poly-glutamine tracts associated with Computational and theoretical approaches Huntington's disease [123] and atomistic methods to As we gain greater knowledge of the types of macromol- understand aggregation of amyloid β [124]. Clearly, in- ecules that undergo phase separation to form liquid-like creased effort in this area is needed to understand the structures both in vitro and in cells, computational molecular basis for phase separation. models are needed to analyze the structural and dynamic While computational approaches face challenges in features, encoded by their amino acid sequences, so as addressing the protein phase separation problem, signifi- to understand their phase separation behavior. A large cant progress has been made in recent years in under- proportion of the proteins, or protein regions, shown to standing relationships between the sequence features of undergo phase separation are intrinsically disordered, IDPs and IDRs and the general conformational features which presents a variety of computational challenges, of IDP ensembles [125–127]. Results from NMR, single- notably conformational sampling and physical accuracy. molecule fluorescence and computational approaches A wide variety of methods are used to address the need have shown that the charge features of IDPs influence to sample the extensive conformational space explored the shape of their dynamic ensembles. Pappu and co- by IDPs/IDRs, including molecular dynamics methods, workers have extended these finding using both compu- often enhanced by approaches such as replica exchange tational and experimental methods to show that not only and related methods [106, 107], and Monte Carlo sam- the faction of charged residues and net charge per resi- pling methods [108, 109]. Many different force fields due within IDPs and IDRs influence their overall con- and variants thereof are available [110–112] and several formational features, but also the distribution of were recently tested and compared [113]. Computations oppositely charged residues within sequences signifi- are often performed without experimental restraints and cantly influences the compaction of IDP ensembles therefore they are reliant on the underlying force fields [128]. These advances have led to the development of a for generation of physically accurate molecular ensem- novel phase diagram based upon net positive and nega- bles. A problem in the past was that computational tive charge per residue values for the classification of models of IDPs were overly compact [114] but this IDP and IDR sequences [129]. These developments pro- issue is being addressed through the method refine- vide a conceptual framework for establishing relation- ment [112, 115–117] and consideration of NMR, SAXS ships between the charge features of IDPs and IDRs, and smFRET data [110, 113, 118]. Another group of ap- their conformational features and their propensities for proaches utilize experimental restraints (e.g., NMR and/or phase separation. Charge features are certainly im- SAXS data) to select conformers for inclusion within IDP portant factors governing protein phase separation Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 16 of 20 behavior; for example, arginine residues are prevalent not related to phase separation per se, this study pro- in low complexity regions known to form liquid-like vides an explanation for how Importin β-bound cargo droplets in vitro and within protein components of can rapidly diffuse through the condensed phase within membrane-less organelles [44, 47]. However, these se- the core of the nuclear pore complex, which is com- quences are often enriched in aromatic and other prised of several FG-Nup proteins, including Nup153. neutral amino acids, indicating that, while electrostatic in- NMR spectroscopy was used to understand the ensem- teractions may play important roles in some cases, other ble averaged conformational and dynamic features of types of molecular interactions are at play in other cases backbone amide groups within disordered Nup153 in [48, 50, 53]. This was born out in a recent study by García the absence and presence of Importin-β and to generate Quiroz and Chilkoti [130] in which they identified the se- a conformational ensemble using the sample-and-select quence features of designed proteins that can undergo approach. This ensemble was validated by back- phase separation due to either a temperature increase calculation of the X-ray scattering profile and compari- (termed LCST sequences) or decrease (termed UCST son with experimental SAXS data, an illustration of sequences). The LCST sequences were enriched in hydro- spanning length scales from amino acids to a whole dis- phobic residues while the UCST sequences were enriched ordered protein. To complement this information, data in charges residues [131]. This study, which involved the- from smFRET and fluorescence lifetime measurements oretical considerations as well as in vitro experimental were used to understand the conformational features of measurements, serves as a model for future studies into many individual molecules under the same conditions the physical basis for phase separation of the growing list while fluorescence correlation spectroscopy was used to of proteins and RNA molecules shown to partition into compare molecular diffusion properties of Nup153 with- the liquid-like or gel-like phase of membrane-less organ- out and with Importin β. Additionally, molecular dy- elles and other cellular bodies. namics and Brownian dynamics computational methods were used to relate insights from the aforementioned Integrative approaches to understand the molecular basis biophysical methods to the mechanism of Nup153/ of phase separation Importin β interaction at atomistic resolution. Finally, None of the individual methods or approaches discussed these various pieces of molecular data were related to above will alone uncover the molecular basis for phase the Importin-β-dependent transport through the NPCs separation by proteins and protein-nucleic acids mix- in live cells using bulk and single-particle fluorescence tures; therefore, there is a need to apply multiple, com- tracking. plementary methods and to integrate results to advance Another example is provided by a recent study of the mechanistic understanding. Integration is needed to ALS-associated protein, FUS, from Fawzi and co- span the broad length scales relevant to membrane-less workers that employed NMR and various fluorescence organelles, ranging from the atomic scale (units of Å) microscopy methods to study the molecular features of relevant to amino acid conformations and their inter- FUS within in vitro liquid-like droplets and its interac- molecular interactions to the overall size of in vitro tions with RNA and the C-terminal domain of RNA Pol liquid-like droplets and cellular membrane-less organ- II. A final example is provided by a recent study of the elles (units of micrometers). Integration is also needed highly abundant nucleolar protein, NPM1, which was across the broad range of relevant time scales, including shown to phase separate into liquid-like droplets with motions of amino acids and their polypeptide chains that other nucleolar proteins and ribosomal RNA [NPM1 in- mediate their conformational heterogeneity and inter- tegrates within the nucleolus via multi-modal interac- molecular interactions on the ns to μs time scale, to the tions with proteins displaying R-rich linear motifs and diffusion of macromoclecules into and out of, and rRNA: Mitrea DM, et al., under review]. NMR, smFRET, within, liquid-like structures on the timescale of seconds and SANS were used to understand the conformational to tens of seconds. A key challenge is to understand the and dynamic features of NPM1 before and after phase relationships between conformational features and mo- separation with a peptide derived from the ribosomal pro- tions of amino acids at the atomic scale and the macro- tein, rpL5, and revealed molecular organization extending scopic properties of these structures (e.g., viscosity, to ~10 nm within liquid-like droplets. In addition, deletion surface tension, macromolecular diffusion rates, etc.). analyses identified the domains of NPM1 required for A few studies have begun to address the challenges as- phase separation in vitro and for localization within nucle- sociated with spanning these broad length and time oli in cells. scales. For example, a recent report addressed the con- The three studies discussed above illustrate approaches formational features of the FG-Nup protein, Nup153, to relate the molecular features of phase-separation-prone and how these features mediate ultra-fast interactions proteins studied with atomic resolution to the macro- the nuclear transport receptor, Importin β [132]. While scopic features of the liquid-like structures that they form. Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 17 of 20 Importantly, two of the studies also integrated results membrane-less organelles to modulate their signaling be- from cellular assays, allowing molecular features to be re- havior are on the horizon. lated to biological function. We are just beginning to Abbreviations understand the physical properties of phase separation- mRNP: messenger ribonucleoprotein; snRNP: small nuclear ribonucleoprotein; prone proteins that are associated with their localization snoRNP: small nucleolar ribonucleoprotein; TEM: transmission electron within membrane-less organelles and eagerly await the re- microscopy; FC: fibrillar centers; DFC: dense fibrillar component; GC: granular component; rRNA: ribosomal RNA; rDNA: ribosomal DNA; RNA Pol I/II: RNA sults of similarly adventurous integrative studies to polymerase I/II; NOR: nucleolar organizing region; SAXS: small angle X-ray broaden our knowledge of these features and, importantly, scattering; SANS: small angle neutron scattering; FRAP: fluorescence recovery how they contribute to the diverse biological processes after photobleaching; FLIP: fluorescence loss in photobleaching; smFRET: single molecule Förster resonance energy transfer. that occur within liquid-like cellular bodies. Competing interests Conclusions The authors declare no competing interests. The compartmentalization of macromolecules within living cells creates heterogenous functional assemblies Authors’ contributions that mediate diverse biological processes. Membrane- DMM and RWK conceived and wrote the manuscript. Both authors read and approved the manuscript. less organelle assembly follows the physical laws of poly- mer condensation and depends upon factors such as Acknowledgements component concentration and temperature (Fig. 1). Con- We apologize to the colleagues whose work, although valuable to the field, densation is triggered by specific, initiating interactions was not mentioned in this manuscript, due to size limitations. The authors thank Chris Stanley of Oak Ridge National Laboratory for helpful comments between multivalent macromolecules and is further ex- on the manuscript. This work was supported by NIH 1R01GM115634 tended by recruitment of additional protein or RNA (to R.W.K.), National Cancer Institute Cancer Center Support Grant P30CA21765 molecules via monovalent or multivalent interactions (to St. Jude Children’s Research Hospital) and ALSAC (to St. Jude Children’s Research Hospital). (Fig. 2). The complex composition of the intra-organelle matrix arises and is maintained by weak, multivalent in- Received: 29 July 2015 Accepted: 29 December 2015 teractions between modular proteins and RNA. Condensation through phase separation of specific pro- teins and nucleic acids into dense liquid- or gel-like struc- References 1. Handwerger KE, Cordero JA, Gall JG. 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Publisher: Springer Journals
Copyright: Copyright © 2016 by Mitrea and Kriwacki.
