Phase-Separated Multienzyme Compartmentalization for Terpene Biosynthesis in a Prokaryote.

A typical human cell is a crowded soup of thousands of different proteins. One way that the cell organizes this complex mix of contents is by creating separate droplets within the cell, like oil in water. These droplets can form through a process known as liquid-liquid phase separation, or LLPS, where specific proteins gather in high concentrations to carry out their cellular roles. The critical role of LLPS in cellular organization means that it is widely studied by biologists. To detect LLPS, researchers often subject the cells to treatments designed to hold all the proteins in place, creating a snapshot of their natural state. This process, known as fixing, allows scientists to easily label a protein with a fluorescent tag, take pictures of the cells, and look at whether the protein forms droplets in its natural state. This is often easier to do than imaging cells live, but it relies on LLPS being well-preserved upon fixation. To test if this is true, Irgen-Gioro, Yoshida et al. looked at protein droplets in live cells, and then fixed the cells to check whether the appearance of the droplets had changed. The images taken showed that fixation could alter the size and number of droplets depending on the protein being studied. To explain why the effects of fixing change depending on the protein, Irgen-Gioro, Yoshida et al. hypothesized that a faster fixation – relative to how quickly proteins can bind and unbind to their droplets – can better preserve the LLPS droplets. They verified their idea using a microscopy technique in which they imaged single molecules, allowing them to see how different fixation speeds relative to protein binding affected the droplets. The work of Irgen-Gioro, Yoshida et al. identifies an important caveat to using fixation for the study of LLPS in cells. Their findings suggest that researchers should be cautious when interpreting the results of such studies. Given that LLPS in cells is an area of research with a lot of interest, these results could benefit a broad range of biological and medical fields. In the future, Irgen-Gioro, Yoshida et al.’s findings could prompt scientists to develop new fixing methods that better preserve LLPS in cells.

