A Framework to Assess Liquid‐Liquid‐Phase‐Separation in Bacterial Cells

Liquid-liquid phase separation (LLPS) in cells is known as a complex physicochemical process causing the formation of membrane-less organelles (MLOs). Cells have well-defined different membrane-surrounded organelles like mitochondria, endoplasmic reticulum, lysosomes, peroxisomes, etc., however, on demand they can create MLOs as stress granules, nucleoli and P bodies to cover vital functions and regulatory activities. However, the mechanism of intracellular molecule assembly into functional compartments within a living cell remains till now not fully understood. in vitro and in vivo investigations unveiled that MLOs emerge after preceding liquid-liquid, liquid-gel, liquid-semi-crystalline, or liquid-crystalline phase separations. Liquid-liquid and liquid-gel MLOs form the majority of cellular phase separation events, while the occurrence of micro-sized crystals in cells was only rarely observed, however can be considered as a result of a preceding protein phase separation event. In vivo, also known and termed as in cellulo crystals, are reported since 1853. In some cases, they have been linked to vital cellular functions, such as storage and detoxification. However, the occurrence of in cellulo crystals is also associated to diseases like cataract, hemoglobin C diseases, etc. Therefore, better knowledge about the involved molecular processes will support drug discovery investigations to cure diseases related to in cellulo crystallization. We summarize physical and chemical determinants known today required for phase separation initiation and formation and in cellulo crystal growth. In recent years it has been demonstrated that LLPS plays a crucial role in cell compartmentalization and formation of MLOs. Here we discuss potential mechanisms and potential crowding agents involved in protein phase separation and in cellulo crystallization.

The limited options for treating patients with drug-resistant cancers have emphasized the need to identify alternative treatment targets. Tumor cells have large super-enhancers (SEs) in the vicinity of important oncogenes for activation. The physical process of liquid-liquid phase separation (LLPS) contributes to the assembly of several membrane-less organelles in mammalian cells. Intrinsically disordered regions (IDRs) of proteins induce LLPS formation by developing condensates. It was discovered that key transcription factors (TFs) undergo LLPS in SEs. In addition, TFs play critical roles in the epigenetic and genetic regulation of cancer progression. Recently, we revealed the essential role of disease-specific TF collaboration changes in advanced prostate cancer (PC). OCT4 confers epigenetic changes by promoting complex formation with TFs, such as Forkhead box protein A1 (FOXA1), androgen receptor (AR) and Nuclear respiratory factor 1 (NRF1), inducing PC progression. It was demonstrated that TF collaboration through LLPS underlying transcriptional activation contributes to cancer aggressiveness and drug resistance. Moreover, the disruption of TF-mediated LLPS inhibited treatment-resistant PC tumor growth. Therefore, we propose that repression of TF collaborations involved in the LLPS of SEs could be a promising strategy for advanced cancer therapy. In this article, we summarize recent evidence highlighting the formation of LLPS on enhancers as a potent therapeutic target in advanced cancers.

Role of protein aggregates in bacteria.

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

Cellular uptake of nickel by NikR is regulated by phase separation

Biomolecular condensates, physically underpinned to a significant extent by liquid-liquid phase separation (LLPS), are now widely recognized by numerous experimental studies to be of fundamental biological, biomedical, and biophysical importance. In the face of experimental discoveries, analytical formulations emerged as a powerful yet tractable tool in recent theoretical investigations of the role of LLPS in the assembly and dissociation of these condensates. The pertinent LLPS often involves, though not exclusively, intrinsically disordered proteins engaging in multivalent interactions that are governed by their amino acid sequences. For researchers interested in applying these theoretical methods, here we provide a practical guide to a set of computational techniques devised for extracting sequence-dependent LLPS properties from analytical formulations. The numerical procedures covered include those for the determination of spinodal and binodal phase boundaries from a general free energy function with examples based on the random phase approximation in polymer theory, construction of tie lines for multiple-component LLPS, and field-theoretic simulation of multiple-chain heteropolymeric systems using complex Langevin dynamics. Since a more accurate physical picture often requires comparing analytical theory against explicit-chain model predictions, a commonly utilized methodology for coarse-grained molecular dynamics simulations of sequence-specific LLPS is also briefly outlined.

