Condensed matter biology: from phase separation to percolated network.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization. https://doi.org/10.7554/eLife.48562.001 Introduction The appropriate spatiotemporal regulation of DNA replication is essential to genetic integrity and cell proliferation. In eukaryotes, the initiation of DNA replication requires the coordinated action of three proteinaceous factors – the Origin Recognition Complex (ORC), Cdc6, and Cdt1 – which co-assemble on DNA origins in the G1 phase of the cell cycle to catalyze loading of the Mcm2-7 replicative helicase onto chromatin. Once activated during the transition to S phase, Mcm2-7 helps promote origin melting, replisome assembly, and translocation of the replication fork. ORC consists of a heterohexameric complex comprising the subunits Orc1-6. Five ORC subunits (Orc1-5), as well as Cdc6 and the six subunits of Mcm2-7, possess an ATPases Associated with diverse cellular Activities (AAA+) domain; Orc6 and Cdt1 are the only non-AAA+ proteins used for Mcm2-7 loading. Despite sharing a high overall degree of conservation across eukaryotes, certain aspects of initiator subunit sequence and function have nonetheless diversified during evolution. For example, S. pombe Orc4 contains a unique domain not found in other ORCs that endows the protein with a preference for A/T rich regions of DNA (Chuang and Kelly, 1999; Lee et al., 2001). Conversely, S. cerevisiae ORC is able to recognize specific origin sequences (Bell and Stillman, 1992; Li et al., 2018), whereas origin specification for metazoan ORCs appears more contextual (Remus et al., 2004; Vashee et al., 2003). S. cerevisiae Cdt1 additionally possesses a catalytically-inactive dioxygenase domain at its N-terminus that is necessary for yeast viability and Mcm2-7 loading (Frigola et al., 2017; Takara and Bell, 2011); this fold is absent in S. pombe and metazoan Cdt1s. Much of our current understanding of initiator mechanism derives from reconstitution studies using budding yeast initiation factors. These efforts have helped define an orchestrated set of ATP-dependent molecular exchanges between ORC, Cdc6, and Cdt1 that culminate in the loading of two copies of Mcm2-7 onto origin DNA in the form of a stable double-hexamer (Duzdevich et al., 2015; Evrin et al., 2009; Remus et al., 2009; Ticau et al., 2015). One aspect of initiation that has been difficult to probe in vitro, however, is the potential for interactions between initiation factors associated with different origins. Evidence for mesoscale coordination of origin activity derives from multiple sources. For example, genomic studies aimed at defining sites of ORC binding and their relation to replication origins have revealed evidence of origin clustering across chromosomes at nucleosome-free regions (Cayrou et al., 2011; Miotto et al., 2016; Vaughn et al., 1990). Cellular patterns of ORC localization are also strikingly non-uniform, often presenting as concentrated foci on chromatin whether it be in D. melanogaster follicle cells (Austin et al., 1999) or human cells in tissue culture (Lidonnici et al., 2004; Prasanth et al., 2010; Shen et al., 2010). Outside of initiation, there exists a well-characterized phenomenon of origin interference that has been taken as evidence of inter-origin communication (Cayrou et al., 2011); the clustering of co-replicating regions into so-called Replication Factories or replication domains (RD) similarly has been observed in both fixed and live cells (Cook, 1999; Xiang et al., 2018). Finally, the replication timing profile of origins, which fire asynchronously throughout S-phase, partition to specific chromosome territories (Cremer and Cremer, 2001; Gilbert et al., 2005; Pope et al., 2014). It is currently unclear whether the clustering patterns observed for ORC binding sites, origin communication, and the timing of replication domains is coincidence or dependent upon an as-yet-discovered set of physiological properties of replication factors. Present-day views of mesoscale organization within cells typically invoke processes such as protein gradients or membrane compartmentalization. Recently, a rapidly expanding body of work has begun to recognize protein/protein and protein/RNA liquid–liquid phase separation (LLPS) as playing critical functions in generating membraneless pseudo-organelles and co-localized bodies (Boeynaems et al., 2018). Both nuclear and cytoplasmic liquid phase condensates (also known as biomolecular condensates) have been observed and implicated in a panoply of functions, including cellular signaling (Li et al., 