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed-cell imaging techniques such as immunofluorescence have been widely used to detect liquid–liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein–protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein–protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Consistent with simulations, live-cell single-molecule imaging experiments showed that a fast overall rate of fixation relative to protein–protein interaction dynamics can minimize fixation artifacts. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact. Editor's evaluation Chemically fixing cells for fluorescence microscopy is a common practice in cell biology. However, fixation artifacts can lead the incorrect interpretations of experimental results. This article presents compelling evidence showing that in the context of liquid condensates formed by liquid–liquid phase separation (LLPS), paraformaldehyde (PFA) fixation creates a number of artifacts – such as changes in the number, appearance, or disappearance of liquid condensates. These important findings will be of great interest not only for those in the LLPS field but for any cell biologists using fixed samples for microscopy. https://doi.org/10.7554/eLife.79903.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest A typical human cell is a crowded soup of thousands of different proteins. One way that the cell organizes this complex mix of contents is by creating separate droplets within the cell, like oil in water. These droplets can form through a process known as liquid-liquid phase separation, or LLPS, where specific proteins gather in high concentrations to carry out their cellular roles. The critical role of LLPS in cellular organization means that it is widely studied by biologists. To detect LLPS, researchers often subject the cells to treatments designed to hold all the proteins in place, creating a snapshot of their natural state. This process, known as fixing, allows scientists to easily label a protein with a fluorescent tag, take pictures of the cells, and look at whether the protein forms droplets in its natural state. This is often easier to do than imaging cells live, but it relies on LLPS being well-preserved upon fixation. To test if this is true, Irgen-Gioro, Yoshida et al. looked at protein droplets in live cells, and then fixed the cells to check whether the appearance of the droplets had changed. The images taken showed that fixation could alter the size and number of droplets depending on the protein being studied. To explain why the effects of fixing change depending on the protein, Irgen-Gioro, Yoshida et al. hypothesized that a faster fixation – relative to how quickly proteins can bind and unbind to their droplets – can better preserve the LLPS droplets. They verified their idea using a microscopy technique in which they imaged single molecules, allowing them to see how different fixation speeds relative to protein binding affected the droplets. The work of Irgen-Gioro, Yoshida et al. identifies an important caveat to using fixation for the study of LLPS in cells. Their findings suggest that researchers should be cautious when interpreting the results of such studies. Given that LLPS in cells is an area of research with a lot of interest, these results could benefit a broad range of biological and medical fields. In the future, Irgen-Gioro, Yoshida et al.’s findings could prompt scientists to develop new fixing methods that better preserve LLPS in cells. Introduction Fixing cells to preserve a snapshot of biomolecular transactions in vivo is a widely used strategy in numerous techniques in biology and medicine. Due to its small size and high reactivity with a wide range of biological entities, paraformaldehyde (PFA) is one of the most commonly used fixatives to create covalent cross-linking between biomolecules, for example, proteins and nucleic acids. PFA nonselectively ‘fixes’ or cross-links molecules in proximity to enable characterization of biomolecular interactions formed in living cells. Examples of popular techniques that use PFA to fix cells include ChIP-sequencing (Robertson et al., 2007; Solomon and Varshavsky, 1985), chromosome conformation capture (3C)-based techniques (Dekker et al., 2002), immunofluorescence (Richter et al., 2018), fluorescence in situ hybridization (FISH) (Moter and Göbel, 2000), cross-linking mass spectrometry (Sutherland et al., 2008), super-resolution expansion microscopy (Chen et al., 2015), and super-resolution localization microscopies such as stochastic optical reconstruction microscopy (STORM) (Rust et al., 2006). Although PFA fixation has been used to faithfully preserve live-cell conditions in many scenarios, a number of studies have uncovered situations in which fixation fails to cross-link DNA–protein interactions formed in living cells. By imaging different transcription factors (TFs) in live and fixed cells, Schmiedeberg et al., 2009 showed that TFs bound to DNA with fast dissociation dynamics (<5 s residence times as determined