Appendix.

Editor's evaluation: Fixation can change the appearance of phase separation in living 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

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.

Liquid and solid phase separation during melt spinning and annealing in melt-spun Cu–Cr ribbons

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

Networking and Dynamic Switches in Biological Condensates

Amyloid fibrils are rigid, elongated protein aggregates that form condensed phases in functional biological assemblies and pathological deposits. From a colloidal physics perspective, two separation pathways can lead to these condensed phases: liquid-liquid phase separation (LLPS), which results from a trade-off between entropy and enthalpy, and liquid-liquid crystalline phase separation (LLCPS), an essentially entropic process in which the condensed phase is a nematic liquid following precise symmetry rules. The interplay between these pathways in amyloid fibril dispersions is debated. Here we show that lysozyme and β-lactoglobulin amyloid fibrils dominantly undergo LLCPS but exhibit a pH-dependent transition to LLPS. Particularly, increasing pH lowers the critical concentration for formation of nematic condensates until, near isoelectric point, where condensates without a coherent nematic order form. This behavior is consistent with the classical picture of rigid rods retaining nematic order once formed, with enthalpic attraction overcoming entropic ordering at low charge densities. Our work establishes that LLCPS and LLPS in amyloid fibrils are separable and provides a framework for controlling fibril organization using simple solution conditions. The competition between LLCPS and LLPS may clarify the mesoscopic organization of amyloid fibrils in condensates in vivo and direct design principles of multiphase amyloid-based materials.

Liquid-liquid phase separation (LLPS) of proteins can be considered an intermediate solubility regime between disperse solutions and solid fibers. While LLPS has been described for several pathogenic amyloids, recent evidence suggests that it is similarly relevant for functional amyloids. Here, we review the evidence that links spider silk proteins (spidroins) and LLPS and its role in the spinning process. Major ampullate spidroins undergo LLPS mediated by stickers and spacers in their repeat regions. During spinning, the spidroins droplets shift from liquid to crystalline states. Shear force, altered ion composition, and pH changes cause micelle-like spidroin assemblies to form an increasingly ordered liquid-crystalline phase. Interactions between polyalanine regions in the repeat regions ultimately yield the characteristic β-crystalline structure of mature dragline silk fibers. Based on these findings, we hypothesize that liquid-liquid crystalline phase separation (LLCPS) can describe the molecular and macroscopic features of the phase transitions of major ampullate spidroins during spinning and speculate whether other silk types may use a similar mechanism to convert from liquid dope to solid fiber.

Metal screens are commonly used as components for fluid handling in spacecraft and rocket tank designs. In most cases the screen performs a passive separation of the gaseous from the liquid propellant phase. This means that the liquid is able to flow through the screen, causing a flow through screen pressure drop, while the gaseous phase is blocked due to the pressure jump across a curved liquid-gas interphase at the small screen pores. As long as the flow through screen pressure drop is smaller than the bubble point pressure, phase separation is possible and allows the provision of gas-free liquid for the spacecraft or rocket engine. The opposite, the separation of the liquid from the gaseous propellant phase, is more challenging. Liquid-gas phase separation means that the gaseous phase is allowed to enter the phase separation device while the liquid phase is blocked. The separation of the liquid from the gas is possible due to a double screen element, as the work of Conrath et al. (Int. J. Multiphase Flow 50, 1–15, 2013) and Behruzi et al. (2013) has shown for storable liquids in Earth’s gravity and microgravity as well as for cryogenic liquids in Earth’s gravity. The liquid-gas phase separation of cryogenic liquids in microgravity however has not been investigated yet. Therefore, an experimental campaign consisting of six drop tests in microgravity using the drop tower at the University of Bremen, has been conducted. The experimental results confirm predicted governing physical phenomena and give evidence about further fluid mechanical and thermodynamical effects.

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.

Abstract. Liquid–liquid phase separation (LLPS) in organic aerosol particles can impact several properties of atmospheric particulate matter, such as cloud condensation nuclei (CCN) properties, optical properties, and gas-to-particle partitioning. Yet, our understanding of LLPS in organic aerosols is far from complete. Here, we report on the LLPS of one-component and two-component organic particles consisting of α-pinene- and β-caryophyllene-derived ozonolysis products and commercially available organic compounds of relevance to atmospheric organic particles. In the experiments involving single-component organic particles, LLPS was observed in 8 out of 11 particle types studied. LLPS almost always occurred when the oxygen-to-carbon elemental ratio (O:C) was ≤0.44 but did not occur when O:C was &gt;0.44. The phase separation occurred by spinodal decomposition as well as the nucleation and growth mechanism, and when LLPS occurred, two liquid phases coexisted up to ∼100 % relative humidity (RH). In the experiments involving two-component organic particles, LLPS was observed in 23 out of 25 particles types studied. LLPS almost always occurred when the average was O:C ≤0.67 but never occurred when the average O:C was &gt;0.67. The phase separation occurred by spinodal decomposition as well as the nucleation and growth mechanism. When LLPS occurred, two liquid phases coexisted up to ∼100 % RH. These results provide further evidence that LLPS is likely a frequent occurrence in organic aerosol particles in the troposphere, even in the absence of inorganic salts.