Subject: Life Sciences; Cell Biology; Protein-Ligand Interactions; Receptors; Cytokines and Growth Factors
eISSN: 1478-811X
DOI: 10.1186/s12964-015-0125-7
pmid: 26727894
Publisher site: See Article on Publisher Site

Abstract

Inside eukaryotic cells, macromolecules are partitioned into membrane-bounded compartments and, within these, some are further organized into non-membrane-bounded structures termed membrane-less organelles. The latter structures are comprised of heterogeneous mixtures of proteins and nucleic acids and assemble through a phase separation phenomenon similar to polymer condensation. Membrane-less organelles are dynamic structures maintained through multivalent interactions that mediate diverse biological processes, many involved in RNA metabolism. They rapidly exchange components with the cellular milieu and their properties are readily altered in response to environmental cues, often implicating membrane-less organelles in responses to stress signaling. In this review, we discuss: (1) the functional roles of membrane-less organelles, (2) unifying structural and mechanistic principles that underlie their assembly and disassembly, and (3) established and emerging methods used in structural investigations of membrane-less organelles. Keywords: Membrane-less organelles, Phase separation, Multivalency, Stress response, RNA metabolism Background specialized in protein sorting and trafficking through the Similar to the division of labor in human societies, the cell. Mitochondria supply the ATP energetic needs of a cellular “workforce”, macromolecules such as proteins, cell, and are enclosed in a double layer membrane, in DNA and RNA, is spatially organized in the cell based contrast to the single lipid bilayer that surrounds the on functional specialization. Subcellular organization of other membrane-bounded organelles. macromolecules underlies vital cellular processes such With the advent of electron microscopy that allowed as development, division and homeostasis, while disrup- visualization of nanometer scale structures [1] and ad- tion of this organization is often associated with disease. vances in fluorescent dyes and light microscopy, it be- A large proportion of the enzymatic and signaling came evident that there is further sub-division and local reactions in biology occurs in aqueous solution. Lipid organization within the nucleus and cytosol in the form bilayers, immiscible with the aqueous phase, enclose the of non-membrane bounded, macromolecular assemblies. water-soluble components of a cell. The plasma mem- Currently characterized membrane-less bodies or or- brane engulfs all the internal components of a cell. ganelles range in size from tens of nm to tens of μm and Membrane-bounded organelles provide the physical sep- were defined as highly dynamic macromolecular assem- aration required for specialized processes to occur in blies, whose components rapidly cycle between the functionally optimized compartments within a cell. organelle and surrounding milieu [2–7]. Nucleoli Thus, the nucleus contains the machinery dedicated for (reviewed in [8]), nuclear speckles (reviewed in [3, 9]), DNA and RNA synthesis, while the cytoplasm houses paraspeckles (reviewed in [2, 10]), and PML (reviewed in components that control protein synthesis and degrad- [11, 12]) and Cajal bodies (reviewed in [4]) are enclosed ation. The endoplasmic reticulum, Golgi apparatus and within the nuclear envelope and are specialized in vari- the lipid vesicles are membrane-bounded compartments ous aspects of gene regulation and RNA metabolism. Cytoplasmic messenger ribonucleoprotein (mRNP) gran- * Correspondence: [email protected] ules, such as P-bodies, germ granules, and stress gran- Department of Structural Biology, St. Jude Children’s Research Hospital, ules (reviewed in [13]) fulfill specific roles in mRNA Memphis, TN 38105, USA Department of Microbiology, Immunology and Biochemistry, University of metabolism and homeostasis. Analogous forms of RNA Tennessee Health Sciences Center, Memphis, TN 38163, USA © 2016 Mitrea and Kriwacki. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 2 of 20 granules have recently been identified in mitochondria component (DFC) and granular component (GC). Dur- with roles in mitochondrial ribosome biogenesis and ing mitosis, the GC dissolves, disrupting nucleolar RNA processing [14]. organization but components of the FC and DFC main- In this review we will present an overview of current tain interactions as diffusible sub-structures. knowledge regarding the structural biology of membrane- Nucleolar assembly (reviewed in [8]) is initiated by less organelles and the molecular mechanisms involved in RNA Polymerase I (RNA Pol I) transcription of clustered regulating their structure and function. ribosomal RNA (rRNA) genes (rDNA) bound to the transcription factor UBF. Ribosome biogenesis occurs Overview of membrane-less organelles vectorially, starting from the FCs, where rDNA is tran- Membrane-less organelles were described as dynamic scribed into rRNA. pre-rRNA molecules transit through structures which often display liquid-like physical prop- the DFC, where they are spliced and the small ribosomal erties [5, 6]. Although it is well established that they are subunit is assembled, then move into the GC where the implicated in important biological processes, their pre- large ribosomal subunit is assembled. Pre-ribosomal par- cise roles remain elusive, often being associated with ticles are then released into the nucleoplasm and subse- more than a single functional pathway. As will be de- quently exported into the cytoplasm where functional scribed in greater detail in the following sections, the ribosomes are assembled. proteinaceous composition of membrane-less organelles p53-dependent stress sensing mechanisms are inte- and their morphology are altered in response to changes grated into the nucleolus, thereby allowing the cell to in the cellular environment. This ability to respond to halt the energetically expensive process of ribosome bio- environmental cues may represent the mechanistic basis genesis under conditions that are unfavorable for growth for the involvement of the membrane-less organelles dis- and proliferation. For example, in response to oncogenic cussed herein in stress sensing [2, 4, 9, 11, 13, 15]. The stress (e.g., activation of Myc), Mdm2, the E3 ubiquitin lack of a lipid-rich barrier to enclose the constituents of ligase responsible for rapid turnover of p53, is immobi- ARF membrane-less organelles presents the advantage that lized in the nucleolus through interactions with p14 changes in the surrounding environment can readily in order to upregulate p53 and its downstream cell cycle alter their internal equilibrium. Release or sequestration arrest effectors [17]. of constituent proteins or RNAs from or within membrane-less organelles alters their concentrations in Paraspeckles the surrounding freely diffusing pool of macromolecules, Paraspeckles are nuclear bodies located in the interchro- thereby sending signals that impinge upon stress re- matin space, with roles in control of gene expression sponse pathways. One example is the accumulation into through nuclear retention of specific RNA molecules, the nucleolus, followed by release into the nucleoplasm marked by adenosine-inosine editing [2]. The proteins ARF of the tumor suppressor p14 in response to DNA that comprise paraspeckles are associated with RNA damage, which activates the p53 tumor suppressor path- Polymerase II (RNA Pol II) transcription and processing way [16]. The nuclear volume is partitioned into mul- of RNA. The DBHS family of splicing proteins, tiple membrane-less organelles, also called nuclear P54NRB/NONO, PSPC1, PSF/SFPQ [2, 10, 18, 19], and bodies. Cytoplasmic bodies further partition the cyto- the long non-coding RNAs (lcnRNA) NEAT1/Men ε/β solic components. Nuclear and cytoplasmic bodies are and Ctn are integral components of paraspeckles [2]. dynamic structures, with well-defined compositions, Paraspeckles are responsive to stress and exchange com- which have the ability to exchange components in re- ponents with the nucleolus in response to environmental sponse to alterations to their environment. In the follow- cues. For example, paraspeckle protein 1 (PSPC1) was ing section we will discuss the functional roles of first identified as a nucleolar protein; however, it was membrane-less organelles and the unique features that later shown that, under conditions of active RNA Pol II- define them. dependent transcription, it partitions into a different nuclear body, the paraspeckles, and only becomes re- Nuclear membrane-less bodies localized to the nucleolus when RNA Pol II activity is The nucleolus suppressed [10, 18]. Interestingly, this re-localization oc- The largest and best studied membrane-less organelle, curs at the peri-nucleolar caps, which are structures that the nucleolus, functions as the center for ribosome bio- appear to be physically associated with nucleoli, but are genesis in eukaryotic cells. The nucleolus exhibits com- not integrated into the nucleolar matrix [10]. This plex, compartmentalized organization in interphase and suggests that either the physical properties of PSPC1- disassembles in mitosis. Three distinct regions can be containing bodies and of the nucleolus are different, pre- observed by transmission electron microscopy (TEM) in cluding fusion, or their dynamic behavior is restricted in intact nucleoli: the fibrillar centers (FC), dense fibrillar response to the signals that inhibit RNA Pol II activity. Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 3 of 20 Nuclear speckles is dispensable with respect to the formation of PML Similar in appearance to paraspeckles and localized adja- bodies [29]. cent to nucleoplasmic interchromatin regions [3], nu- clear speckles, also referred to as snurposomes, are a Cytosolic membrane-less bodies distinct class of dynamic organelles [1]. The compos- Dynamic membrane-less organelles were also described ition of nuclear speckles, enriched in pre-mRNA spli- in the cytoplasm. They are generally referred to as cing factors, such as small nuclear ribonucleoproteins mRNP granules, are involved in mRNA metabolism and (snRNPs) and serine/arginine-rich (SR) proteins [20], homeostasis, and include structures such as P-bodies, and poly(A) RNA[21],aswell astheir spatialprox- stress granules and germ granules (reviewed in [13, 30]). imity to sites of active transcription, suggest they may Several different types of mRNP granules share protein playarole inregulatinggeneexpressionby supplying and mRNA components and it has been demonstrated or storing factors associated with the splicing of pre- that they have the ability to physically interact with one mRNAs [22]. another in vivo, undergoing docking and fusion events [13]. These observations suggest that not only are these Cajal bodies membrane-less organelles functionally related, but under Although not fully elucidated, the role of the Cajal bod- certain conditions they exhibit similar physico-chemical ies is linked to regulation of snRNPs and small nucleolar properties that allow for their structural miscibility. The ribonucleoprotein particles (snoRNPs) [4]. Time lapse major types of mRNP granules are discussed below. experiments monitoring fluorescently tagged coilin and survival of motor neurons (SMN) proteins, two well de- P-bodies scribed markers of Cajal bodies, showed that they are Processing or P-bodies are ubiquitous to all types of cells dynamic structures within the nucleus that undergo fu- and contain proteins involved in mRNA transport, sion and fission events [23]. Similar to other nuclear modification and translation (reviewed in [31]). Studies membrane-less organelles, Cajal bodies are responsive to in yeast demonstrated that deletion of any single protein stress conditions. The tumor suppressor p53 associates component was not sufficient to fully abrogate the as- with Cajal bodies under conditions of UV-irradiation sembly of P-bodies [32], but highlighted the importance and chemotoxic stress [24], while coilin re-localizes to of partner-specific interactions to the accumulation of a nucleolar caps, along with fibrillarin and components of number of proteins into the organelle [33, 34]. For ex- the RNA Pol I machinery [25]. Furthermore, similar to ample, recruitment of the Dcp1 decapping enzyme to the nucleolus, the structural integrity of Cajal bodies is the organelle is mediated by interactions with its co- cell cycle