by fluorescence recovery after photobleaching [FRAP]) are not cross-linked to DNA upon PFA fixation. Using live-cell single-molecule imaging, Teves et al., 2016 showed that TFs stay bound to chromosome during mitosis and fixing cells can artificially deplete transiently bound TFs from mitotic chromosomes. These studies exemplify the fact that fixation, with limited reaction rates, cannot provide an instantaneous snapshot and may miss or obfuscate biomolecular interactions that happen either at or faster than the timescale of fixation. What further complicates the result of cell fixation is that the reactivity and reaction rates of PFA are variable and dependent on its biomolecule substrates (Gavrilov et al., 2015; Shishodia et al., 2018). For example, the efficiency and rates at which PFA reacts with proteins can vary by orders of magnitude (Kamps et al., 2019) and are dependent on their amino acid sequences (Kamps et al., 2019; Metz et al., 2004; Sutherland et al., 2008) and tertiary structures (Hoffman et al., 2015). Among the numerous biomolecular transactions investigated using fixed-cell imaging is liquid–liquid phase separation (LLPS), a long-observed behavior of polymers in solution (Gibbs, 1879; Graham, 1861; Hyman et al., 2014) that has recently generated much excitement in biological research communities due to its proposed roles in cellular organization and functions (Banani et al., 2017; Boeynaems et al., 2018; Mitrea and Kriwacki, 2016; Shin and Brangwynne, 2017). LLPS is driven by excessive levels of transient, selective, and multivalent protein–protein interactions mediated by intrinsically disordered regions (IDRs) within the proteins of interest (Chong et al., 2018; Kato and McKnight, 2018; Li et al., 2012). Whereas rigorous characterization of LLPS in vivo has been challenging and remains a question under active investigation (McSwiggen et al., 2019b), detection of discrete puncta that have a spherical shape, undergo fusion and fission, and dynamically exchange biomolecules with the surrounding according to FRAP is often considered evidence of putative LLPS in living cells. While such diverse measurements have been widely used for studying proteins under overexpression conditions, far fewer approaches are available to probe LLPS under physiological conditions. Detecting local high-concentration regions or puncta of an endogenously expressed protein using immunofluorescence of fixed cells has been used in many studies as evidence of LLPS (Boija et al., 2018; Guo et al., 2019; Owen et al., 2021; Xie et al., 2022; Yang et al., 2020). Not only is the detection of puncta an inconclusive metric for establishing LLPS, whether a punctate distribution observed in fixed cells actually represents the live-cell scenario remains unclear as fixation has only been assumed, but not directly shown, to faithfully preserve multivalent interactions and LLPS formed in living cells. This knowledge gap motivated us to image cells that overexpress various known IDR-containing proteins before and after fixation to evaluate the ability of PFA fixation to preserve LLPS behaviors. We found that, interestingly, fixation can significantly alter the appearance of droplet-like puncta in cells. Our quantitative image analysis suggests that depending on the LLPS-driving protein, fixing cells can either enhance or diminish the apparent LLPS behaviors in vivo. In certain cases, fixation can even cause droplet-like puncta to artificially appear in cells that have a homogeneous protein distribution and no detectable puncta in the live condition. Conversely, fixation can also cause droplet-like puncta in living cells to completely disappear. Combining experiments that modulate fixation rates, live-cell single-molecule imaging that quantifies protein binding dynamics, and simulations based on a kinetic model, we further demonstrated that protein localization in fixed cells depends on an intricate balance of protein–protein interaction dynamics, the overall rate of fixation, and the difference between protein fixation rates in and out of droplet-like puncta. Our work urges caution in the interpretation of previous claims of in vivo phase separation based solely on immunofluorescence imaging of fixed cells and serves to guide future judicious application of PFA fixation. Results Fixation enhances the LLPS appearance of FET family proteins To investigate the effect of PFA fixation on the appearance of LLPS, we first compared confocal fluorescence images of live and fixed U2OS cells that transiently express an IDR tagged with EGFP and a nuclear localization sequence (NLS). We focused on the FET family protein IDRs (AA2-214 of FUS, AA47-266 of EWS, and AA2-205 of TAF15) that are reported to undergo putative LLPS in cells upon overexpression (Altmeyer et al., 2015; Chong et al., 2018; Wang et al., 2018). Figure 1, Video 1, and Figure 1—figure supplement 1 compare the same cells before and after treatment of 4% PFA for 10 min unless otherwise noted, a typical condition utilized for fixed-cell imaging techniques