Fabrication of Anisotropic Polymer Materials by Polymerization-Induced Phase Separation (PIPS) Under External Fields: Computational Analysis

In this study, the self-condensation polymerization of a tri-functional monomer in a monomer-solvent mixture and the phase separation of the system were simultaneously modeled and simulated. Nonlinear Cahn–Hilliard and Flory–Huggins free energy theories incorporated with the kinetics of the polymerization reaction were utilized to develop the model. Linear temperature and concentration gradients singly and in combination were applied to the system. Eight cases which faced different ranges of initial concentration and/or temperature gradients in different directions, were studied. Various anisotropic structural morphologies were achieved. The numerical results were in good agreement with published data. The size analysis and structural characterization of the phase-separated system were also carried out using digital imaging software. The results showed that the phase separation occurred earlier in the section with a higher initial concentration and/or temperature, and, at a given time, the average equivalent diameter of the droplets <dave> was larger in this region. While smaller droplets formed later in the lower concentration/temperature regions, at the higher concentration/temperature side, the droplets went through phase separation longer, allowing them to reach the late stage of the phase separation where particles coarsened. In the intermediate stage of phase separation, <dave> was found proportional to , where was in the range between and for the cases studied and was consistent with published results.

&lt;p&gt;In this study, the self-condensation polymerization of a tri-functional monomer in a monomer-solvent mixture and the phase separation of the system were simultaneously modeled and simulated. Nonlinear Cahn-Hilliard and Flory-Huggins free energy theories incorporated with the kinetics of the polymerization reaction were utilized to develop the model. Linear temperature and concentration gradients singly and in combination were applied to the system. Eight cases which faced different ranges of initial concentration and/or temperature gradients in different directions, were studied. Various anisotropic structural morphologies were achieved. The numerical results were in good agreement with published data. The size analysis and structural characterization of the phase-separated system were also carried out using digital imaging software. The results showed that the phase separation occurred earlier in the section with a higher initial concentration and/or temperature, and, at a given time, the average equivalent diameter of the droplets was larger in this region. While smaller droplets formed later in the lower concentration/temperature regions, at the higher concentration/temperature side, the droplets went through phase separation longer, allowing them to reach the late stage of the phase separation where particles coarsened. In the intermediate stage of phase separation, was found proportional to t*(alpha), where alpha was in the range between 1/3 and 1/2 for the cases studied and was consistent with published results. &lt;/p&gt;

The dynamics of phase separation for polymer blends is important in determining the final morphology and properties of polymer materials; in practical applications, this phase separation can be controlled by coupling to polymerization reaction kinetics via a process called 'polymerization-induced phase separation'. We develop a phase-field model for a polymer melt blend using a polymerizing Cahn-Hilliard (pCH) formalism to understand the fundamental processes underlying phase separation behavior of a mixture of two species independently undergoing linear step-growth polymerization. In our method, we explicitly model polydispersity in these systems to consider different molecular-weight components that will diffuse at different rates. We first show that this pCH model predicts results consistent with the Carothers predictions for step-growth polymerization kinetics, the Flory-Huggins theory of polymer mixing, and the classical predictions of spinodal decomposition in symmetric polymer blends. The model is then used to characterize (i) the competition between phase separation dynamics and polymerization kinetics, and (ii) the effect of unequal reaction rates between species. For large incompatibility between the species (i.e. high χ), our pCH model demonstrates that the strength for phase separation directly corresponds to the kinetics of phase separation. We find that increasing the reaction rate k̃, first induces faster phase separation but this trend reverses as we further increase k̃ due to the competition between molecular diffusion and polymerization. In this case, phase separation is delayed for faster polymerization rates due to the rapid accumulation of slow-moving, high molecular weight components.