dependent; they are intact during interphase factor, Dcp2 [34], while Dcp2 directly interacts with the and dissolve during mitosis [26]. scaffold protein Edc3 [33, 34]. As with other membrane- less organelles, RNA plays a central role in the assembly PML bodies of P-bodies. Elevated levels of non-translating mRNA, Localized primarily in the nucleus, PML bodies are char- achieved by inhibition of translation initiation or stress, acterized by the presence of promyelocytic leukemia is correlated with an increase in the size and number of (PML) protein. A member of the TRIM family of pro- P-bodies [35]. Conversely, entrapment of mRNA into teins, PML contains a RING domain, two B-box do- polysomes by inhibiting the elongation step or enzymatic mains and a predicted coiled-coil domain, all of which degradation of mRNA correlated with dissolution of have been shown to be required for proper assembly of P-bodies [31, 35]. PML bodies. The exact role of these organelles is yet to be fully elucidated. Evidence that transcriptional regula- Stress granules tors such as p53, CBP and Daxx are transiently targeted Stress granules, as the name suggests, assemble in re- and retained in PML bodies suggests that they function sponse to stress signals to sequester transcriptionally as a storage compartment and thus regulate pathways silent mRNA molecules and transcription factors involved in tumor suppression, viral defense and apop- (reviewed in [30]). Translation initiation factors and tosis [12]. As with other membrane-less organelles, the components of the small ribosomal subunit are amongst number and structural integrity of PML bodies are influ- the proteins enriched within stress granules [13]. Re- enced by cell cycle phase and stress stimuli [27]. In sen- moval of the stress signals and re-initiation of mRNA escent cells, PML bodies become enlarged and associate translation caused stress granules to disassemble [36]. with the nucleolar caps [28]. Newly synthesized RNA Similarly to P-bodies, sequestration of non-translating accumulates at the periphery of PML bodies, support- mRNA molecules in polysomes inhibited formation of ing a role in RNA metabolism. However, unlike the stress granules [36], thus suggesting that mRNA is re- other membrane-less organelles described herein, RNA quired in their assembly. P-bodies and stress granules in Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 4 of 20 yeast exhibit extensive compositional overlap, but dis- the stress granule microenvironment, where high local tinct physical properties [37]. Furthermore, yeast strains protein concentrations are achieved [37, 42, 44, 45]. Fur- deficient in formation of P-bodies were also unable to ef- thermore, genetic mutations within the prion-like do- ficiently form stress granules. The formation of P-bodies mains of these proteins known to be associated with ALS in yeast was not affected in mutant strains that were accelerated formation of amyloid-like fibrils and inhibited deficient in stress granules assembly. Together, these stress granule clearance in vivo, thereby disrupting mRNA observations suggested that pre-assembly of mRNA/ homeostasis [41–44]. These findings suggest that the protein complexes in P-bodies is a pre-requisite for the highly dense environment of mRNP granules facilitates fi- formation of stress granules [32], highlighting a functional bril formation by the proteins noted above, especially connection between the two types of membrane-less when their aggregation propensity is enhanced by muta- organelles. tion. Further, these studies establish correlations between ALS-associated mutations in mRNP granule proteins, and Germ granules heightened fibril formation and altered mRNA metabol- The term, germ granules, encompasses a class of non- ism. Additional research is needed, however, to under- membrane bounded organelles found in the specialized stand how these changes to mRNP granule structure and germ cells that generate sexual cells upon meiosis in the function are related to neuropathogenesis. developing embryo and are referred to as P-granules, In the next section we will discuss the common germinal bodies or Nuage bodies, depending on the or- physico-chemical features of membrane-less organelles ganism of origin (reviewed in [38]). Significant advances and unifying mechanistic insights that describe their have been made in understanding both the biology and assembly into multicomponent dense phases. the biophysics of P-granules in the nematode, C. elegans. P-granules are enriched in mRNA, RNA helicases and Common features of membrane-less organelles RNA modifying enzymes and are involved in the post A hallmark of the membrane-less organelles described transcriptional regulation of mRNA in primordial germ above is that their composition and physical properties cells [38]. For example, nos-2 RNA is asymmetrically vary depending upon cellular factors such as cell cycle segregated during C. elegans larval development [39]. stage, growth stimuli and stress conditions. In addition, P-bodies physically dock, but do not fuse with germ they exhibit dynamic structural features. Brangwynne granules in C. elegans embryos. This physical association and colleagues demonstrated that the nucleolus [5] and between the two types of organelles allows P-bodies to P-granules [6] exhibit liquid-like behavior in vivo and segregate within the germline blastomere, a property that this fluid organization arises from phase separation borrowed from the germ granules. Furthermore, these of their molecular components. This concept is sup- P-bodies that are associated with germ granules fail ported by a growing body of evidence identifying pro- to undergo maturation into organelles that degrade teins, sometimes co-mixed with nucleic acids, that phase mRNA [40]. Collectively, these observations exemplify separate in vitro into dense liquid-like [46–49] or hydro- how distinct physico-chemical properties preserve or- gel [50, 51] structures (reviewed in [52]). The proteins ganelle integrity and suggest inter-organelle interac- and nucleic acids are concentrated ~ 10-100-fold in the tions as a novel mechanism for regulating function. dense phase [46, 48], where they can reach concentra- tions in the millimolar range [53]; the dilute phase is mRNP granules in neurodegenerative disease maintained at the critical phase separation concentra- Debilitating neurodegenerative diseases such as amyo- tion. Experimentally, the two physical states, liquid and trophic lateral sclerosis (ALS), multisystem proteinopathy hydrogel, are distinguished by their ability to flow when (MSP) and frontotemporal lobar degeneration (FTLD) are their surfaces are subjected to shear stress. The liquid- characterized by formation of pathological mRNP inclu- like features of membrane-less organelles and in vitro sions and disruption of normal mRNA metabolism phase separated protein and protein/RNA droplets, have (reviewed in [41]). These pathological inclusions are been demonstrated based upon measurements of their formed through aggregation of proteins found in en- viscoelastic properties [5, 6, 44, 47, 54, 55]. For example, dogenous mRNP granules. Interestingly, many of the pro- liquid-like P-bodies [37] and P-granules [6] adopted teins associated with pathological inclusions contain a spherical shapes in the cytoplasm that were governed by prion-like domain in their amino acid sequence, which surface tension, and coalesced and fused into larger promotes their assembly into amyloid-like fibrils. Several droplets that returned to spherical shapes. Additionally, proteins known to localize within stress granules, includ- P-granules became reversibly deformed when they en- ing FUS [42], hnRNPA1 [43–45] and hnRNPA2 [43], were countered a physical barrier (i.e. “dripped” on the sur- found in ALS-associated pathological inclusions. Interest- face of the nucleus) [6]. In contrast, hydrogels do not ingly, fibril formation by these proteins is promoted within exhibit flow under steady-state conditions [50, 51, 56]. Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 5 of 20 Microrheology analysis indicated that liquid-like mem- composition regulated in response to stress signals?In the brane-less organelles [5, 6] and protein and protein/RNA next section we address the molecular principles that droplets prepared in vitro are characterized by high vis- underlie phase separation and the structural organization cosity. Strikingly, the measured values for viscosity of membrane-less organelles. We also discuss current evi- varies widely, over a range of three orders of magni- dence that suggests how their dynamic structure and tude, from ~ 1 Pa · s for P-granules to ~ 10 Pa · s for compositions are regulated. nucleoli [5, 6, 47, 54, 55]. Although not necessarily a direct indicator of liquid-like behavior, macromolecules Structural and compositional features of proteins resident within membrane-less organelles ([7, 37, 44, 46]) and within membrane-less organelles liquid-like droplets [42, 44, 46, 53, 55] recover after photo- Results from knock-down and knock-out studies [32, 39, bleaching on a timescale of seconds to tens of seconds. 61–63] showed that the structural integrity of several This indicates rapid exchange of molecules within the membrane-less organelles depends upon heterogeneous liquid-like phase, or with the surrounding milieu, when interactions amongst multiple components. Knock-down the object is photobleached in part or in full, respectively. or genetic deletion of single proteins, such as NPM1 Membrane-less organelles exhibit compositions of [61] or nucleolin [62] in the nucleolus or PGL-1 and varied complexity. For example, P-granules are com- PGL-3 [63] in germ granules, altered organelle morph- prised of approximately 40 proteins [57] while mass ology but did not prevent other, unaltered organelle spectrometry has shown that human nucleoli contain a components from assembling into punctate structures. staggering ~4500 proteins [58]. Furthermore, the protein These observations are consistent with redundancy of composition of membrane-less organelles can vary de- the sequence features of proteins found within various pending upon cellular conditions. Notably, the nucleolar membrane-less organelles (Table 1). proteome is significantly altered under stress conditions and the alterations are specific to particular forms of stress Basic principles of phase separation by polymers; from [59, 60]. These observations raise two important ques- chemical polymers to proteins tions: (1) how is the specific molecular composition of Phase separation of organic polymers in solution has membrane-less organelles achieved and (2) how is their been extensively studied and can be described by Table 1 Protein and RNA composition of membrane-less organelles Organelle Biological role Protein Domains/Motifs RNA Nucleolus Ribosome biogenesis in nucleus Fibrillarin RGG box [133] rRNA [8] Nucleolin RRMs; RGG box [67] Paraspeckles Regulation of gene expression PSPC1 RRMs; Coil [2] ncRNA NEAT1 (Menε/β); Ctn [2, 19] in nucleus NONO/P54NRB RRMs; Coil [2] SFPQ/PSF RRMs; Coil [2] Nuclear speckles Regulation of gene expression via SRSF1 RRMs; RS [134] Poly(A) RNA; lncRNA MALAT1 [3, 134] storage of splicing factors Cajal bodies Regulation of snRNP maturation Coilin Coiled-coil [23] snRNA; snoRNA [4, 135] SMN Coiled-coil [23] PML bodies Regulation of transcription and PML Coiled-coil [12] None [11, 29] protein storage Germ granules Regulation of mRNA translation in GLH-1, GLH-2, FG [74] Developmentally regulated maternal the cytoplasm of germ cells GLH-4 mRNAs (nos-1, pos-1, mex-1, skn-1 , gld-2) [74, 136] PGL-1, PGL-3 RGG [63] DDX4 FG; RG [48] LAF-1 RGG box [47] P bodies mRNA processing and decay Pdc1 HLM; Coiled-coil [49] mRNA [31] Dcp2 HLM [49] Edc3 LSm; FDF [49] Stress granules Storage of translationally stalled FUS RRM; RGG box; [G/S]Y[G/S] [50, 137] Poly-(A) mRNA associated with PABP [30] mRNA and proteins of the hnRNPA1 RRM; RGG box; [G/S]Y[G/S] [50] translational machinery Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 6 of 20 simplified mathematical thermodynamic models. Flory- behavior of bimolecular and unimolecular protein sys- Huggins theory describes the free energy of mixing of a tems. However, the sequence complexity of protein poly- polymer with solvent, wherein polymers are treated as mers, in contrast with compositionally more simple simplified arrays of modules that represent their repeti- chemical polymers, provides the opportunity for add- tive segments. Liquid-liquid