such as immunofluorescence. At high enough expression levels, all three IDRs are able to form discrete and spherical puncta in the live cell nucleus, which show fusion and fission behaviors and are thereby consistent with LLPS droplets (Alberti et al., 2019; Banani et al., 2017). Interestingly, after fixation, the puncta of all three IDRs appear to increase in their numbers, sizes, and contrast compared with the dilute phase. In particular, PFA fixation was able to artificially turn a cell with EGFP-EWS(IDR) homogeneously distributed in the nucleus without any puncta into one with many discrete puncta (Figure 1). We quantified the fixation-induced changes of LLPS appearance by calculating three parameters from the fluorescence images of cells, including the number of puncta, surface roughness, and punctate percentage, and found a significant increase in all three parameters after fixation (Figure 1D–F, Figure 1—source data 1). The number of puncta and punctate percentage (percentage of intranuclear fluorescence intensity in the concentrated phase) are indicators of the propensity to phase separate (Berry et al., 2015). The surface roughness (standard deviation of pixel intensities across the nucleus) quantifies the uneven distribution of a fluorescently labeled protein in the nucleus, allowing for detection of puncta appearance or disappearance without the need for an algorithm to identify individual puncta in the cell. Figure 1 with 4 supplements see all Download asset Open asset Fixation can change the apparent liquid–liquid phase separation (LLPS) behaviors of proteins. (A) EGFP-EWS(IDR), (B) EGFP-FUS(IDR), and (C) EGFP-TAF15(IDR) are transiently expressed in U2OS cells and imaged before and after fixation using confocal fluorescence microscopy. A schematic of each protein construct is shown on the left. A maximum z-projection of a representative live cell expressing its respective protein is shown next to that of the same cell after 10 min of fixation with 4% paraformaldehyde (PFA). The inserts show a zoomed-in region of the cell. (D–F) Quantification of percentage change of LLPS parameters after fixation. The values are averaged from 34 (D), 17 (E), or 24 (F) cells measured in 3 (D), 2 (E), or 2 (F) independent transfection and imaging sessions. Error bars represent standard errors. Asterisks indicate a significant difference compared with 0 (p<0.05, Wilcoxon signed-rank test). Figure 1—source data 1 Quantification of puncta parameters used to generate the bar plots. https://cdn.elifesciences.org/articles/79903/elife-79903-fig1-data1-v3.xlsx Download elife-79903-fig1-data1-v3.xlsx Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Real-time imaging of a U2OS cell expressing EGFP-FUS(IDR) during paraformaldehyde (PFA) fixation. We next tested how the fixation artifact is dependent on the length of PFA treatment, PFA concentration, and the type of fixatives. We performed real-time imaging of live cells expressing EGFP-FUS(IDR) and found that their morphology and LLPS appearance start to change immediately upon PFA treatment and reach a steady state after ~100 s of treatment (Video 1, Figure 1—figure supplement 2). We treated cells expressing EGFP-EWS(IDR) with different concentrations of PFA (1, 2, 4, and 8%) and observed statistically significant changes to the above three LLPS-describing parameters upon fixation at all the concentrations (Figure 1—figure supplement 3). PFA in combination with glutaraldehyde (GA) has been shown to reduce fixation artifacts in imaging the distribution of cell membrane receptors (Stanly et al., 2016). However, we still observed statistically significant fixation-induced changes to the apparent LLPS behavior of EGFP-EWS(IDR) using 4% PFA and 0.2% GA in combination (Figure 1—figure supplement 4). We next compared the intracellular distribution of TAF15(IDR) tagged with different fluorescent tags, including, EGFP, DsRed2, and HaloTag, before and after fixation with 4% PFA. The LLPS behavior of DsRed2-TAF15(IDR) is enhanced upon fixation like EGFP-TAF15(IDR) (Figure 2A), but the enhancement has a different appearance. Whereas there is not a significant change to the large preformed DsRed2-TAF15(IDR) puncta, thousands of smaller puncta emerge in the dilute phase within the nucleus (Figure 2B). In contrast, Halo-TAF15(IDR) displays a diminished LLPS behavior after fixation, with its puncta becoming smaller and dimmer or completely disappearing (Figure 2C, Figure 2—figure supplement 1). Quantification of the number of puncta, surface roughness, and punctate percentage of the TAF15(IDR) LLPS systems before and after fixation further confirmed these observations (Figure 2D–F, Figure 2—source data 1). The fact that different phase-separating proteins can have bifurcating behaviors upon fixation is interesting. While it is known that EGFP and DsRed2 can dimerize and HaloTag cannot (Costantini et al., 2012; Sacchetti et al., 2002), it is unclear whether and how the dimerization potential might contribute to the proteins’ bifurcating responses to PFA