PDE11A is a little-studied phosphodiesterase sub-family that breaks down cAMP/cGMP, with the PDE11A4 isoform enriched in the memory-related hippocampal formation. Age-related increases in PDE11A expression occur in human and rodent hippocampus and cause age-related cognitive decline of social memories. Interestingly, age-related increases in PDE11A4 protein ectopically accumulate in spherical clusters that group together in the brain to form linear filamentous patterns termed "PDE11A4 ghost axons". The biophysical/physiochemical mechanisms underlying this age-related clustering are not known. Here, we determine if age-related clustering of PDE11A4 reflects liquid-liquid phase separation (LLPS; biomolecular condensation), and if PDE11A inhibitors can reverse this LLPS. We show human and mouse PDE11A4 exhibit several LLPS-promoting sequence features, including intrinsically disordered regions, non-covalent pi-pi interactions, and prion-like domains that were particularly enriched in the N-terminal regulatory region. Further, multiple bioinformatic tools predict PDE11A4 undergoes LLPS. Consistent with these predictions, aging-like PDE11A4 clusters in HT22 hippocampal neuronal cells were membraneless spherical droplets that progressively fuse over time in a concentration-dependent manner. Deletion of the N-terminal intrinsically disordered region prevented PDE11A4 LLPS despite equal protein expression between WT and mutant constructs. 1,6-hexanediol, along with tadalafil and BC11-38 that inhibit PDE11A4, reversed PDE11A4 LLPS in HT22 hippocampal neuronal cells. Interestingly, PDE11A4 inhibitors reverse PDE11A4 LLPS independently of increasing cAMP/cGMP levels via catalytic inhibition. Importantly, orally dosed tadalafil reduced PDE11A4 ghost axons in old mouse ventral hippocampus by 50%. Thus, PDE11A4 exhibits the four defining criteria of LLPS, and PDE11A inhibitors reverse this age-related phenotype both in vitro and in vivo.

The regulation of bone physiology and pathophysiology is intricately controlled by a complex interplay of cellular and molecular mechanisms. In these processes, the precise spatiotemporal coordination of biological activities in bone-resident cells plays a central role. Recently, liquid-liquid phase separation (LLPS), a mechanism underlying membraneless biomolecular condensate formation, has emerged as a transformative area of research. Liquid-liquid phase separation refers to the phase transition of biomolecules under specific conditions, leading to the formation of biomolecular condensates, which orchestrate diverse cellular functions. In this review, we provide a comprehensive synthesis of how LLPS influences bone turnover, focusing on its role in regulating bone homeostasis and its dysregulation in bone disease pathogenesis. Furthermore, aside from addressing the current challenges and limitations in this nascent field, we explore the implications of LLPS in bone regeneration, preventive strategies, and precision medicine. Despite LLPS research being in its early stages, its rapid advancement underscores its crucial role in bone biology and highlights the urgent need to integrate LLPS insights with translational approaches to advance therapeutic interventions for bone disorders.

Membraneless biomolecular condensates formed by liquid-liquid phase separation (LLPS) are crucial for many spatiotemporal biological functions. Designing synthetic mimics to emulate and understand LLPS is an active area of research, which has led to the development of coacervate droplets through elegant bioinspired designs. However, recent interest in this field has shifted toward designing programmable coacervates to impart spatiotemporal control over these liquid phases. Herein, we demonstrate the programming of LLPS in synthetic systems by employing concepts of competitive binding and reaction-coupled assembly involving dynamic covalent bonds. Our results utilize small building blocks that follow a simple coacervation mechanism, distinguishing this approach from previously reported programmable complex coacervates, which often rely on reaction-controlled generation of one of the components. We introduce these concepts using dynamic covalent bonds (boronate esters) and small chromophoric building blocks appended with terminal boronic acid groups. Upon reaction with substrates (monosaccharides), these building blocks form molecular structures resembling "sticker-and-spacer" designs for coacervation, leading to a reaction-driven, temporally controlled LLPS process. The differential reactivity of various monosaccharides, combined with the reversibility of dynamic bonds, enables competitive binding-driven control over the growth, inhibition, and dissolution of the coacervation process, offering new strategies for programmable LLPS that are reminiscent of protein-induced inhibition in biomolecular condensates. Detailed spectroscopic probing and kinetic analyses provide mechanistic insights into the reaction-coupled and autocatalytic growth processes, revealing the glucose-selective nature of this coacervation system. Finally, coupling dynamic covalent reactions with temporal pH modulation results in transient coacervation, which can be visualized by using confocal microscopy. We anticipate that this approach will pave the way for designing coacervate droplets with novel biorelevant emergent properties.