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

The surface-directed phase separation (SDPS) phenomena of a model binary polymer blend quenched into the unstable region of its binary symmetric upper critical solution temperature phase diagram is numerically investigated using a mathematical model composed of the nonlinear Cahn–Hilliard (CH) theory for phase separation along with the Flory–Huggins–de Gennes (FHdG) free energy functional. The SDPS occurs in a square domain with a linear temperature gradient along the horizontal direction and with all sides having short range surface potential h1. The effects of different quench depth, diffusion coefficient, surface potential, and temperature gradient were studied numerically. The numerical results indicate that there is a simultaneous competition between the four surfaces in attracting the preferred polymer. The side with a higher surface potential would win the competition against the side with a lower surface attraction in the case of a uniform quench. The numerical results also indicated a later transition time for higher values of h1. As surface potential increased, the transition time from complete wetting to partial wetting occurred at a later time on the surface. The impact of different temperature gradient ΔT*/Δx* values on the surface enrichment rate with fixed temperature at one surface and higher temperature at the opposite surface was studied for the first time within a multiple surface potential set up. The results showed that higher values of ΔT*/Δx* increased the growth rate of the preferred polymer on the surface adding to the thickness of the wetting layer. The transition time from complete wetting to partial wetting occurred slightly later at the lower temperature side.

Photo-enforced stratification of liquid crystal / monomer mixtures : principle, theory and analysis of a paintable LCD concept

A computational study of short-range surface-directed phase separation in polymer blends under a linear temperature gradient

Inspired by the Cyphochilus scale with extreme whiteness, fluoropolymer is used as the matrix to prepare highly scattering porous polymer films through polymerization‐induced phase separation (PIPS) method. The mixture used for the PIPS technique contains a fluoride monomer, a UV photoinitiator, and porogens (cyclohexanol and perfluorooctanol). By carefully tuning the mixture parameters (cyclohexanol–perfluorooctanol and monomer–porogen weight ratios), the porous morphology can be well tailored, which induces different scattering performances. With an optimized formulation (50 wt% monomer, 25 wt% cyclohexanol, and 25 wt% perfluorooctanol), the porous film with a thickness of 55 µm can achieve an average total reflectance of ≈90%, featuring a measured transport mean free path of 1.3–1.7 µm (comparable to the 1.5 µm of the benchmark Cyphochilus scale). Further, the porous films are applied to the light‐emitting diode (LED) device and it is demonstrated that they can effectively improve the light extraction efficiency of the LED and thus enhance the luminous performance. Due to the simplicity, cost‐effectiveness, and scalability of the PIPS method, the as‐developed porous fluoropolymer films with such excellent scattering ability can find many potential applications not only in the field of optoelectronics, but also in various complex scenarios, such as daytime passive radiative cooling.

Contribution of monomer functionality and additives to polymerization kinetics and liquid crystal phase separation in acrylate‐based polymer‐dispersed liquid crystals (PDLCs)

The a-D-glucose was chemically modified with an allyl isocyanate (MG) and introduced into the polymer matrix for holographic polymer-dispersed liquid crystal (HPDLC), and the effects were studied in terms of morphology, grating formulation dynamics and electro-optical and biodegradable properties. Phase separation and diffraction efficiency increased at low content of (MG ≤ 4 wt%), while a rapid increase in crosslink density entrapped the LC droplets within the polymer to give poor phase separation, small droplet size, and low diffraction efficiency at high content. The HPDLC film was driven only with the addition of MG due to the increased droplet size with a minimum driving voltage of 18 V at 6.0 wt% MG. With the addition and an increasing amount of MG, the biodegradation of the composite film in a buffer solution was significantly increased in proportion to its amount.

A Polymer Dispersed Liquid Crystal composite based upon a smectic liquid crystal and reactive crosslinkable epoxide-amine monomers was studied. The composite material is obtained by a polymerization induced phase separation process. The polymerization and the phase separation kinetics are both monitored. The polymerization induced phase separation is detected and quantitatively monitored by X-ray diffraction. The normalized X-ray intensity is used to calculate a phase separation rate and to define the phase separation kinetics which is compared to the polymerization rate and to the polymerization kinetics. The rate difference accounts for changes in morphology.

We have used light scattering to study the kinetics of polymerization-induced phase separation in a liquid-crystal--polymer mixture. The evolution of the structure factor is compared with scaling predictions for thermally quenched systems. We have also observed a cascading phenomenon where phase separated domains become unstable and undergo phase separation for a second time.