fixation. We note that the fixation-induced changes to LLPS appearance can affect the physical characterization of in vivo LLPS systems based on fixed-cell imaging, such as the Gibbs energy of transfer between dilute and concentrated phases (Riback et al., 2020) and how far from the critical concentration a system is (Bracha et al., 2018), potentially affecting the interpretation of the functional role of LLPS in cellular processes. Moreover, the fact that PFA fixation can artificially promote puncta formation even in cells without detectable puncta in the live condition presents an important caveat in fixation-based approaches that have been commonly used for characterizing LLPS under physiological conditions, for example, immunofluorescence. Figure 2 with 1 supplement see all Download asset Open asset Paraformaldehyde (PFA) fixation can both enhance and diminish liquid–liquid phase separation (LLPS) appearance. U2OS cells expressing (A) EGFP-TAF15(IDR), (B) DsRed2-TAF15(IDR), and (C) Halo-TAF15(IDR), ligated with the JFX549 Halo ligand, are imaged using confocal fluorescence microscopy before and after 10 min of fixation with 4% PFA. Schematics of the protein constructs are shown on the left. Live- and fixed-cell images are compared. (D–F) Quantification of LLPS parameters after fixation. The values are averaged from 24 (D), 23 (E), or 10 (F) cells measured in 2 (D), 2 (E), or 3 (F) independent transfection and imaging sessions. Error bars represent standard errors. Asterisks indicate a significant difference compared with 0 (p<0.05, Wilcoxon signed-rank test). Figure 2—source data 1 Quantification of puncta parameters used to generate the bar plots. https://cdn.elifesciences.org/articles/79903/elife-79903-fig2-data1-v3.xlsx Download elife-79903-fig2-data1-v3.xlsx Furthermore, to examine whether all phase-separating proteins show the fixation artifact, we compared live- and fixed-cell images of EGFP-tagged full-length FUS (FUS(FL)). Full-length FUS is reported to have a greater LLPS propensity in vitro than its IDR alone (Wang et al., 2018). We found that EGFP-FUS(FL) overexpressed in live U2OS cells forms many small puncta throughout the nucleus, and we did not observe a significant change of this behavior after PFA fixation (Figure 3A, Figure 3—source data 1). We also fused Halo-tagged TAF15(IDR) to FTH1 that forms a 24-mer (Bellapadrona and Elbaum, 2014 and Bracha et al., 2018) to make an artificial protein with a high LLPS propensity. We found that TAF15(IDR)-Halo-FTH1 overexpressed in live U2OS cells forms large droplet-like puncta and the appearance of LLPS does not significantly change after PFA fixation (Figure 3B, Figure 3—source data 1). In addition, we looked into a native IDR-containing protein, EWS::FLI1, an oncogenic TF causing Ewing sarcoma (Grünewald et al., 2018) and known to form local high-concentration hubs at target genes associated with GGAA microsatellites (Chong et al., 2018). Although there is no convincing evidence that EWS::FLI1 undergoes LLPS under physiological conditions, the formation of its hubs is mediated by the homotypic multivalent interactions of EWS(IDR) within the protein. Excessive levels of such multivalent interactions often result in LLPS (Li et al., 2012). We previously Halo-tagged endogenous EWS::FLI1 in an Ewing sarcoma cell line A673 using CRISPR/Cas9-mediated genome editing (Chong et al., 2018). Here, we compared live and fixed A673 cell images of endogenous EWS::FLI1-Halo and did not observe a significant difference in its distribution (Figure 3C, Figure 3—source data 1). This result suggests that PFA fixation does not change the intracellular distribution of all proteins that have a LLPS potential. Figure 3 Download asset Open asset Not all puncta-forming proteins show the fixation artifact. U2OS cells expressing (A) EGFP-FUS(FL) and (B) TAF15(IDR)-Halo-FTH1, and (C) an A673 cell expressing endogenous EWS::FLI1-Halo are imaged using confocal fluorescence microscopy before and after 10 min of fixation with 4% paraformaldehyde (PFA). Halo-tagged proteins are ligated with the JFX549 Halo ligand before imaging. Schematics of the protein constructs are shown on the left. Live- and fixed-cell images are compared. (D–F) Quantification of puncta parameters after fixation. The values are averaged from 21 (D), 16 (E), or 15 (F) cells measured in 1 (D), 4 (E), or 2 (F) independent transfection and imaging sessions. Error bars represent standard errors. NS: not significant difference compared with 0 (p<0.05, Wilcoxon signed-rank test). None of the examined proteins show significant changes in their liquid–liquid phase separation (LLPS) or hub appearance in the fixed-cell image as compared to the live-cell image. Figure 3—source data 1 Quantification of puncta parameters used to generate the bar plots. https://cdn.elifesciences.org/articles/79903/elife-79903-fig3-data1-v3.xlsx Download elife-79903-fig3-data1-v3.xlsx Switching between enhancing and diminishing the LLPS appearance depends on fixation kinetics To understand what factors are underlying