Biomolecular condensates provide a mechanism for compartmentalization of biomolecules in eukaryotic cells. These liquid-like condensates are formed via liquid–liquid phase separation, by a plethora of interactions, and can mediate several biological processes in healthy cells. Expansions of dipeptide repeat proteins, DPRs, in which arginine rich DPRs like poly-proline-arginine (PR), and poly-glycine-arginine (GR), partition RNA into condensates can however induce cell toxicity. Here, we use (GR)20 as a model for biological poly-GR and condense it using either excluded volume interactions with polyethylene glycol (PEG) as a crowder or direct electrostatic interactions with RNA oligomers. Using two-dimensional infrared (2D IR) spectroscopy, we observe that (GR)20 condensed through an excluded volume forms β-sheet structures, whereas (GR)20 condensed with RNA forms loops. We also investigate local hydrogen-bond dynamics in the condensate and compare the measurements with molecular dynamics simulations. Hydrogen bond lifetimes undergo a marked slowdown compared to dynamics in the dilute phase. This is representative of confined water within the percolated networks inside the condensate due to the interaction present in the condensate disrupting H-bond networks. Overall, our results show that both protein structure and dynamics are inherently dependent on the type of interactions that stabilize the condensates.

Intrinsically disordered proteins (IDPs) are unable to adopt a unique 3D structure under physiological conditions and thus exist as highly dynamic conformational ensembles. IDPs are ubiquitous and widely spread in the protein realm. In the last decade, compelling experimental evidence has been gathered, pointing to the ability of IDPs and intrinsically disordered regions (IDRs) to undergo liquid–liquid phase separation (LLPS), a phenomenon driving the formation of membrane-less organelles (MLOs). These biological condensates play a critical role in the spatio-temporal organization of the cell, where they exert a multitude of key biological functions, ranging from transcriptional regulation and silencing to control of signal transduction networks. After introducing IDPs and LLPS, we herein survey available data on LLPS by IDPs/IDRs of viral origin and discuss their functional implications. We distinguish LLPS associated with viral replication and trafficking of viral components, from the LLPS-mediated interference of viruses with host cell functions. We discuss emerging evidence on the ability of plant virus proteins to interfere with the regulation of MLOs of the host and propose that bacteriophages can interfere with bacterial LLPS, as well. We conclude by discussing how LLPS could be targeted to treat phase separation-associated diseases, including viral infections.

RNA viruses encode multifunctional proteins to overcome limited genomic capacity and mediate diverse processes in viral replication and host cell modulation. The rabies virus P gene encodes full-length P1 protein and the truncated isoform, P3, which acquires phenotypes absent from P1, including interactions with cellular membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS). This gain-of-function suggests that isoform multifunctionality arises not only from discrete functions of protein modules/domains, but also from conformational regulation involving interactions of the globular C-terminal domain and N-terminal intrinsically disordered regions (IDRs). The precise mechanisms underlying gain-of-function, however, remain unresolved. Here, we compare the structure and function of P1 and P3, identifying isoform-specific long-range intra-protomer interactions between the IDRs and C-terminal domain that correlate with conformational states, LLPS behavior, and subcellular localization. Mutations in P3 that alter MLO interactions correspondingly modulate these interactions. P1 and P3 can interact with similar/overlapping sets of MLO-associated proteins and have similar LLPS capacity, but only P3 binds RNA, and this interaction correlates with gain-/loss-of-function mutations. Our findings reveal that conformational differences in isoforms regulate LLPS behavior and contribute to protein-RNA interactions, which controls access to host LLPS structures, uncovering a previously unrecognized strategy in P protein multifunctionality.