phase separation into a itional inter-molecular interactions that can “tune” the polymer-rich phase and a polymer-poor phase occurs phase separation phenomenon. These results provide a when a critical concentration or temperature threshold foundation for understanding the phase separation behav- is crossed, whereupon the polymer becomes a better ior of more complex systems in vitro in the future. Fur- solvent for itself than is the buffer it is dissolved in thermore, they provide a foundation for in depth study of (reviewed in [64]; Fig. 1). the behavior of membrane-less organelles in cells. Rosen and colleagues reported that multivalent, repeti- tive domains from two signaling proteins that regulate Protein elements associated with phase separation; low actin polymerization, NCK and N-WASP, phase separate complexity sequences and folded domains in vitro and that the phase separation threshold depends Proteins associated with membrane-less organelles often on the protein concentration and valency of each indi- exhibit multivalent features which are manifested struc- vidual interaction partner [46]. Employing a simplified turally in different ways. Folded domains are proteins protein representation akin to that used for organic segments which adopt discrete and stable secondary and polymers, the authors used an adaptation of the Flory- tertiary structures. Disordered regions, also referred to Huggins formalism to describe the phase transition be- as intrinsically disordered protein regions (IDRs), are havior of the binary NCK/N-WASP system. The model protein segments that do not adopt stable secondary and included four parameters: association/dissociation pa- tertiary structure and are conformationally heterogenous rameters, and diffusion and crowding coefficients. Quali- and dynamic. Some proteins within membrane-less or- tatively, this formalism, which assumed structural ganelles contain folded domains but may also contain uncoupling between individual binding domains, pre- IDRs, while others are entirely disordered (termed in- dicted the effect of varying valency on the concentration trinsically disordered proteins or IDPs). A subset of threshold for phase separation [46]. A similar adaptation disordered protein regions, termed low complexity re- of this model was used to describe the phase separation gions, exhibit compositional bias towards a small set behavior of the unimolecular RNA helicase, Ddx4 [48]. of amino acids. Interestingly, low complexity se- While the general phenomenology can be described quences and disorder [47, 48, 50, 56] are overrepre- using this simplified model, a recent report involving sented in proteins shown to phase separate in vitro. the binary NCK/N-WASP system demonstrated that These features provide a high degree of conform- charged residues within the disordered linker connect- ational flexibility which is required for binding events ing SH3 domain binding modules caused weak self- to remain uncoupled [46]. NMR analysis of proteins association of NCK and reduction of the critical con- within the liquid-like phase after phase separation did centration for phase separation [65] (Fig. 1). Thus, not provide evidence of folding-upon-binding, thereby Flory-Huggins theory describes the basic phase separation suggesting that the disordered low complexity regions Low High PTMs, temperature, component component ionic strength, etc. concentration concentration Decreased threshold, assembly is enhanced Increased threshold, disassembly is promoted Disassembly Phase separation/ Critical assembly concentration for phase separation (M) Fig. 1 Macromolecular condensation mediates the formation of membrane-less organelles. Membrane-less organelles are dynamic structures formed via a polymer-condensation-like, concentration-dependent phase separation mechanism. The critical concentration threshold (grey line) for phase separation can be tuned within a range of concentrations (shaded green box) through physico-chemical alterations to the system (i.e., posttranslational modifications to domains and/or motifs that alter the affinity of their interactions, changes in temperature, altered ionic strength, etc.). These changes can drive phase separation and assembly of membrane-less organelles, or their disassembly Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 7 of 20 preserve their conformational flexibility within the to form membrane-less organelles? Given the large liquid-like phase [48, 53]. The detailed interpretation differences in critical concentration measured for the of these data is complicated, however, by the possibil- various systems, one possible answer is that compo- ity for organizational heterogeneity of the protein mol- nents with the lowest critical concentration phase ecules outside and possibly within liquid-like droplets, separate first, thus increasing the local concentration and the influence of inter-molecular interactions and above the critical concentration for phase separation apparent molecular size on resonance line widths and of other components which subsequently become in- intensities. corporated into the dense phase. Both folded domains Multivalent interactions are likely to contribute to the and disordered/low complexity regions have been re- dynamic, liquid-like properties of phase separated uni- ported to initiate phase separation in vitro and in cellulo. molecular assemblies [47, 48], as well as of more com- The folded domains are often implicated in specific plex assemblies [46, 49]. Amongst proteins associated protein-nucleic acid [67–69] and protein-protein [19, 70] with phase separation in membrane-less organelles, interactions and may provide an organizational scaffold multivalency is achieved through repetitive display of for the assembly of a membrane-less organelle. Low com- two types of protein modules: i) folded domains and plexity domains, on the other hand, provide a means for ii) low complexity disordered segments (summarized more dynamic interactions with a potentially broader in Tables 1 & 2; Fig. 2). In vitro studies had shown range of binding partners (Fig. 2). A compelling example that one of the two types of multivalency is necessary of such a synergistic cooperation between multivalent and sufficient for protein phase separation. The pro- folded domains and their respective connecting flexible tein concentrations associated with phase separation linkers was reported by Bajade et al., on the Nck/N- varied over several orders of magnitude for different WASP/nephrin system [65]. Nck constructs that are systems, ranging from sub-micromolar [44, 47] to divalent in SH3 motifs bind to PRM motifs in N-WASP hundreds of micromolar [44, 46, 48, 53]. Membrane- with micromolar to millimolar affinity and undergo phase less organelles are multicomponent systems and their separation. Through weak, largely electrostatically driven assembly, as demonstrated for the nucleolus, depends interactions, the disordered linker connecting the SH3 on the total concentration of their constituents [66]. domains in Nck promotes self-assembly, effectively Given the observations noted above that the accumu- lowering the critical concentration for phase separ- lation of components with nucleoli is temporally de- ation. Furthermore, addition of a disordered region of fined (reviewed in [8]) and occurs at pre-formed Nephrin containing multiple phospho-tyrosine resi- nucleolar organizing regions (NORs) raises an import- dues, which bind to a folded SH2 domain within Nck, ant question. Are some components more important enhances multivalent interactions and further lowers the others for initiating the phase separation process the critical concentration for phase separation. Thus, multivalent display of folded domains and low com- plexity sequences with disordered regions within pro- teins enables synergy between the various components Table 2 Examples of protein regions involved in phase of complex liquid-like droplets. Similar synergy be- separation and their functional roles tween multivalent components is likely to promote Domains Sequence/Structural Role features formation of membrane-less organelles in cells. FG FG/GFGG low complexity Association of P granules to repeats the NPC [74] Initiation events in the assembly of membrane-less RRM Folded domain RNA binding [19, 68] organelles Many of the proteins that participate in the formation of Coiled-coil Coiled-coil fold Homo/hetero-dimerization [12] membrane-less organelles exhibit segments with low RS RS low complexity repeats RNA binding; protein-protein interactions (Reviewed in complexity sequence features, often containing multiple [138, 139]) motifs enriched in the amino acids arginine, serine, gly- RGG RGG low complexity RNA binding (Reviewed in cine, glutamine, asparagine and/or aromatic residues repeats [140, 141]) (Tables 1 & 2). However, despite the low complexity of HLM Short helical leucine-rich LSm domains binding in their sequences, these proteins are often associated with motif P granules [49, 75] specific membrane-less organelles. What is the basis for SH3 Folded domain PRM motif finding [46] the incorporation of particular proteins and nucleic acid SH2 Folded domain Phosphorylated tyrosine molecules within particular membrane-less organelles? recognition [46] The emerging solution to this conundrum, at least in PRM Proline-rich short linear SH3 domain binding [46] some cases, is that specific protein-nucleic acid or motif protein-protein interactions initiate the assembly of Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 8 of 20 Catalytic Low domain complexity region Binding Low domain complexity region Active transcription, Multivalent RNA levels increase interactions Binding Catalytic domain domain Stress signals, altered Dimerization domain critical concentration threshold, reduced Dimerization Low domain complexity RNA level region Structural modularity and Pre-initiation Phase separation multivalency in soluble proteins intermediates Fig. 2 Molecular basis for membrane-less organelles assembly. The proteins enriched within the matrices of membrane-less organelles commonly exhibit multiple modules that create multivalency, including folded binding domains (red) and low complexity regions (purple). Valency is often amplified by domains that enable homo-, or hetero-oligomerization (orange). Interactions between proteins containing different combinations of these interaction modules provide a framework for building a heterogeneous, infinitely expandable network within membrane-less organelles. Formation of this type of network drives phase separation when the critical concentration threshold is reached. For many of the examples discussed herein, active RNA transcription is needed for membrane-less organelle assembly. We hypothesize that expression of RNA in excess of a critical concentration threshold is needed to nucleate interactions with specific, multi-modular proteins, and for nucleating formation of membrane-less organelles. Stress signals can alter the multivalent interactions that drive phase separation and lead to partial or complete disassembly of the organelle membrane-less organelles, which then create a micro- scattering (SAXS) to study the polymerization of DBHS environment that is conducive to phase separation of family of splicing factors, localized to and enriched in additional components (Fig. 2). This concept was de- paraspeckles [19, 70]. Extended coiled-coil interaction scribed for the nucleolus, which assembles around motifs within the polymerization domain of these pro- NORs, stable nucleolar precursors, comprised of clus- teins provided the structural scaffold for formation of tered arrays (i.e. multivalency) of the genes for rRNA, extended polymers of indefinite length. Weak, polar bound to the transcription factor UBF [71]. Notably, contacts stabilize the coiled-coil interactions and are UBF contains an array of six HMG box domains that ex- thought to be advantageous in maintaining the solubility hibit a broad range of binding affinities for DNA [69]. of unpaired extended helical structures [70]. The valency RNA Pol I is recruited to the NORs to transcribe pre- of the molecular assembly is enhanced by an additional rRNA, which initiates the assembly of the nucleolus. In dimerization domain which mediates homo- and hetero- the case of germ granules [63] and PML bodies [12], dimerization between DBHS family proteins, such as their formation is initiated by self-association of the PSPC1 and NONO [19] or SFPQ and NONO [70]. Fur- coiled-coil