the diverging fixation artifact of in vivo LLPS we performed the fixation imaging with to live cells to PFA fixation. is with and is commonly used to the formation of protein–protein cross-linked by quickly and cross-linked (Hoffman et al., 2015). We utilized to generate a fixation reaction in the cell protein–protein fixation. We found that to live U2OS cells that overexpress DsRed2-TAF15(IDR) the punctate percentage from to from 23 an increase in the of LLPS. Although the underlying mechanism of such increase is we this might be because that an important role in TAF15(IDR) LLPS et al., are enhanced by the presence of of the fixation effect on the LLPS behavior of Whereas PFA fixation in the of enhances the LLPS appearance (Figure Figure in the presence of glycine, fixation many of the smaller puncta formed in live cells to completely and preformed puncta to turn into a shape, with the of the puncta still but the of the protein (Figure None of these fixed-cell images are of live cells, but it that the critical parameters that the artifact of PFA fixation. The that the appearance of droplet-like puncta in fixed cells can be by the presence of that the kinetics of fixation can an essential role in the appearance of LLPS in fixed cells. Figure 4 Download asset Open asset fixation creates a fixation artifact. (A) Fixing U2OS cells that express DsRed2-TAF15(IDR) in the of many small puncta to (B) Fixing cells in the presence of results in a in the number of puncta, with large puncta In both (A) and cells are imaged using confocal fluorescence microscopy before and after 10 min of fixation with 4% paraformaldehyde (PFA). the fixation artifact Given that fixation kinetics are critical to the appearance of LLPS in fixed cells, we a kinetic model et al., 2006). shown in Figure and the model on one protein of interest which before fixation can either be in state – or – of molecules are dynamically in and out of puncta, the percentage of is at an determined by the of the binding and the dissociation These are the exchange rates between and and do not the potential in the rates at the For example, individual molecules at the surface and of a might with different rates, but model does not these We the that PFA is as and fixed of which are cross-linked to proteins within with a fixation rate of and cross-linked to proteins with a fixation rate of fixing to both and are when the cell is fixed after of PFA there is no any concentration in and The fixation artifact of an LLPS system can be as the change in punctate percentage, or the of to after Figure Download asset Open asset bifurcating fixation artifacts. (A) that fixation of a phase-separating protein of interest in the cell. (B) The kinetic model with associated kinetic rates the different (C) of the fixation artifact as a of the punctate percentage and the relative fixation rate the overall fixation rate as as overall protein binding and dissociation rates are fixation liquid–liquid phase separation (LLPS) behavior to be fixation LLPS behavior to be of the fixation artifact as a of the punctate percentage and the relative overall fixation rate individual fixation rates are overall fixation rate compared with protein–protein interaction dynamics the fixation artifact. (C) and punctate from to are on (C) and We hypothesized that the balance between interaction and fixation dynamics in a LLPS system the fixation artifact and tested the by calculating as a of various kinetic and is that the dilute and concentrated phases of an LLPS system have different protein and concentrations and 2022; et al., et al., 2015; et al., The rate of fixation is known to vary with both factors by orders of with the timescale of fixation from to (Hoffman et al., 2015; et al., 2019; Metz et al., Metz et al., protein–protein interactions that LLPS are dynamic with binding residence times in the range of to of (Chong et al., 2018), fixation with either or rates than protein binding and We first examined whether different fixation rates of in and out of puncta can cause a fixation artifact, the overall fixation rates are than protein binding and and how the fixation artifact may on protein–protein interaction we as a of the punctate percentage and the relative fixation rate when the relative overall fixation rate is (Figure In the scenario where the rate of fixation is the same in and out of the puncta the live-cell is in fixed cells of the punctate percentage However, when one fixation rate is faster than the we observe a bifurcating the fixation rate the puncta is greater than the puncta the fixed cell will have a punctate percentage than the live cell, that fixation enhances the apparent LLPS behaviors. the balance is the fixed cells will have diminished apparent LLPS behaviors than in the live cell. For where the punctate percentage is or due to significantly different binding and the dissociation rates or no significant change to LLPS appearance after fixation In suggests that fixation rates in and out of puncta is to cause a fixation artifact of LLPS systems a