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.

Liquid–liquid phase separation (LLPS) is a thermodynamically driven process that occurs in mixtures of low miscibility material. LLPS is an important process in chemical, biological, and environmental systems. In atmospheric chemistry, LLPS in aerosol containing internally-mixed organic and inorganic particles has been an area of significant interest, with particles separating to form organic-rich and aqueous phases on dehydration. This alters the optical properties of the particles, has been connected to changes in the cloud nucleation ability of the aerosol, and potentially changes the reactivity of particles towards gas-phase oxidants. Although the chemical systems that undergo LLPS have become quite well-characterized, the properties and processes of LLPS particles are quite poorly understood. In this work, we characterize LLPS in aerosol particles containing ammonium sulfate and triethylene glycol (3EG), a semi-volatile organic molecule. We explore the relative humidity (RH) conditions under which LLPS occurs and characterize the rate of evaporation of 3EG from well-mixed and LLPS particles as a function of RH. We show that the evaporation rates vary with RH due to changes in chemical activity, however no clear change in the dynamics following LLPS are observed. We interpret our observations using a thermodynamic model (AIOMFAC) coupled with an evaporation model and show that a significant increase in the activity coefficient of 3EG as the RH decreases, required for LLPS to occur, obscures a clear step-change in the evaporation rates following LLPS. By characterizing the evaporation rates, we estimate the composition of the organic-rich phase and compare our results to thermodynamic predictions. This study is the first to explore the connection between LLPS and the chemical evolution of aerosol particles via the evaporation of semi-volatile organic material. Ultimately, we reveal that the thermodynamics of non-ideal mixing are primarily responsible for the controlling both the rate of evaporation and the onset of LLPS, with LLPS itself having limited impact on the rate of evaporation in a fluid system. These results have significant implications for understanding and predicting the lifetime of aerosol particles, their effect on cloud formation, and the chemical evolution of multiphase systems by particle-gas partitioning and heterogeneous reactions.

Spring viremia of carp virus (SVCV), a highly pathogenic rhabdovirus prevalent in fish, causes substantial mortality by evading host antiviral immunity; however, the underlying mechanisms remain incompletely understood. This study reveals a novel immune evasion strategy whereby the SVCV phosphoprotein (SVCV-P) hijacks the TBK1-IRF3 signaling axis via liquid-solid phase transition (LSPT), sequestering interferon regulatory factor 3 (IRF3) and inhibiting interferon (IFN) production. Upon stimulation, IRF3 facilitates TBK1 into functional liquid-liquid phase separation (LLPS) condensates, spatially enhancing IRF3 phosphorylation and downstream IFN responses. IRF3 acts as a scaffold via its DNA-binding domain (DBD) and intrinsically disordered region (IDR), while TBK1 incorporates via its kinase domain (KD), ubiquitin-like domain (ULD), and scaffold dimerization domain (SDD). Conversely, SVCV-P, driven by its IDRs and central domain (CD), undergoes robust LLPS, competitively recruiting TBK1 into SVCV-P-TBK1 condensates. These condensates merge with IRF3-TBK1 droplets, forming SVCV-P-TBK1-IRF3 ternary condensates. These subsequently undergo LSPT, immobilizing IRF3 and preventing its nuclear translocation. In vitro reconstitution and domain-deletion assays confirmed key domain roles in mediating LLPS and LSPT. Disrupting SVCV-P LLPS restored IFN expression and reduced viral replication in vitro. Zebrafish infection models demonstrated SVCV-P-mediated LLPS impaired IFN signaling and increased mortality. Phase-separation-deficient mutants (SVCV-PΔIDR) lost immunosuppressive activity; this defect was rescued by chimeric SVCV-P proteins with heterologous LLPS domains. This study unravels a novel LLPS-dependent mechanism for TBK1-IRF3 signalosome regulation and demonstrates how SVCV hijacks phase separation to remodel host complexes into pathological aggregates, providing a paradigm for viral immune evasion and suggesting new antiviral targets.IMPORTANCEUnderstanding interferon (IFN) signaling regulation and viral evasion is central to host-pathogen interactions. The discovery of liquid-liquid phase separation (LLPS) in cellular activities provides a new perspective for such investigations. Spring viremia of carp virus (SVCV), a severe fish pathogen, has potent IFN evasion capabilities, making it an attractive research model. Here, we demonstrate that LLPS spatially enhances IFN production by concentrating interferon regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK1) into functional droplets, thereby boosting IRF3 activation. However, the SVCV phosphoprotein (SVCV-P) disrupts this via dual phase-separation mechanisms. First, SVCV-P undergoes LLPS to hijack TBK1 into viral-host condensates, sequestering it from IRF3. Second, these droplets merge with host defense droplets, trapping IRF3 within ternary aggregates. This paralyzes IRF3, blocking its nuclear translocation and IFN production. These findings provide new insights into how viruses exploit phase separation to block innate immune signaling, highlighting LLPS as a promising cross-species antiviral target.

Associative properties of rapeseed napin and pectin: Competition between liquid-liquid and liquid-solid phase separation