domains of the proteins PGL-1/3 and PML, thermore, multivalent interactions with RNA are medi- respectively. In these examples, structured domains me- ated by tandem RRM domains present in NONO, diate specific interactions to form assemblies that serve PSPC1 and SFPQ [19, 70]. These studies exemplify how as scaffolds for further assembly of components of modular, multivalent proteins can mediate the formation membrane-less organelles. Some of the proteins that of heterogeneous, dynamic molecular assemblies, thereby promote assembly contain both structured domains and providing the structural basis for formation of a low complexity segments that mediate multivalent inter- membrane-less organelle (Fig. 2). actions. The formation of membrane-less organelles may thus involve hierarchical assembly of specific, higher Forces that mediate the interactions associated with protein affinity protein-nucleic acid complexes followed by the phase separation recruitment of additional components through weaker, As discussed above, proteins that undergo phase separ- multivalent interactions. ation commonly contain segments with low sequence The assembly behavior of proteins associated with complexity. Further, these regions are often enriched in paraspeckles provides another example of how initiation charged and aromatic amino acids, highlighting the im- events can mediate the recruitment of components portance of electrostatic and hydrophobic interactions in within a membrane-less organelle. Bond and co-workers the process of phase separation. For example, disordered used X-ray crystallography and small angle X-ray segments of the DEAD-box helicases Ddx4 [48] and Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 9 of 20 LAF-1 [47], as well as hnRNPA1 [44] that mediate phase variable contributions of the different types of intermo- separation are enriched in arginine residues within their lecular interactions that promote phase separation deter- low complexity RGG box and RRM domains. Due to mine selective accumulation of specific proteins within their overall positive charge, the formation of liquid-like specific types of membrane-less organelles. droplets by these proteins is highly sensitive to the ionic strength of the surrounding solution. Numerous other Mechanisms involved in achieving local organization and proteins associated with nuclear bodies and mRNP gran- compositional complexity in membrane-less organelles ules are enriched in arginine residues (e.g. RGG and SR The localization of specific macromolecules within par- domains; see Table 1). For example, the low complexity ticular membrane-less organelles is achieved through SR repeats common to the SR family of splicing factors specific interactions with the molecular network that ex- were identified as targeting signals for nuclear speckle tends from the nucleating region. As discussed above, a localization [72, 73]. These observations strongly suggest large proportion of the proteins known to associate with that electrostatic interactions play a key role in the phase membrane-less organelles exhibit multivalency through separation of a subset of proteins (Fig. 1). the display of repeated low complexity motifs (e.g., SR, Electrostatics are not, however, the only interactions RGG or FG motifs) and/or of multiple copies of folded that promote the formation of the protein-rich phase domains, such as RRM domains. Through combinatorial separated state. Low complexity regions that are rich in utilization of a finite number of intermolecular inter- aromatic residues (i.e. phenylalanine, tyrosine) are over- action modules, complex mixtures of proteins and nu- represented in proteins that reside within membrane- cleic acids can thus be recruited into the condensed less organelles [48, 74] and other phase separated phase. For example, the formation of P-granules is initi- matrixes, as is the case for the FUS protein in mRNP ated by self-association of the coiled-coil domains of granules [50, 53] and the FG-Nups in the nuclear pore PGL-1 and PGL-3 proteins, which further bind mRNA complex [51]. Interestingly, mutations of F to Y, but not via their low complexity RGG domains. Vasa-related F to S, within the FG repeat domain preserved in vitro helicases GLH-1, 2, 3 and 4 that contain FG repeats are hydrogel formation by the yeast nucleoporin Nsp1p [51], then incorporated to facilitate P-granule association with demonstrating the importance of aromatic residues in nuclei, through interactions with and expansion of the assembly phenomena associated with the nuclear pore nuclear pore complex hydrogel matrix [74]. The pres- complex. Furthermore, the critical concentration for for- ence of homo- and hetero-oligomerization domains fur- mation of in vitro FUS liquid droplets was lowered by ther enhances the degree of multivalency and promotes increasing the ionic strength of the solution, consistent integration within membrane-less organelles (Fig. 2). with the interpretation that salting out the hydrophobic The PML protein forms homo- and hetero-oligomers via interactions reduced the solubility threshold for the pro- its coiled-coil domain, but valency can be increased by tein in buffer [53]. Nott et al., noted that evolutionarily homo-dimerization through the RING domain. Muta- conserved clustering of similarly-charged amino acid tions in either the coiled-coil or RING domains led to residues and regular spacing between the RG and FG disruption of PML bodies [12]. Components of the motifs are required for the phase separation of a Ddx4 mRNA decapping machinery found in P-bodies, includ- construct [48]. These studies highlight the roles of ing Pdc1, Dcp2 and Edc3, assemble into liquid-like drop- cation-π [48] and π-π [50, 51] interactions in phase sep- lets in vitro. Two LSm domains in dimeric Edc3 interact aration phenomena. with Dcp2 and Pdc1, which both contain multivalent In the absence of a lipid membrane barrier, the move- HLM motifs. Edc3 binds to various HLM motifs with af- ment of molecules into and out of membrane-less or- finities within the low micromolar to millimolar range ganelles is diffusion limited [1], and their accumulation [49]. The valency of the HLM motifs in Pdc1 is in- is mainly dependent on retention based on interactions creased through oligomerization via a central coiled-coil with the organelle matrix. Interestingly, the diffusion domain [49, 75]. These examples illustrate how multiva- barrier for exogenous macromolecules such as dextrans, lent interaction modules and oligomerization domains is dictated by the physical properties of the membrane- can cooperate to initiate phase separation in the context less organelle matrix [1]. The DFC of the nucleolus is of different types of membrane-less organelles. Add- less permissive to accumulation of dextrans compared to itional domains within these proteins, which are not dir- the surrounding GC, consistent with the observations ectly involved in the mechanism of phase separation, that the DFC is denser than the GC [1]. Furthermore, can mediate the recruitment of additional components the dynamic features of components specifically retained into the liquid phase. For example, the helicase Ddx6/ within membrane-less organelles vary based on the Dhh1 and mRNA can be recruited to P-bodies via the nature of their interactions with other constituents of FDF domain of Edc3 and the RNA binding domain of the matrix [7, 23]. Together, these results suggest that the helicase, respectively [49]. We thus distinguish Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 10 of 20 between two basic types of components of membrane- induced by DRB, a small molecule that selectively in- less organelles: (i) multivalent macromolecules that dir- hibits RNA Pol II, caused dissolution of paraspeckes be- ectly participate in interactions involved in the process fore a significant decrease in the total Mem ε/β lncRNA of phase separation and underlie the structural features levels could be measured [77]. This finding suggests that of the liquid phase and (ii) other macromolecules that a currently unknown regulatory mechanism controls the are recruited via specific interactions with the phase sep- structural integrity of paraspeckles and that there is a arated assembly, which lack multivalent interaction sharp and sensitive threshold for sensing and responding elements, but perform specialized functions within the li- to cellular stress. This raises an important general ques- quid phase (i.e., enzymes that catalyze specific biochemical tion: how are changes in environmental conditions, for reactions). However, the capability for assembly/phase example in response to different types of stress, transmit- separation and biochemical functionality can be embodied ted to the membrane-less organelle matrix and mani- within a single protein, as is seen with Ddx4, which har- fested as changes in structure and function? This topic is bors a helicase domain and a multivalent, low complexity discussed in the next section. RGG domain that mediates phase separation [48]. Structural and dynamic regulation of phase separated RNA within membrane-less organelles structures While much attention has been given to understanding The lack of a lipid bilayer barrier between membrane- the roles of multivalent proteins in the formation of less organelles and their surroundings circumvents the membrane-less organelles, the primary functions of need for active transport of macromolecules across many of these organelles are different aspects of RNA membranes and enables rapid signal transduction. Stress metabolism and, consequently, RNA is also involved in signals influence the structural integrity of membrane- their assembly and structural integrity. The assembly of less organelles, providing a mechanism for organelle- the nucleolus at the exit of mitosis is initiated by mediated stress responses. We next discuss various transcriptional activation of RNA Pol I [8, 76] and the factors that influence the structure and function of structural integrity of paraspeckles is dependent upon membrane-less organelles. transcriptional activity of RNA Pol II [2]. Proteins cap- able of undergoing phase separation often contain simi- Chemical and other environmental factors lar sets of folded and low complexity multivalent Changes in temperature [27, 48], ionic strength [47, 48], domains, giving rise to structural redundancy and the and chemotoxic and DNA damage [27, 59, 60, 78, 79] potential, under certain conditions, to promiscuously are environmental changes known to disrupt phase sepa- localize within more than one types of membrane-less rated cellular bodies and in vitro liquid droplets. The organelle. In contrast, the different types of organelles stiffness of nucleoli isolated from HeLa cells was de- generally contain specific types of RNA (summarized in creased or increased upon RNA Polymerase or prote- Table 1), suggesting that the RNA components are the asome inhibition, respectively, based on atomic force principal determinants of organelle identity. In support microscopy measurements [79]. Thus, stress signals of this hypothesis, disruption of RNA transcription affect the viscoelastic properties of nucleoli and conse- causes re-localization of the protein components of dif- quently modulate their functions. ferent nuclear and cytoplasmic bodies [25, 59]. For ex- Membrane-less organelles form, disassemble and func- ample, Mao et al., demonstrated that the lncRNA Mem tion in an intracellular environment crowded with mac- ε/β was required for the recruitment of specific protein romolecules. The high cumulative concentration of and RNA molecules to paraspeckles [77]. Additionally, macromolecules in the cell, which correlates with a high immobilization of PSP1, a modular, paraspeckle protein percentage of excluded volume (~20–30 % of the total shown to homo- and hetero-oligomerize [18], was able cell volume), affects the kinetics and thermodynamics of to recruit some paraspeckle protein components, but most biochemical processes [80]. In vitro, molecular was unable to recapitulate complete assembly of the or- crowding agents promote assembly of recombinant ganelle [77]. Recruitment of the full complement of pro- hnRNPA1 into protein dense liquid-like droplets at tein and RNA components of paraspeckles, coupled with lower critical concentrations than observed in buffer exclusion of macromolecules associated with nuclear alone [44, 45]. Thus, the increase in excluded volume speckles, was achieved only under conditions of active