Liquid–liquid phase separation (LLPS) forms biomolecular condensates or coacervates in cells. Metabolic enzymes can form phase‐separated subcellular compartments that enrich enzymes, cofactors, and substrates. Herein, we report the construction of synthetic multienzyme condensates that catalyze the biosynthesis of a terpene, α‐farnesene, in the prokaryote E. coli. RGGRGG derived from LAF‐1 was used as the scaffold protein to form the condensates by LLPS. Multienzyme condensates were then formed by assembling two enzymes Idi and IspA through an RIAD/RIDD interaction. Multienzyme condensates constructed inside E. coli cells compartmentalized the cytosolic space into regions of high and low enzyme density and led to a significant enhancement of α‐farnesene production. This work demonstrates LLPS‐driven compartmentalization of the cytosolic space of prokaryotic cells, condensation of a biosynthetic pathway, and enhancement of the biosynthesis of α‐farnesene.

Networking and Dynamic Switches in Biological Condensates

Membrane-less organelles and RNP granules are enriched in RNA and RNA-binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear-cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C-terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid-liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane-less organelles, aggregation within phase-separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS-mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS-mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.

Human LDL undergoes a reversible thermal order-disorder phase transition associated with the cholesterol ester packing in the lipid core. Structural changes associated with this phase transition have been shown to affect the resistance of LDL to oxidation in vitro studies. Previous electron cryo-microscopy studies have provided image evidence that the cholesterol ester is packed in three flat layers in the core at temperatures below the phase transition. To study changes in lipid packing, overall structure and particle morphology in three dimensions (3D) subsequent to the phase transition, we cryo-preserved human LDL at a temperature above phase transition (53°C) and examined the sample by electron microscopy and image reconstruction. The LDL frozen from 53°C adopted a different morphology. The central density layer was disrupted and the outer two layers formed a "disrupted shell"-shaped density, located concentrically underneath the surface density of the LDL particle. Simulation of the small angle X-ray scattering curves and comparison with published data suggested that this disrupted shell organization represents an intermediate state in the transition from isotropic to layered packing of the lipid. Thus, the results revealed, with 3D images, the lipid packing in the dynamic process of the LDL lipid-core phase transition.

Tracer diffusion coefficients have been measured for water in liquid and supercritical carbon dioxide (CO2) from 10 to 35 °C in the pressure range from 130 to 300 bar. The measurements were performed by means of pulsed field gradient NMR (PFG-NMR) methods incorporating compensation for electrical eddy currents and mass convection. In the NMR active volume, the sample was contained in a 1.4 mm i.d. PEEK tube with provisions for recirculation and external sample loading. The diffusion coefficients are consistent with the Stokes−Einstein equation with “slip” boundary conditions and a hydrodynamic radius of 1.7 A for water in the high temperature and low density region. In the low temperature and high density region, the diffusion coefficients indicate either a trend toward “stick” boundary conditions or the dynamic clustering of water molecules.

Aerosol Optical Tweezers Constrain the Morphology Evolution of Liquid-Liquid Phase-Separated Atmospheric Particles

In recent years, there has been a jarring awakening that liquid-liquid phase separation (LLPS) of key protein and nucleic acid scaffolds underpins the biogenesis of diverse membraneless organelles, including P granules and stress granules in the cytoplasm and nucleoli and paraspeckles in the nucleus. These biomolecular condensates are proposed to be critical organizers of subcellular biochemistry and to control the flow of information from genotype to phenotype. Despite clear biological utility, LLPS can also have deleterious outcomes. Phase-separated compartments can concentrate specific RNA-binding proteins (RBPs), such as TDP-43 and fused in sarcoma (FUS), that through low-complexity, prion-like domains have an intrinsic tendency to form self-templating fibrils that are closely tied to fatal neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. This series of reviews illuminates the molecular language underlying membraneless organelle biogenesis, how prion-like domains and post-translational modifications regulate phase behavior, how cells exploit the phase-separation process for adaptive modalities, and how phase separation is corrupted in neurodegenerative diseases. Collectively, these pieces provide a cutting-edge view of our functional and mechanistic understanding of phase separation in physiology and disease.

Understanding how protein rich condensates formed upon liquid-liquid phase separation (LLPS) evolve into solid aggregates is of fundamental importance for several medical applications, since these are suspected to be hot-spots for many neurotoxic diseases. This requires developing experimental approaches to observe in real-time both LLPS and liquid-solid phase separation (LSPS), and to unravel the delicate balance of protein and water interactions dictating the free energy differences between the two. We present a vibrational THz spectroscopy approach that allows doing so from the point of view of hydration water. We focus on a cellular prion protein of high medical relevance, which we can drive to undergo either LLPS or LSPS with few mutations. We find that it is a subtle balance of hydrophobic and hydrophilic solvation contributions that allows tuning between LLPS and LSPS. Hydrophobic hydration provides an entropic driving force to phase separation, through the release of hydration water into the bulk. Water hydrating hydrophilic groups provides an enthalpic driving force to keep the condensates in a liquid state. As a result, when we modify the protein by a few mutations to be less hydrophilic, we shift from LLPS to LSPS. This molecular understanding paves the way for a rational design of proteins.