Biostabilization processes, based on desiccation, cooling or freezing, are surface-induced and thus are inevitably governed by scale-dependent kinetic constraints. During processing, these kinetic constraints induce heterogeneity (solute, and solvent concentration gradients, thermal gradients, and eventually, phase separation in the medium). During freezing, ice crystallization induces thermal (due to latent heat release), and concentration gradients (due to solute rejection) causing phase separation in the medium (i.e., formation of regions at different thermodynamic states). For example, during a typical cryopreservation application initially the bulk water freezes, increasing the concentration of the solutes in the rest of the unfrozen solution. Upon further cooling, the freeze-concentrated solution may also vitrify (or some constituents may crystallize), creating at least two distinct regions within the same medium with two distinct states.

We have investigated experimentally the structure formation processes during phase separation via spinodal decomposition above and below the spinodal line in a binary polymer blend system exposed to in-plane stationary thermal gradients using phase contrast optical microscopy and temperature gradient hot stage. Below the spinodal line there is a coupling of concentration fluctuations and thermal gradient imposed by the temperature gradient hot stage. Also under the thermal gradient annealing phase-separated domains grow faster compared with the system under homogeneous temperature annealing on a zero-gradient or a conventional hot stage. We suggest that the in-plane thermal gradient accelerates phase separation through the enhancement in concentration fluctuations in the early and intermediate stages of spinodal decomposition. In a thermal gradient field, the strength of concentration fluctuation close to the critical point (above the spinodal line) is strong enough to induce phase separation even in one-phase regime of the phase diagram. In the presence of a temperature gradient the equilibrium phase diagrams are no longer valid, and the systems with an upper critical solution temperature can be quenched into phase separation by applying the stationary temperature gradient. The in-plane temperature gradient drives enhanced concentration fluctuations in a binary polymer blend system above and below the spinodal line.

In state-of-the-art lithium ion batteries, separators (microporous membranes) play a passive yet critical role – hosting liquid electrolyte and maintaining physical separation of the electrodes. However, as the demands on lithium ion batteries increase, with an emphasis on greater energy density, longevity (cycle/calendar life), and safety, engineering separators to take a on more active role in the cell (electro)chemistry is expected to be an important strategy. Myriad membrane materials and separator designs have been developed to impart additional functionality, for example, acid and/or transition metal scavenging, temperature responsiveness, enhanced thermal stability, increased ion dissociation, combustion suppression, and mechanical strength.In this talk, I will preset my group’s approach to additive manufacturing of next-generation lithium ion battery separators. Our approach is based on polymerization induced phase separation (PIPS), wherein polymerizable monomers (or prepolymer resins) are mixed with porogen. Through rapid, low-cost, readily scalable photopolymerization, the monomers are converted to a crosslinked polymer network, which results in the porogen becoming immiscible and phase separating through spinodal decomposition. By tuning the thermodynamics of the polymer-porogen mixture and photopolymerization kinetics, the porosity and pore size of the resulting polymeric phase can be tuned. We have shown that ethylene carbonate (EC) mixed with common acrylate monomers, such as 1,4-butanediol diacrylate, is an effective porogen. Most importantly because EC is an indispensable component in liquid electrolytes, it does not need to be extracted from the separator prior to incorporation into the electrochemical cell.By controlling the ratio of the 1,4-butanediol diacrylate (BDDA) monomer to EC, monolithic microporous membranes are readily prepared with 25 µm thickness and pore sizes and porosities ranging from 6.8 to 22nm and 15.4% to 38.54%, respectively. The optimal poly(1,4-butanediol diacrylate) (pBDDA) separator has a porosity of 38.5% and average pore size of 22 nm; uptakes 127% liquid electrolyte by mass, and has an ionic conductivity of 1.98 mS/cm, which is higher than that of Celgard 2500. Lithium ion battery half cells consisting of LiNi0.5Mn0.3Co0.2O2 cathodes and pBDDA separators were shown to undergo reversible charge/discharge cycling with an average discharge capacity of 142 mAh/g and a capacity retention of 98.4% over 100 cycles - comparable to cells using state-of-the-art separators. Furthermore, the pBDDA separators were shown to be thermally stable to 400°C, lack low temperature thermal transitions that can compromise cell safety, and exhibits no thermal shrinkage up to 150°C. I will also discuss my group’s efforts to engineer separators with additional functionality to improve cell performance under abuse conditions.