2012; Su et al., 2016), centrosome assembly (Woodruff et al., 2017), and chromatin/heterochromatin assembly and maintenance (Larson et al., 2017; Strom et al., 2017; reviewed in Maeshima et al., 2016). A functional role for biological condensates is only beginning to emerge; such entities may help sequester and/or co-localize certain factors to modulate biochemical output and response (Shin and Brangwynne, 2017). Intrinsically disordered amino acid regions (IDRs) are often found in proteins that phase separate and can underpin multivalent interactions that help drive phase separation (Li et al., 2012). Many phase-separating IDRs are additionally enriched for certain amino acids and are hence referred to as low-complexity domains (LCD) (Hennig et al., 2015; Kato et al., 2012). Although multiple nuclear events are now being scrutinized through the lens of phase separation, how these processes integrate and communicate with other LLPS or dispersive-state pathways is currently unknown. Here we report that the Drosophila melanogaster replication initiation factors ORC, Cdc6, and Cdt1 possess N-terminal IDRs that facilitate DNA-dependent liquid-liquid phase separation in vitro. Bioinformatic analyses reveal that these initiator IDRs possess a high-complexity sequence signature that is preserved in metazoan homologs, but that is distinct from other condensate-promoting IDRs in other cellular pathways. Biochemical studies show that these IDRs drive the selective co-assembly and enrichment of Drosophila initiation factors into liquid phases in the presence of DNA, while simultaneously excluding non-partner proteins that also phase separate. Although Drosophila Mcm2-7 does not appear to phase separate on its own, initiation factors can recruit the complex into condensates in a DNA-, ATP-, and ORC/Cdc6/Cdt1-dependent manner. In addition, cellular and genetic studies establish that the Orc1 IDR is critical for its recruitment to mitotic chromosomes and is essential for viability. Collectively, our observations not only reveal a new class of high-complexity sequences that undergo phase separation, but also provide a model for how these elements promote physical interactions between replication initiation factors that could help promote long-range chromosomal communication observed in replication programs. Results D. melanogaster Cdt1 undergoes DNA-dependent liquid-liquid phase separation (LLPS) The architecture of D. melanogaster Cdt1 (DmCdt1) is markedly different from its budding yeast counterpart (Figure 1A). Sequence analysis with the disorder prediction server DISOPRED (Jones and Cozzetto, 2015) reveals that the N-terminal sequence of DmCdt1 is predicted to be an extended intrinsically disordered region (IDR) (Figure 1A). Although the sequence of this region is not conserved per se, an N-terminal IDR is present in other metazoan Cdt1s (Table 1). Figure 1 with 1 supplement see all Download asset Open asset D. melanogaster Cdt1 undergoes DNA-dependent phase separation. (A) Architecture of D. melanogaster and S. cerevisiae Cdt1. The per-residue DISOPRED (Jones and Cozzetto, 2015) disorder prediction score is shown in the plot below each gene, with a cutoff value of 0.5 indicated by the dashed line. Residues scored above this cutoff are predicted to be disordered. (B) dsDNA-coupled (‘DNA Beads’) and control (‘Ctrl Beads’) agarose beads were used to pull-down DmCdt1 and ScCdt1. DmCdt1 but not ScCdt1 bound to the DNA-coupled beads. (C) Fluorescence anisotropy measurements of Cdt1 binding to a Cy5-labeled duplex oligonucleotide. ScCdt1 showed no evidence of binding. DmCdt1 bound with a Kd, app = 83 ± 17 nM. (D) EMSA analysis of DmCdt1 binding to duplex DNA. The complex between DNA and DmCdt1 is heterogeneous and large, and is fully well-shifted at the highest concentrations. In good agreement with anisotropy measurements, the calculated Kd, app for DmCdt1 is ~100 nM. (E) Mixing concentrated DmCdt1 with duplex DNA results in a visible increase in solution turbidity that can be reversed with the addition of KCl. (F) DIC microscopy analysis of solutions of Cdt1, DNA, and a Cdt1/DNA mixture (scale bar = 5 µm). Phase-separated droplets were evident when Cdt1 was mixed with DNA. Gel images are representative from three independent experiments. https://doi.org/10.7554/eLife.48562.002 Table 1 Conservation of initiator IDR sequence features. Analysis of eukaryotic initiator homologs for the presence of an N-terminal IDR, as well as IDR sequence features (‘pI’=isoelectric point; ‘FCR’=fraction charged residues). https://doi.org/10.7554/eLife.48562.004 Orc1Cdc6Cdt1D. melanogaster