caused by macromolecular crowding increases the local transcription of the Mem ε/β lncRNA. While the obser- concentration of individual protein species, thereby de- vations summarized above clearly indicate the dominant creasing the effective concentration threshold for phase role of RNA in the molecular makeup of certain separation (Fig. 1). membrane-less organelles, other factors can also influ- Alterations in the morphology and viscoelastic proper- ence their structural integrity. For example, stress signals ties of mRNP granules, due to mutations in resident Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 11 of 20 proteins (e.g. hnRNPA1, FUS) are associated with debili- that ATP-hydrolyzing enzymes regulate the dynamics of tating neurodegenerative diseases [13, 42, 44, 45]. In vitro, macromolecules within membrane-less organelles. Simi- both FUS and hnRNPA1 phase separate into liquid-like larly, several other types of ATP-dependent enzymes, in- droplets [42, 44, 45, 53] or hydrogels [42, 44, 50], de- cluding kinases and DEAD-box helicases [47–49, 78], pending on protein concentration and experimental which are incorporated into these organelles, may be in- conditions. The low complexity regions in the two volved in maintaining their liquid-like physical proper- proteins, along with the RRM domains [44, 45, 53], ties. Helicases may modulate RNA structure as well as contribute to phase separation. Mutations within Q/N- protein-RNA interactions and, thereby, actively control rich low complexity regions, termed prion-like domains, the viscoelastic properties of membrane-less organelles. are associated with defects in mRNP granules and neuro- pathogenesis [42, 44]. These defects are attributed to a Role of posttranslational modifications in regulating kinetically slow step (tens of minutes to hours time scale) membrane-less organelle structure and dynamics that occurs in the dense liquid-like phase, referred to as The assembly of components within many of the phase “droplet aging” [42], wherein the liquid-like phase trans- separated systems we have discussed is electrostatically forms into a solid-like state. Phenomenological observa- driven. Therefore, posttranslational modifications that tions suggest that this physical transformation is a result alter the charge features of amino acids within the do- of a slow structural re-organization of the dense, liquid- mains and low complexity segments of proteins provide like phase. The reorganization leads to decreased dy- a means to modulate their multivalent interactions and namics within the phase separated state and culmi- phase separation behavior (Fig. 1). nates in a transition from a liquid-like state to a The importance of electrostatic interactions is illus- hydrogel or solid-like state. The transition between the trated by the phase separation behavior of LAF-1 [47], two physical states is accompanied by morphological hnRNPA1 [44, 45] and Ddx4 [48], whose ability to form changes, from nearly spherical droplets, shaped by surface liquid-like droplets is strongly influenced by the salt tension, to elongated, fibril-like structures [42, 44, 45]. A concentration of the surrounding buffer. The phase sep- similar transition was observed in vitro and in vivo aration concentration threshold for both scaled linearly droplets containing Whi3, a protein encoding a polyQ with ionic strength as the NaCl concentration was in- tract [55]. A potential underlying mechanism is that creased. In addition, methylation of arginine residues in under the conditions of the high local protein con- the RGG domain of Ddx4 increased the phase separation centration within the dense, liquid-like phase, new, threshold in vitro [48]. less dynamic interactions occur, perhaps between the Phosphorylation plays a crucial role in many signal low complexity prion-like domains. In time, these in- transduction pathways and also modulates the structural teractions may become dominant over the more dy- integrity and dynamics of membrane-less organelles. For namic, multivalent electrostatic interactions that give example, tyrosine phosphorylation of nephrin stimulates rise to the liquid-like state. We speculate that the balance the phase separation of the ternary system nephrin/ of the thermodynamic favorability of these two types of in- NCK/N-WASP [46]. Interestingly, a common feature of teractions may influence the physical nature of the phase certain well-characterized membrane-less organelles is separated state (i.e., liquid, hydrogel/solid) and determine that they incorporate kinases and phosphatases within the different propensities of wild-type and mutant proteins their matrixes [39, 78, 82]. Active phosphorylation/ to undergo the transition for the liquid-like to solid-like dephosphorylation cycles have been linked to regula- structural state. tion of organelle structural integrity. The activity of the nucleolar kinase CK2 controls the structural connect- Energy-dependent control of membrane-less organelle ivity between the GC and the DFC regions within the nu- dynamics cleolus [78] and increases the dynamics of NPM1 We have emphasized that the physical properties of exchange between the nucleolar and nucleoplasmic com- membrane-less organelles depend upon their protein partments [83]. Furthermore, phosphorylation of MEG-3 and RNA composition. In addition, however, the nucle- and MEG-4 proteins by MBK-2/DYRK kinase and de- PPTR-1/PPTR2 olus requires ATP in order to maintain its liquid-like phosphorylation by PP2A phosphatase regu- behavior, a physical state termed an “active liquid” [5]. It lates P-granule disassembly and assembly, respectively, is currently unclear what specific ATP-dependent pro- during mitosis in C. elegans in association with embryo- cesses are involved in maintaining this active liquid- genesis [39]. state. Furthermore, the activity of ATP-dependent chap- Assembly and disassembly of membrane-less organ- erones, such as Hsp70/Hsp40, which accumulate within elles provides a mechanism for controlling the concen- stress granules, is required for their disassembly upon tration and associated signaling behavior of freely recovery from stress [81]. These observations suggest diffusing molecules within the membrane-bounded Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 12 of 20 compartments of the cell. For example, the dynamic paraspeckles depends upon the concentrations of their properties of stress granules are coupled with mTORC1 constituent RNAs, which are controlled by the transcrip- signaling by immobilization of mTORC1 within the tional activity of RNA polymerases [2, 8], suggesting that granules, while phosphorylation-mediated dissolution of transcriptional control of RNA concentration may be a these organelles liberates mTORC1, activating down- general mechanism to tune the physical properties of stream signaling [82]. As another example, Wippich et al. membrane-less organelles (Fig. 1). [82], demonstrated that the kinase DYRK3 condenses in Many membrane-less organelles are involved in cellu- cytoplasmic granules via its low complexity N-terminal lar responses to various types of stress and the sensitivity domain, in a concentration dependent manner, and local- of their structural integrity to protein and RNA concen- izes to stress granules under osmotic and oxidative stress. trations provides a mechanism for rapidly responding to Inactive DYRK3 condensed into stress granules, together stress signals that affect these levels. For example, inhib- with components of the mTORC1 pathway. Activation of ition of Pol I-, II- and III-dependent RNA transcription DYRK3 and downstream phosphorylation of PRAS40, an by Actinomycin D was associated with re-organization mTORC1 inhibitor, results in dissolution of stress gran- of constituents of both nuclear and cytoplasmic ules and disruption of the inhibitory PRAS40/mTORC1 membrane-less organelles [59]. After Actinomycin D interaction. treatment, NPM1, a major component of the GC of the Further evidence for the role of posttranslational mod- nucleolus, becomes delocalized to the nucleoplasm and ifications in regulation of the features of membrane-less cytoplasm due to inhibition of RNA Pol I-dependent organelles is provided by the observation that the amino transcription of rRNA. Under these conditions, cytoplas- acids arginine, serine and tyrosine are overrepresented mic NPM1 was found to interact with components of in the low complexity sequences of proteins within stress granules, such as mRNA, and the proteins them. These amino acids can be posttranslationally hnRNPU and hnRNPA1 [84]. modified, arginines by methylation and serines and tyro- Also under conditions of Actinomycin D treatment, sines by phosphorylation, providing general mechanisms protein and RNA components associated with para- for modulating protein condensation thresholds and speckles, and PML and Cajal bodies, re-localize to nucle- consequently the signaling pathways downstream of olar caps. Interestingly, while proteins from the GC are components sequestered within the phase separated ejected from the nucleolus, proteins from the DFC, such fraction. as fibrillarin, re-localize to nucleolar caps [25]. These ob- servations suggest that environmental changes can alter Component concentration as a factor in membrane-less the equilibria that maintain the integrity of membrane- organelle assembly/disassembly less organelles, thereby altering the concentrations of Another important factor in phase separation-dependent their components in the freely diffusing pools of mac- formation of membrane-less organelles is the local romolecules within the nucleoplasm and cytoplasm concentration of components (Fig. 1). For example, and allowing their redistribution within various other regulation of P-granules during the oocyte-to-embryo organelles. transition, when they transit from the perinuclear region to the cytoplasm, is regulated by a concentration gradi- Emerging methods for the study of phase separated ent, which causes dissolution of the perinuclear droplets structures and re-condensation in the cytoplasm. A similar mech- Detailed analysis of the structural features of membrane- anism is employed during the asymmetric segregation of less organelles and their underlying macromolecular as- P-granules into the germline founder cell [6]. Recently, semblies presents challenges not encountered in other Brangwynne and colleagues demonstrated that the levels areas of structural biology. Interactions relevant to the of RNA in LAF-1 droplets, a minimalistic in vitro model phase separation phenomenon occur over multiple of P-granules, tunes the viscosity and molecular dynam- length scales, from sub-nanometer to tens of microme- ics within the liquid-like phase [47]. The viscoelastic ters, thereby making any single analytical technique properties of liquid-like droplets containing Whi3 are insufficient for the study of phase separated macromol- also modulated by RNA concentration. While Whi3 is ecular assemblies. For example, while liquid-like droplets able to phase separate in a unimolecular fashion under exceed the size limitations associated with analysis by certain conditions, the presence of RNA is required for NMR spectroscopy, the structural and dynamic features the process to occur at physiological salt concentrations. of flexible components within them have been character- Furthermore, an increase in the RNA concentration ized [53]. However, the dynamic features of these correlates with an increase in droplet viscosity and a systems are incompatible with X-ray crystallography. Al- decrease in Whi3 dynamics of recovery after photo- though the macromolecular assemblies formed are read- bleaching [55]. In addition, assembly of nucleoli and ily observable by conventional microscopy techniques, Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 13 of 20 the interactions responsible for assembly occur on dynamics that characterize IDPs are often an advantage length scales that are below the resolution limit of detec- for NMR studies because they cause resonance narrow- tion. Additionally, these systems are highly heteroge- ing and enhance detection. However, some IDPs experi- neous and therefore, integrative solutions that combine ence motions on time scales that cause resonance complementary methods are needed in order to under- broadening and can hamper NMR studies. Despite these stand their structural features. limitations, NMR has already been demonstrated to pro- vide unique insights into the conformational and dynamic Atomic-resolution