Complex coacervation refers to the liquid-liquid phase separation (LLPS) process occurring between charged macromolecules. The study of complex coacervation is of great interest due to its implications in the formation of membraneless organelles (MLOs) in living cells. However, the impacts of the crowded intracellular environment on the behavior and interactions of biomolecules involved in MLO formation are not fully understood. To address this knowledge gap, we investigated the effects of crowding on a model protein-polymer complex coacervate system. Specifically, we examined the influence of sucrose as a molecular crowder and polyethylene glycol (PEG) as a macromolecular crowder. Our results reveal that the presence of crowders led to the formation of larger coacervate droplets that remained stable over a 25-day period. While sucrose had a minimal effect on the physical properties of the coacervates, PEG led to the formation of coacervates with distinct characteristics, including higher density, increased protein and polymer content, and a more compact internal structure. These differences in coacervate properties can be attributed to the effects of crowders on individual macromolecules, such as the conformation of model polymers, and nonspecific interactions among model protein molecules. Moreover, our results show that sucrose and PEG have different partition behaviors: sucrose was present in both the coacervate and dilute phases, while PEG was observed to be excluded from the coacervate phase. Collectively, our findings provide insights into the understanding of crowding effects on complex coacervation, shedding light on the formation and properties of coacervates in the context of MLOs.

Eukaryotic cells contain distinct organelles, but not all of these compartments are enclosed by membranes. Some intrinsically disordered proteins mediate membraneless organelle formation through liquid-liquid phase separation (LLPS). LLPS facilitates many biological functions such as regulating RNA stability and ribonucleoprotein assembly, and disruption of LLPS pathways has been implicated in several diseases. Proteins exhibiting LLPS typically have low sequence complexity and specific repeat motifs. These motifs promote multivalent connections with other molecules and the formation of higher-order oligomers, and their removal usually prevents LLPS. The intrinsically disordered C-terminal domain of TAR DNA-binding protein 43 (TDP-43), a protein involved in motor neuron disease and dementia lacks a dominant LLPS motif, however, and how this domain forms condensates is unclear. Using extensive mutagenesis of TDP-43, we demonstrate here that three tryptophan residues and, to a lesser extent, four other aromatic residues are most important for TDP-43 to undergo LLPS. Our results also suggested that only a few residues may be required for TDP-43 LLPS because the α-helical segment (spanning ∼20 residues) in the middle part of the C-terminal domain tends to self-assemble, reducing the number of motifs required for forming a multivalent connection. Our results indicating that a self-associating α-helical element with a few key residues regulates condensate formation highlight a different type of LLPS involving intrinsically disordered regions. The C-terminal domain of TDP-43 contains ∼50 disease-related mutations, with no clear physicochemical link between them. We propose that they may disrupt LLPS indirectly by interfering with the key residues identified here.

Liquid-liquid phase separation (LLPS) drives the formation of various membraneless organelles, which are crucial for biological processes and disease development. Despite the significant regulatory effects on LLPS of protein post-translational modifications (PTMs), specific data resource and predictor are still lacking. First, we constructed a well-curated database of PTM regulation on liquid-liquid Phase Separation (PTMPhaSe) (https://ptmphase.sjtu.edu.cn) that contains manually curated complete experimental evidence. Second, we developed graph neural network-based deep learning model (named PhosLLPS) to predict functional phosphorylation sites regulating LLPS, which achieved better identification performance (AUC = 0.9116) than four baseline models and the existing FuncPhos-SEQ method. Meanwhile, human proteome-scale predictions for functional phosphorylation sites were performed with PhosLLPS. PhosLLPS is now freely available in web server (https://ptmphase.sjtu.edu.cn/Predictor). By bridging the gap between PTM regulation and LLPS, these resources could contribute to a better understanding of the molecular function of LLPS and facilitate further drug development for LLPS-related diseases.