N-term IDR length: pI: FCR:362 aa 10.2 0.32246 aa 9.4 0.35294 aa 10.1 0.32Human N-term IDR length: pI: FCR:300 aa 10.7 0.33136 aa 10.6 0.29175 aa 10.6 0.29Mouse N-term IDR length: pI: FCR:298 aa 10.2 0.34141 aa 10.0 0.31178 aa 9.8 0.24X. laevis N-term IDR length: pI: FCR:327 aa 9.7 0.33140 aa 10.8 0.26248 aa 10.1 0.31C. elegans N-term IDR length: pI: FCR:242 aa 9.1 0.39172 aa 9.9 0.38194 aa 10.3 0.35D. rerio N-term IDR length: pI: FCR:391 aa 9.8 0.31156 aa 11.1 0.22309 aa 9.9 0.31S. cerevisiae N-term IDR length: pI: FCR:143 aa 4.7 0.4931 aa 6.2 0.32N/A N/A N/AS. pombe N-term IDR length: pI: FCR:117 aa 10.6 0.36133 aa 10.2 0.22N/A N/A N/A The N-terminal IDR of metazoan Cdt1 contains multiple conserved short linear motifs (SLiMs) (Davey et al., 2012) necessary for Cdt1 regulation, such as a PCNA interacting peptide (PIP) box and kinase consensus sequences (Pozo and Cook, 2016). However, known SLiMs only account for a small fraction (<25%) of the total length of the metazoan Cdt1 N-terminal IDR, so we asked whether this domain might have more general functional significance. The IDR of DmCdt1 has a predicted pI that is relatively basic (pI = 10.2); this feature, combined with the presence of tandem WH domains in the protein, suggested that DmCdt1 might bind DNA. We found that agarose beads coupled with a random 60 bp double-stranded DNA (dsDNA) were able to efficiently pull down DmCdt1, whereas no such interaction was observed for ScCdt1 (Figure 1B). Quantitative analysis of dsDNA binding using a fluorescence polarization (FP)-based assay also revealed DNA binding by DmCdt1 (Kd, app = 83 ± 17 nM) but not ScCdt1 (Figure 1C). Mouse and S. pombe Cdt1 have similarly been shown to bind DNA in vitro (Houchens et al., 2008; Yanagi et al., 2002). Electrophoretic mobility assays confirmed DNA binding by DmCdt1 (Kd, app ≈ 100 nM) but also showed that the complex is highly heterogenous (Figure 1D). Indeed, elevated concentrations of the protein (>190 nM) resulted in a well-shifted species, suggestive of increasingly higher-order assemblies; however, size-exclusion chromatography showed that purified DmCdt1 is monodisperse in solution (Figure 1—figure supplement 1A). Interestingly, in the course of conducting these studies, we found that upon mixing 20 µM DmCdt1 with stoichiometric amounts of a 60 bp duplex oligo, the solution became visibly turbid, and that this turbidity was reversible by the addition of salt (400 mM KCl) (Figure 1E). Inspection of the turbid solution by differential interference contrast (DIC) microscopy unexpectedly revealed the presence of phase-separated droplets up to 4 µm in diameter (Figure 1F). These droplets were fully absent from the DNA alone sample and barely detectable in both number and size in the DmCdt1 alone sample. Notably, Cdt1 phase separation was dependent on the length, but not the sequence, of the dsDNA substrate, with maximal LLPS occurring in the presence of oligonucleotides longer than 25 basepairs (Figure 1—figure supplement 1B–C). To confirm that the affinity determined for Cdt1 in binding DNA was not confounded by the phase transition event, we determined whether a rapid binding/exchange equilibrium was maintained in the FP DNA-binding assay using a competition assay that titrated ‘cold’ dsDNA against pre-formed Cdt1/DNA complexes (Figure 1—figure supplement 1D). The unlabeled oligonucleotide was able to fully reduce DNA binding by Cdt1 to background levels, with an inhibition constant (Kc, app = 62 ± 4 nM) comparable to the observed Kd, app (83 nM), demonstrating that the labeled DNA in droplets is freely exchangeable and therefore in equilibrium between bound and unbound states. Together, these data demonstrate not only that DmCdt1 binds DNA, but that DNA-binding in turn induces the protein to phase separate. DNA-dependent phase separation of DmCdt1 requires the N-terminal IDR and occurs at physiologic concentrations The DNA-dependency of Cdt1 phase separation predicts that droplets should be enriched for both protein and nucleic acid. To test this prediction, DmCdt1 was expressed and purified with an N-terminal enhanced Green Fluorescent Protein (eGFP) tag (eGFP-Cdt1) (Figure 2A). A Cy5-labeled duplex oligonucleotide (Cy5-dsDNA) was then mixed with an equimolar amount of protein (5:5 µM) and imaged by two-color fluorescence imaging (Figure 2B). When eGFP-Cdt1 was mixed with Cy5-dsDNA, droplets up to 3 µm in diameter appeared, all containing both protein and nucleic acid. Samples with eGFP-Cdt1 alone did not show such droplets, nor were they observed for samples with Cy5-dsDNA only. These data demonstrate