structure determination methods features of phase separation-prone IDPs both before and Several studies utilizing classical structural methods, in- after phase separation; several exemplary studies are dis- cluding solution NMR [46, 48, 49, 67–69] and X-ray cussed below under “Integrative approaches to understand crystallography [19, 70], have provided detailed insights the molecular basis of phase separation”. into the molecular interactions that mediate the network structure that drives phase separation of modular pro- Methods to study molecular interactions associated with teins within membrane-less organelles. However, due to phase separation technological limitations, these studies were performed Classical methods for characterization of biomolecular with truncated forms of the proteins and nucleic acids interactions, such as ITC [49] and SPR [68, 69], have corresponding to individual interaction modules. These been employed to characterize the wide range of binding traditional methods will be useful in the future for deter- affinities associated with the different types of interac- mining the structural basis of interactions between tions that occur within liquid-like droplets and/or folded domains within multi-domain phase separation- membrane-less organelles. NMR can also be used to prone proteins and their interaction partners, including characterize macromolecular interactions and is particu- peptides corresponding to short linear motifs and seg- larly well suited in studies of weak interactions that ments of RNA. However, because many phase separation- present challenges for other methods. For example, prone proteins exhibit low complexity and disordered chemical shift perturbations observed during titrations sequence features, these methods for determining discrete of an unlabeled binding partner into an isotope-labeled protein structure are likely to receive limited application protein can be quantitatively analyzed to report residue- in this emerging field. specific and global K values for interactions associated with phase separation [NPM1 integrates within the NMR spectroscopy; a versatile tool in studies of phase nucleolus via multi-modal interactions with proteins separation-prone proteins displaying R-rich linear motifs and rRNA: Mitrea DM, NMR spectroscopy offers unique capabilities in studies et al., under review]. However, the multivalent features of disordered proteins, by providing insights into confor- of phase separation-prone proteins can give rise to com- mations and dynamics of individual amino acids plex, multi-step interaction mechanisms, which compli- throughout the polypeptide chain. Measurements of cate the analysis of data from the methods discussed chemical shift values for nuclei of backbone atoms re- above. Therefore, experiments are often performed with port on secondary structure propensities and dynamics truncated macromolecules of reduced multivalency and can be probed on ps to ns, and μs to ms timescales using therefore do not address interactions under the conditions a variety of relaxation methods [85]. Furthermore, long- of phase separation. Despite these limitations, these bio- range structure within disordered proteins can be stud- physical methods provide important insights into the ied using paramagnetic relaxation enhancement (PRE) binding features of the individual elements within multi- methods and through the measurement of residual di- valent macromolecules that undergo phase separation. polar couplings [86]. The former method, however, re- quires that proteins be engineered to include single Scattering methods to probe structural features before and cysteine residues for labeling with a paramagnetic probe. after phase separation A limitation of these NMR approaches is that rapid con- Dynamic light scattering and small angle X-ray scatter- formational fluctuations of disordered polypeptides ing (SAXS) [19, 46] have been employed to gain insight causes ensemble averaging of NMR parameters. A sec- into the overall size and shape of the macromolecular ond limitation is that the structural and dynamic infor- assemblies. In particular, SAXS has been used to mation gained reports on the features of individual sites characterize the shapes (e.g., radius of gyration) of en- within a protein on a very limited length scale (Å or tens sembles of disordered proteins [88]. However, scattering of Å in the case of PRE measurements). An exception is methods can also detect long-range order within so- the use of pulsed field gradient methods to study protein called soft materials and uniquely provide insights into diffusion [87] but this has not yet been used in studies the structural makeup of these materials. Small-angle of proteins within liquid-like droplets. The extensive neutron scattering (SANS) has previously been employed Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 14 of 20 in the structural analysis of polymer blends [89–91] and High resolution and single-molecule microscopy polymeric soft nanomaterials [92] and has great potential Electron microscopy can extend into the length scale in studies of membrane-less organelles to provide infor- gap between the two sets of techniques described above mation about the spatial organization of macromolecules and has been extensively utilized to study cellular ultra- within the condensed state. One recent study used SANS structure [1]. A significant limitation of this technique is to characterize the regular spacing of molecules within the low certainty with which specific molecules can be droplets comprised of the nucleolar protein, nucleophos- identified based upon the greyscale contrast of images min (NPM1), and a peptide derived from the ribosomal [96]. The emerging field of correlated light and electron protein, rpL5, on length scales from 5.5 to 11.9 nm microscopy (CLEM; reviewed in [96]) presents the op- [NPM1 integrates within the nucleolus via multi-modal portunity of directly connecting dynamic information interactions with proteins displaying R-rich linear motifs obtained via live fluorescence microscopy methods with and rRNA: Mitrea DM, et al., under review]. SANS has ultrastructural detail acquired by electron microscopy. the advantage of allowing detection of scattering from Significant advances were made in the last decade in specific components within heterogenous, phase sepa- super resolution microscopy methods (reviewed in [97]) rated states through selective protonation and/or deu- and were successfully applied to decipher chromosomal teration and solvent contrast matching [93]. architecture [98]. Lattice sheet microscopy coupled with Furthermore, time-resolved SANS has been used in structured illumination microscopy, a method that the past in studies of mutant huntingtin exon 1 phase returns 3D images with resolution ~ 200 nm x 200 nm separation into amyloid fibers to determine the mech- in the x/z plane that exceeds the diffraction limit, was anism of macromolecular assembly and the geometry applied to study the ultrastructural organization of germ of monomer packing within the fibrils [94]. We envision granules in C. elegans [39]. The internal structure ob- that SAXS and SANS may be able to reveal the spacing of served in several membrane-less organelles suggests that partially ordered macromolecules within the liquid-like the condensed macromolecules are not homogenously structure of droplets prepared in vitro and possibly within distributed, but further partition into phase separated membrane-less organelles if technical issues associated fractions with distinct physical properties. These methods with sample preparation can be addressed. We envision provide opportunities to reveal the heterogenous ultra- that these scattering methods will be powerful tools in the structure of membrane-less organelles in the future. characterization of biological structures that arise from Single-molecule fluorescence microscopy holds great phase separation in the future. potential in the analysis of proteins within liquid-like droplets in vitro and membrane-less organelles in cells. For example, single-molecule fluorescence correlation Light microscopy spectroscopy (FCS) [99] and Förster resonance energy Light microscopy methods (reviewed in [95]) have been ex- transfer (smFRET) [100] have been used to study the tensively utilized to visualize the subcellular localization of structural and dynamic features of aggregation-prone fluorescently tagged molecules. Live imaging coupled with intrinsically disordered proteins in vitro (reviewed in fluorescence recovery after photobleaching (FRAP) or [101]). In addition, single-molecule FRET and other fluorescence loss in photobleaching (FLIP) methods probe methods have been applied to a wide range of disordered the dynamics of macromolecules within membrane-less or- proteins with varied charged residue compositions ganelles inside living cells [7, 46, 48, 77] and phase sepa- and distributions (reviewed in [102]). We envision rated states reconstituted in vitro [46–48, 50]. that these methods will be applied in the future to The information obtained from structural biology disordered proteins within liquid-like droplets to re- −10 −9 methods is on length scales of 10 –10 m, while the veal their structural and dynamics features. Further- classical light microscopy techniques provide informa- more, smFRET and fluorescence lifetime imaging have −7 −3 tion on much greater length scales, from 10 to 10 m. revealed the conformational features of a disordered This situation creates a gap corresponding to two orders protein within HeLa cells [103], providing opportunities of magnitude on the length scale in our understanding in the future for studies of phase-separation-prone pro- of the structural and dynamic features of micron-sized teins within membrane-less organelles in their natural cel- membrane-less organelles. Macromolecular interactions lular setting. that occur on the length scale of this gap are responsible for the structural organization that gives rise to phase Additional physical characterization methods separation and the liquid-like and/or gel-like properties Density [1], viscosity [5, 6, 47] and stiffness [79] are a few of membrane-less organelles and related structures. We of the physical properties that have been measured for next discuss structural methods that can peer into this bona fide membrane-less organelles or in vitro reconsti- length scale gap. tuted liquid droplets. Interferometer microscopy was Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 15 of 20 utilized to measure the density of nuclear membrane- ensembles—the so-called “sample-and-select” methods less organelles in isolated Xenopus laevis germinal ves- [88, 119–121]. Complementary computational methods icles, oocyte nuclei [1]. This method provided important have been developed for generating IDP ensembles based insights into the physical properties of refractory sub- on SAXS data [122]. The development of physically accur- cellular bodies in a quasi-natural environment. A few con- ate molecular ensembles with atomistic detail for IDPs is siderations when interpreting these data, however, are that important because, with the exception of single-molecule the results are based on the simplified assumptions that fluorescence methods, the experimental methods used to the organelles are spherical in shape and are exclusively characterize IDPs are subject to ensemble averaging. composed of homogenously mixed water, proteins and Therefore, computationally generated ensemble models of low molecular weight solutes [1]. IDPs enable examination of the features of large numbers Atomic force microscopy provides the advantage of of individual molecules. However, these approaches are performing surface scans of membrane-less organelles only beginning to be applied to proteins that undergo which produce topological maps with resolution in the phase separation. nanometer range. Also, this method provides a means to A key challenge in computational studies of phase measure other key biophysical properties, such as struc- separation-prone proteins is to gain insight into the tural stiffness, as done for nucleoli [79]. inter-molecular interactions that are the basis for self- Microrheology methods, traditionally used in the association and phase separation. Regarding this goal, characterization of viscoelastic properties of polymers and the field is in its infancy. However, methodologies complex fluids [104], were applied to the characterization applied to understand