We introduce a new 2-D hexagon technique to probe the topological structure of the universe, in which we map regions of the sky with high and low galaxy densities onto a 2-D lattice of hexagon unit cells, We define filled cells as corresponding to high density regions and empty cells as corresponding to low density region. The number of filled cells and empty cells are kept same by controlling the size of the cells. By analyzing the six neighbors of each hexagon we can get and compare statistical topological properties of the high density and low density regions in the universe, in order to have a better understanding of the evolution of the universe. We apply this hexagon method on 2MASS data and discover significant topological differences between the high density and low density regions. Both regions have significant (>5 Sigma) topological shifts from the binomial distribution or the random distribution.

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.

This chapter presents the exact results for the grand partition function of the hard hexagon model in both low and high density regions are presented. In the low density region, these results are used to derive the first 25 virial coefficients of the virial expansion. The analyticity of the pressure in the density plane in both the low and high density regions are then presented. The general theory of the chiral Potts model as a two-dimensional statistical model is presented and the eigenvalues of the three-component superintegrable case are computed in detail. The order parameter is discussed and the phase diagram of the general three-component integrable chiral Potts spin chain is given. Open questions are discussed about Q operators, eight-vertex model degeneracies, and conjectures for correlations functions of the superintegrable chiral Potts model.

The cellular response to mechanical stimuli depends largely on the structure of the cell itself and the abundance of intracellular cytomechanical proteins also plays a key role in the response to the stimulation of external mechanical signals. Liquid-liquid phase separation (LLPS) is the process by which proteins or protein-RNA complexes spontaneously separate and form two distinct "phases", ie, a low-concentration phase coexisting with a high-concentration phase. According to published findings, membrane-free organelles form and maintain their structures and regulate their internal biochemical activities through LLPS. LLPS, a novel mechanism for intracellular regulation of the biochemical reactions of biomacromolecules, plays a crucial role in modulating the responses of cytomechanical proteins. LLPS leads to the formation of highly concentrated liquid-phase condensates through multivalent interactions between biomacromolecules, thereby regulating a series of intracellular life activities. It has been reported that a variety of cytomechanical proteins respond to external mechanical signals through LLPS, which in turn affects biological behaviors such as cell growth, proliferation, spreading, migration, and apoptosis. Herein, we introduced the mechanisms of cytomechanics and LLPS. In addition, we presented the latest findings on cytomechanical protein phase separation, covering such issues as the regulation of focal adhesion maturation and mechanical signal transduction by LIM domain-containing protein 1 (LIMD1) phase separation, the regulation of intercellular tight junctions by zonula occludens (ZO) phase separation, and the regulation of cell proliferation and apoptosis by cytomechanical protein phase separation of the Hippo signaling pathway. The proposition of LLPS provides an explanation for the formation mechanism of intracellular membraneless organelles and supplies new approaches to understanding the biological functions of intracellular physiology or pathology. However, the molecular mechanisms by which LLPS drives focal adhesions and cell-edge dynamics are still not fully understood. It is not clear whether LLPS under in vitro conditions can occur under physiological conditions of organisms. There are still difficulties to be overcome in using LLPS to explain the interactions of multiple intracellular molecules. Researchers should pursue answers to these questions in the future.

We compare the process of Liquid-Liquid Phase Separation (LLPS) of flexible macromolecular solutions, with the Liquid-Liquid Crystalline Phase Separation (LLCPS) of semiflexible polymers and rigid filamentous colloids, which involves the formation of a liquid phase that possesses a directional alignment. Although the observed phase separation follows a similar dynamic path, namely nucleation and growth or spinodal decomposition separating two phases of dilute and concentrated compositions, the underlying physics that defines the theoretical framework of LLCPS is completely different from the one of LLPS. We review the main theories that describe the phase separation processes and relying on thermodynamics and dynamical arguments, we highlight the differences and analogies between these two phase separation phenomena, attempting to clarify the inner mechanisms that regulate those two processes. A particular focus is given to metastable phases, as these intermediate states represent a key element in understanding how phase separation works.