that phase separation by Cdt1 is driven, at least in part, by dsDNA-induced coacervation, and further show that dye-labeled oligonucleotides can serve as a proxy for labeled protein when assessing DNA-dependent protein phase separation by fluorescence microscopy. Figure 2 with 1 supplement see all Download asset Open asset DmCdt1 phase separation is facilitated by an N-terminal IDR. (A) SDS-PAGE analysis and Coomassie stain of purified eGFP-Cdt1. (B) Samples containing eGFP-Cdt1, Cy5-dsDNA (60 bp), and a mixture of eGFP-Cdt1 and Cy5-dsDNA (‘Mixed’) were prepared and analyzed by two-color fluorescent microscopy. Droplets were observed in the Mixed sample enriched for both protein (green) and nucleic acid (red). (C) Cy5-dsDNA was imaged alone or mixed with either wild-type Cdt1 (WT) or a Cdt1 construct lacking the N-terminal IDR (∆IDR). Only WT Cdt1 could induce droplet formation. (D) Schematic of a condensate depletion assay, a method for assessing phase separation (see Materials and methods for details). (E) The depletion assay was utilized to assess the role of the Cdt1 IDR in phase separation. DNA-induced the depletion of WT Cdt1 but not ∆IDR. (F) Phase diagram for Cdt1 in the presence of equimolar amounts of sixty basepair dsDNA (filled markers = phase separation observed, unfilled markers = phase separation not observed). (G) Depletion assay to assess phase separation at sub-physiological concentrations of Cdt1. DNA-induced phase separation of Cdt1 is seen at the lowest concentration tested (50 nM). Gel images are representative of three independent experiments. https://doi.org/10.7554/eLife.48562.005 Given the known importance of protein IDRs in facilitating phase separation (Mitrea and Kriwacki, 2016), we hypothesized that the IDR of Cdt1 might likewise be critical for the condensates we observed. We therefore purified an N-terminal IDR deletion of Cdt1 (Cdt1ΔIDR) and determined whether, upon mixing with DNA, a condensed phase could form (Figure 2C). No droplets were observed when Cdt1ΔIDR was mixed with Cy5-dsDNA, even at elevated levels (10 µM protein, 10 µM DNA). To confirm these data with unlabeled DNA, we devised a simple depletion assay (Figure 2D). In this approach, protein is incubated with or without DNA, after which the denser, phase-separated material is pelleted by centrifugation, and the degree to which protein is depleted from the supernatant is assessed by SDS-PAGE. In agreement with the microscopy data, no depletion of full-length Cdt1 was observed relative to the load control in the absence of DNA (Figure 2E), indicating that no phase separation occurred. Moreover, the full-length Cdt1 signal was fully lost from the supernatant in the presence of DNA, indicating that a near complete partitioning of Cdt1 into the condensed (pelleted) phase took place. When the DmCdt1 IDR was removed, the protein was again fully retained in the supernatant, regardless of whether DNA was present or not. Notably, a construct containing only the Cdt1 N-terminal IDR residues (Cdt1IDR) bound DNA (Kd, app = 158 ± 32 nM) and underwent DNA-dependent phase separation on its own (Figure 2—figure supplement 1A–C), demonstrating that the Cdt1 IDR is necessary and sufficient for and that it with yeast Cdt1, which has an IDR (Table nor binds DNA (Figure did not phase separation in vitro (Figure 2—figure supplement However, all other metazoan Cdt1 homologs possess an N-terminal with human Cdt1 revealed to DmCdt1, it also can undergo DNA-induced liquid phase (Figure 2—figure supplement Collectively, these data demonstrate the essential role of metazoan Cdt1 IDRs in facilitating phase separation. replication in the D. melanogaster Cdt1 concentration at (Figure 2—figure supplement Given the concentration of Cdt1, it was unclear whether our biochemical which were at protein Cdt1 in To this we used fluorescence microscopy to a phase diagram for DmCdt1, protein and salt and for the presence or absence of a condensed phase (Figure Droplets were observed down to the lowest concentration of Cdt1 tested nM) when salt concentrations were set at or below physiological levels salt concentration to physiological levels the concentration of protein necessary to see phase separation to nM. When the concentration of was to no phase separation was observed for concentration of Cdt1 The of Cdt1 to phase separate at concentrations was confirmed by the depletion assay, stoichiometric of DmCdt1 and duplex DNA were prepared from to and assessed for DNA-dependent depletion of protein in the supernatant (Figure DNA-coupled of Cdt1 signal was seen at all concentrations