protein aggregation and fibril for- of membrane-less organelles [5, 6, 42, 105] and in vitro mation can be leveraged to understand the types of in- formed protein and protein-RNA liquid droplets [47, 55]. teractions that drive protein phase separation and In particular, the tracer bead technology provided import- possibly, in the future, protein-nucleic acid phase separ- ant insights into the effect of RNA onto the viscoelastic ation. In the protein aggregation field, course-grained properties of in vitro liquid droplets [47, 55]. computational methods have been applied to understand the aggregation of poly-glutamine tracts associated with Computational and theoretical approaches Huntington's disease [123] and atomistic methods to As we gain greater knowledge of the types of macromol- understand aggregation of amyloid β [124]. Clearly, in- ecules that undergo phase separation to form liquid-like creased effort in this area is needed to understand the structures both in vitro and in cells, computational molecular basis for phase separation. models are needed to analyze the structural and dynamic While computational approaches face challenges in features, encoded by their amino acid sequences, so as addressing the protein phase separation problem, signifi- to understand their phase separation behavior. A large cant progress has been made in recent years in under- proportion of the proteins, or protein regions, shown to standing relationships between the sequence features of undergo phase separation are intrinsically disordered, IDPs and IDRs and the general conformational features which presents a variety of computational challenges, of IDP ensembles [125–127]. Results from NMR, single- notably conformational sampling and physical accuracy. molecule fluorescence and computational approaches A wide variety of methods are used to address the need have shown that the charge features of IDPs influence to sample the extensive conformational space explored the shape of their dynamic ensembles. Pappu and co- by IDPs/IDRs, including molecular dynamics methods, workers have extended these finding using both compu- often enhanced by approaches such as replica exchange tational and experimental methods to show that not only and related methods [106, 107], and Monte Carlo sam- the faction of charged residues and net charge per resi- pling methods [108, 109]. Many different force fields due within IDPs and IDRs influence their overall con- and variants thereof are available [110–112] and several formational features, but also the distribution of were recently tested and compared [113]. Computations oppositely charged residues within sequences signifi- are often performed without experimental restraints and cantly influences the compaction of IDP ensembles therefore they are reliant on the underlying force fields [128]. These advances have led to the development of a for generation of physically accurate molecular ensem- novel phase diagram based upon net positive and nega- bles. A problem in the past was that computational tive charge per residue values for the classification of models of IDPs were overly compact [114] but this IDP and IDR sequences [129]. These developments pro- issue is being addressed through the method refine- vide a conceptual framework for establishing relation- ment [112, 115–117] and consideration of NMR, SAXS ships between the charge features of IDPs and IDRs, and smFRET data [110, 113, 118]. Another group of ap- their conformational features and their propensities for proaches utilize experimental restraints (e.g., NMR and/or phase separation. Charge features are certainly im- SAXS data) to select conformers for inclusion within IDP portant factors governing protein phase separation Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 16 of 20 behavior; for example, arginine residues are prevalent not related to phase separation per se, this study pro- in low complexity regions known to form liquid-like vides an explanation for how Importin β-bound cargo droplets in vitro and within protein components of can rapidly diffuse through the condensed phase within membrane-less organelles [44, 47]. However, these se- the core of the nuclear pore complex, which is com- quences are often enriched in aromatic and other prised of several FG-Nup proteins, including Nup153. neutral amino acids, indicating that, while electrostatic in- NMR spectroscopy was used to understand the ensem- teractions may play important roles in some cases, other ble averaged conformational and dynamic features of types of molecular interactions are at play in other cases backbone amide groups within disordered Nup153 in [48, 50, 53]. This was born out in a recent study by García the absence and presence of Importin-β and to generate Quiroz and Chilkoti [130] in which they identified the se- a conformational ensemble using the sample-and-select quence features of designed proteins that can undergo approach. This ensemble was validated by back- phase separation due to either a temperature increase calculation of the X-ray scattering profile and compari- (termed LCST sequences) or decrease (termed UCST son with experimental SAXS data, an illustration of sequences). The LCST sequences were enriched in hydro- spanning length scales from amino acids to a whole dis- phobic residues while the UCST sequences were enriched ordered protein. To complement this information, data in charges residues [131]. This study, which involved the- from smFRET and fluorescence lifetime measurements oretical considerations as well as in vitro experimental were used to understand the conformational features of measurements, serves as a model for future studies into many individual molecules under the same conditions the physical basis for phase separation of the growing list while fluorescence correlation spectroscopy was used to of proteins and RNA molecules shown to partition into compare molecular diffusion properties of Nup153 with- the liquid-like or gel-like phase of membrane-less organ- out and with Importin β. Additionally, molecular dy- elles and other cellular bodies. namics and Brownian dynamics computational methods were used to relate insights from the aforementioned Integrative approaches to understand the molecular basis biophysical methods to the mechanism of Nup153/ of phase separation Importin β interaction at atomistic resolution. Finally, None of the individual methods or approaches discussed these various pieces of molecular data were related to above will alone uncover the molecular basis for phase the Importin-β-dependent transport through the NPCs separation by proteins and protein-nucleic acids mix- in live cells using bulk and single-particle fluorescence tures; therefore, there is a need to apply multiple, com- tracking. plementary methods and to integrate results to advance Another example is provided by a recent study of the mechanistic understanding. Integration is needed to ALS-associated protein, FUS, from Fawzi and co- span the broad length scales relevant to membrane-less workers that employed NMR and various fluorescence organelles, ranging from the atomic scale (units of Å) microscopy methods to study the molecular features of relevant to amino acid conformations and their inter- FUS within in vitro liquid-like droplets and its interac- molecular interactions to the overall size of in vitro tions with RNA and the C-terminal domain of RNA Pol liquid-like droplets and cellular membrane-less organ- II. A final example is provided by a recent study of the elles (units of micrometers). Integration is also needed highly abundant nucleolar protein, NPM1, which was across the broad range of relevant time scales, including shown to phase separate into liquid-like droplets with motions of amino acids and their polypeptide chains that other nucleolar proteins and ribosomal RNA [NPM1 in- mediate their conformational heterogeneity and inter- tegrates within the nucleolus via multi-modal interac- molecular interactions on the ns to μs time scale, to the tions with proteins displaying R-rich linear motifs and diffusion of macromoclecules into and out of, and rRNA: Mitrea DM, et al., under review]. NMR, smFRET, within, liquid-like structures on the timescale of seconds and SANS were used to understand the conformational to tens of seconds. A key challenge is to understand the and dynamic features of NPM1 before and after phase relationships between conformational features and mo- separation with a peptide derived from the ribosomal pro- tions of amino acids at the atomic scale and the macro- tein, rpL5, and revealed molecular organization extending scopic properties of these structures (e.g., viscosity, to ~10 nm within liquid-like droplets. In addition, deletion surface tension, macromolecular diffusion rates, etc.). analyses identified the domains of NPM1 required for A few studies have begun to address the challenges as- phase separation in vitro and for localization within nucle- sociated with spanning these broad length and time oli in cells. scales. For example, a recent report addressed the con- The three studies discussed above illustrate approaches formational features of the FG-Nup protein, Nup153, to relate the molecular features of phase-separation-prone and how these features mediate ultra-fast interactions proteins studied with atomic resolution to the macro- the nuclear transport receptor, Importin β [132]. While scopic features of the liquid-like structures that they form. Mitrea and Kriwacki Cell Communication and Signaling (2016) 14:1 Page 17 of 20 Importantly, two of the studies also integrated results membrane-less organelles to modulate their signaling be- from cellular assays, allowing molecular features to be re- havior are on the horizon. lated to biological function. We are just beginning to Abbreviations understand the physical properties of phase separation- mRNP: messenger ribonucleoprotein; snRNP: small nuclear ribonucleoprotein; prone proteins that are associated with their localization snoRNP: small nucleolar ribonucleoprotein; TEM: transmission electron within membrane-less organelles and eagerly await the re- microscopy; FC: fibrillar centers; DFC: dense fibrillar component; GC: granular component; rRNA: ribosomal RNA; rDNA: ribosomal DNA; RNA Pol I/II: RNA sults of similarly adventurous integrative studies to polymerase I/II; NOR: nucleolar organizing region; SAXS: small angle X-ray broaden our knowledge of these features and, importantly, scattering; SANS: small angle neutron scattering; FRAP: fluorescence recovery how they contribute to the diverse biological processes after photobleaching; FLIP: fluorescence loss in photobleaching; smFRET: single molecule Förster resonance energy transfer. that occur within liquid-like cellular bodies. Competing interests Conclusions The authors declare no competing interests. The compartmentalization of macromolecules within living cells creates heterogenous functional assemblies Authors’ contributions that mediate diverse biological processes. Membrane- DMM and RWK conceived and wrote the manuscript. Both authors read and approved the manuscript. less organelle assembly follows the physical laws of poly- mer condensation and depends upon factors such as Acknowledgements component concentration and temperature (Fig. 1). Con- We apologize to the colleagues whose work, although valuable to the field, densation is triggered by specific, initiating interactions was not mentioned in this manuscript, due to size limitations. The authors thank Chris Stanley of Oak Ridge National Laboratory for helpful comments between multivalent macromolecules and is further ex- on the manuscript. This work was supported by NIH 1R01GM115634 tended by recruitment of additional protein or RNA (to R.W.K.), National Cancer Institute Cancer Center Support Grant P30CA21765 molecules via monovalent or multivalent interactions (to St. Jude Children’s Research Hospital) and ALSAC (to St. Jude Children’s Research Hospital). (Fig. 2). The complex composition of the intra-organelle matrix arises and is maintained by weak, multivalent in- Received: 29 July 2015 Accepted: 29 December 2015 teractions between modular proteins and RNA. Condensation through phase separation of specific pro- teins and nucleic acids into dense liquid- or gel-like struc- References 1. Handwerger KE, Cordero JA, Gall JG. 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Phase separation in biology; functional organization of a higher order

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