Together, these data demonstrate that DmCdt1 undergoes DNA-dependent phase separation at physiological protein and salt concentrations. ORC and Cdc6 also partition into DNA-dependent liquid phases Given the role of the DmCdt1 N-terminal IDR in phase separation, we whether other replication initiation factors might possess disordered regions of DISOPRED (Jones and Cozzetto, we calculated the of predicted disordered as well as the disordered for each Drosophila protein for and to predicted or high disorder (Figure In addition to Cdt1, Cdc6 and two subunits of ORC and were found to possess a high of disordered as a of their total length, and each a region of disorder longer than amino Conversely, the Mcm2-7 contain than predicted disordered sequence with no disordered region amino patterns were seen for other metazoan replication initiation but not S. cerevisiae proteins (Table 1). Figure 3 with 2 see all Download asset Open asset and undergo DNA-dependent phase separation. (A) of the disorder for each Drosophila replication initiation Cdc6, and Cdt1 each contain IDRs by the each and a high of overall disordered sequence by (B) Analysis of ORC phase separation by the depletion ORC nM) phase in a DNA-dependent in the presence and absence of (C) Cy5-dsDNA µM) was imaged alone and as a mixture with ORC In the presence of ORC, phase-separated droplets (D) ORC Orc1 and have N-terminal The IDR of Orc1 is longer and is enriched for (E) Analysis of nM) and nM) phase separation by depletion of the Orc1 IDR phase separation but of the IDR has no (F) Droplets form when Cy5-dsDNA µM) is mixed with µM) but not when mixed with (G) Cdc6 phase separation was assessed by depletion assay at and 5 µM concentrations. DNA Cdc6 phase separation but this was in the presence of 1 mM Fluorescence imaging reveals phase-separated droplets when Cy5-dsDNA µM) is mixed with Cdc6 Phase separation analysis for a Cdc6 construct lacking the N-terminal IDR nM) no depletion in the presence of DNA nM). µM) is to induce droplet as assessed by fluorescence microscopy with Cy5-dsDNA Gel images are representative of three independent experiments. For proteins with a high fraction of disordered we assessed the of the disordered regions relative to known protein domains (Figure supplement 1A). In all the regions of disorder N-terminal to the of the domain of the of the Cdt1 WH domains and of the and WH domains of and Cdc6 possesses the N-terminal IDR amino and Orc1 the residues). also regions of disorder that serve as sequences between tandem such as between the and WH domain of Cdc6 amino acid or the two WH domains of Cdt1 amino acid The that Cdc6 and two subunits of ORC possess N-terminal IDRs suggested that these Cdt1, might also undergo phase separation in a DNA-dependent and that this might promote their functional within a condensed was tested by the of to phase separate using the depletion assay (Figure to the of DmCdt1 when it ORC was found to be depleted from the supernatant in the but not in the of DNA. The of did not have an on phase separation by ORC (Figure and fluorescence microscopy with Cy5-dsDNA confirmed that the DNA-dependent depletion of ORC from the supernatant was to phase separation (Figure no droplets are observed for DNA ORC phase separation a of protein concentrations (50 to nM) revealed ORC depletion when DNA was present but not when it was absent (Figure supplement 1B). Depletion regardless of oligonucleotide sequence (Figure supplement Cdt1, maximal phase separation with oligonucleotides longer than 25 basepairs (Figure supplement 1D). Cdt1, ORC is able to undergo LLPS at physiological concentrations in the presence of a of DNA without DNA sequence We whether to undergo phase separation could be to the IDR of a specific or whether the IDRs of both Orc1 and are for this In of length, the Orc1 IDR, at amino is 100 residues longer than the IDR (Figure of sequence between Orc1 and IDRs – by the relative of charged and residues – show that the IDRs of Orc1 and are highly in with the for Orc1 and within for each amino acid Interestingly, Orc1 and IDR amino acids are near across and charged this of residues is the predicted of these which is within the is often enriched in protein for both Orc1 and is than for such a region et al., 2018). Although the Orc1 and IDRs are highly in of amino acid they show a in their the Orc1 IDR (pI = is enriched for residues and whereas the IDR (pI = is enriched for residues and that this in might be for facilitating LLPS by ORC, we two with the Orc1 IDR and the other lacking the IDR Phase separation for both was then assessed by the depletion assay and fluorescence imaging with showed no evidence for phase separation in either assay, whereas the construct lacking the IDR wild-type in both assays (Figure These demonstrate that Drosophila ORC phase using interactions that the N-terminal IDR of Orc1 but not Given the predicted of in the Orc1 IDR we were to that this region is relatively across the Drosophila Figure supplement and that the length, and pI of this region is conserved across the metazoan (Table 1). Although S. cerevisiae Orc1 possesses an N-terminal IDR, this region is than observed in and also has an pI (Table 1). with this budding yeast ORC does not phase separate (Figure supplement 1E). We asked whether Cdc6 can undergo DNA-dependent to budding yeast Cdc6 et al., was found to with DNA by dsDNA-coupled agarose pull-down the presence of no on Cdc6 DNA-binding (Figure supplement 1F). whether Cdc6 phase in the presence of DNA using the depletion assay (Figure we observed a DNA-dependent depletion of Cdc6 from the supernatant (Figure however, in contrast to ORC, this depletion was by the presence of (Figure A report that can function as a to protein may provide an for its on Cdc6 phase separation et al., 2017). the assay at concentrations of that DmCdt1, which to fully partition into phases at concentrations (Figure 2E), Cdc6 was only depleted from the supernatant (Figure When a of protein concentrations and DNA sequences (Figure supplement Cdc6 showed phase separating the protein into the Notably, Cdc6 underwent complete partitioning into

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Keywords: biopolymer, gelatine, dextran, PEO, phase separation, polydispersity, molar mass distribution, SEC-MALLS, CSLM The temperature-composition phase diagram of aqueous solutions of gelatine and dextran, which show liquid/liquid phase segregation, were explored at temperatures above the gelation temperature of gelatine. It turned out that the compositions of coexisting phases show practically no dependence on temperature between 40oC and 80oC. Also, the total polymer concentration at which phase separation occurred was found to be nearly independent on temperature. These observations suggest an entropy driven phase separation. Phase separation is found to be accompanied by strong fractionation of the molar mass distribution in the two phases. Molar mass distributions in coexisting phases were investigated using Size Exclusion Chromatography with MultiAngle Laser Light Scattering. The molar mass of the native material and concentration appeared to be the only factors that affected the final molar mass distributions, temperature having no effect. The results show that in the molar mass range where fractionation is the strongest, i.e. roughly below the maximum in the distribution, fractionation is governed by a Boltzmann factor , where DG denotes the free energy involved in transferring a polymer with a certain length from the enriched to the depleted phase, and in this case turns out to be proportional to the molar mass. The phase behaviour of aqueous mixtures of poly(ethylene oxide) (PEO) and dextran is also studied. From the temperature dependence we conclude that the phase separation between PEO and dextran is partly caused by sterical interactions. From the equilibrium phase volumes of the phase separated mixture and the shape of the temperature-composition phase diagram of PEO and dextran we conclude that also the decrease of solvent quality of water for PEO at increasing temperatures is involved. It is suggested that the characteristics of the PEO-water interaction can affect the degree of fractionation. This suggestion is based on the observation that the degree of fractionation is not a simple exponential function of the molar mass. Adsorption of high molar mass dextran at the interface between gelatine and dextran during the phase separation of aqueous mixtures of these two polymers is also studied. This adsorption is observed after macroscopic phase separation as well as during the phase separation. In the last case, the system is studied with Confocal Scanning Light Microscopy. For this purpose, the dextran is covalently labelled with fluorescein 5-isothiocyanate (FITC). It turns out that the adsorption of high molar mass dextran is highly affected by the labelling. The adsorption of labelled dextran leads to a stable film between the two coexisting phases, thus preventing dextran droplets to coalesce. If the degree of labelling increases above a certain level, the labelled dextran does not lead to a stable film, but is preferentially present in the gelatine-rich phase.

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Cell fate transition is a fascinating process involving complex dynamics of three-dimensional (3D) chromatin organization and phase separation, which play an essential role in cell fate decision by regulating gene expression. Phase separation is increasingly being considered a driving force of chromatin folding. In this review, we have summarized the dynamic features of 3D chromatin and phase separation during physiological and pathological cell fate transitions and systematically analyzed recent evidence of phase separation facilitating the chromatin structure. In addition, we discuss current advances in understanding how phase separation contributes to physical and functional enhancer-promoter contacts. We highlight the functional roles of 3D chromatin organization and phase separation in cell fate transitions, and more explorations are required to study the regulatory relationship between 3D chromatin organization and phase separation.Graphical 3D chromatin organization (shown by Hi-C contact map) and phase separation are highly dynamic and play functional roles during early embryonic development, cell differentiation, somatic reprogramming, cell transdifferentiation and pathogenetic process. Phase separation can regulate 3D chromatin organization directly, but whether 3D chromatin organization regulates phase separation remains unclear.

The northwestern caldera wall of Brothers volcano in the southern Kermadec arc features several clusters of hydrothermal venting in a large area that extends from near the caldera floor (~1700 mbsl) almost up to the crater rim (~1300 mbsl). Abundant black smoker-type hydrothermal chimneys and exposed stockwork mineralization in this area provide an excellent archive of hydrothermal processes that form seafloor massive sulfide deposits. Using sulfate precipitates from chimneys and stockwork recently recovered by remotely operated vehicles, we conducted fluid inclusion microthermometry and Sr isotope studies to determine the role of phase separation and mixing between vent fluid and seawater. The variability in the vast majority of fluid inclusion salinities (i.e., 0.1–5.25 wt.% NaCl eq.) and entrapment temperatures of up to 346°C are indicative of phase-separated hydrothermal fluids. Large salinity variations in samples with entrapment temperatures mostly below the boiling temperature for the sample’s depth show that the majority of fluids ascending below the NW Caldera are phase separating in the subsurface and cooling, prior to discharge. In several samples, entrapment temperatures of over 343°C suggest that phase-separating fluids have at least sporadically exited the seafloor at the NW Caldera site. Isobaric-isenthalpic mixing trends between coexisting phase-separated vapors and brines with seawater are consistent with phase-separated fluids at near-seafloor pressures of ~170 bar and suggest that the vast majority of the ascending fluids continue to phase separate to within tens to hundreds of meters below seafloor prior to mixing with seawater. A small subset of the most saline fluid inclusions (up to 18.6 wt.% NaCl eq.) is unlikely formed by near-seafloor phase separation and is considered to be produced either by supercritical phase separation or by the contribution of a magmatic brine from near the magmatic-hydrothermal interface. 87Sr/86Sr values of sulfate samples range from 0.7049 (i.e., near hydrothermal end-member) to 0.7090 (i.e., near seawater) and show that the crystals grew from vapor- and brine-derived fluids in a hydrothermally dominated mixing regime. Our work provides new insights into mineral growth conditions, mixing regimes, and in particular, the extent and character of subseafloor phase separation during the formation of hydrothermal vents and their underlying stockwork in seawater-dominated, arc-related hydrothermal systems.

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