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 systems, we performed the above-described fixation imaging assay with glycine added to live cells prior to PFA fixation. Glycine is highly reactive with formaldehyde and is commonly used to quench the formation of protein–protein cross-linked complexes by quickly forming protein–glycine and glycine–glycine cross-linked adducts instead (Hoffman et al., 2015). We thus utilized additional glycine to generate a competitive fixation reaction in the cell against protein–protein fixation. We found that adding 25 mM glycine to live U2OS cells that overexpress DsRed2-TAF15(IDR) increases the starting punctate percentage from 18 ± 1.92 to 36 ± 3.82% (quantified from 23 cells), indicating an increase in the degree of LLPS. Although the underlying mechanism of such increase is unclear, we speculate this might be because hydrophobic intermolecular contacts that play an important role in TAF15(IDR) LLPS (Patel et al., 2017) are enhanced by the presence of hydrophobic glycine. Importantly, addition of glycine dramatically reversed the fixation effect on the LLPS behavior of DsRed2-TAF15(IDR). Whereas PFA fixation in the absence of additional glycine enhances the LLPS appearance (Figure 2B, Figure 4A), in the presence of 25 mM glycine, fixation causes many of the smaller puncta formed in live cells to disappear completely and larger, preformed puncta to turn into a ‘donut’ shape, with the outline of the puncta still visible but the interior devoid of the protein (Figure 4B). None of these fixed-cell images are good representations of live cells, but it appears that glycine affects the critical parameters that control the divergent artifact of PFA fixation. The observation that the appearance of droplet-like puncta in fixed cells can be dramatically modified by the presence of glycine competition emphasizes that the kinetics of fixation can play an essential role in the appearance of LLPS in fixed cells. Figure 4 Download asset Open asset Competitive fixation pathway creates a reversed fixation artifact. (A) Fixing U2OS cells that express DsRed2-TAF15(IDR) in the absence of additional glycine causes many small puncta to appear. (B) Fixing cells in the presence of 25 mM additional glycine results in a reduction in the number of puncta, with large puncta forming ‘donut’ shapes. In both (A) and (B), cells are imaged using confocal fluorescence microscopy before and after 10 min of fixation with 4% paraformaldehyde (PFA). Kinetic modeling explains the fixation artifact Given our observation that fixation kinetics are critical to the appearance of LLPS in fixed cells, we numerically simulated a four-state kinetic model (Hoops et al., 2006). As shown in Figure 5A and B, the model focuses on one protein of interest (POI), which before fixation can either be in state S1 – ‘in puncta’ or S2 – ‘out of puncta.’ Because POI molecules are dynamically exchanged in and out of puncta, the in-puncta percentage (punctate percentage) of POI is at an equilibrium determined by the ratio of the binding rate, k1 , and the dissociation rate, k2 (Pollard, 2010). These are the average exchange rates between S1 and S2 and do not concern the potential spatial inhomogeneity in the rates at the molecular level. For example, individual POI molecules at the surface and interior of a punctum might dissociate with different rates, but our model does not differentiate these molecules. We define the moment that PFA is added as time zero (t=0) and introduce two fixed states of POI, which are S3 (POI cross-linked to proteins within puncta) with a fixation rate of k3 and S4 (POI cross-linked to proteins outside puncta) with a fixation rate of k4 . Because fixing to both S3 and S4 states are irreversible, when the cell is fully fixed long after addition of PFA (t=∞), there is no longer any concentration in S1 and S2 . The fixation artifact of an LLPS system can be represented as the absolute change in punctate percentage, or the ratio of in-puncta POI to total POI, after fixation: (1) ΔPunctate Percentage=Final Punctate Percentage−Initial Punctate Percentage=([S3 ]t=∞[S3 ]t=∞+[S4 ]t=∞−[S1 ]t=0[S1 ]t=0+[S2 ]t=0)∗100 Figure 5 Download asset Open asset Kinetic simulation explains bifurcating fixation artifacts. (A) Schematic that describes fixation of a phase-separating protein of interest (POI) in the cell. (B) The four-state kinetic model with associated kinetic rates connecting the different states. (C) Simulation of the fixation artifact as a function of the starting punctate percentage and the relative in-puncta fixation rate k3:k4 , assuming the overall fixation rate as well as overall protein binding and dissociation rates are constant (k3+k4=0.2, k1+k2=1). Faster in-puncta fixation causes liquid–liquid phase separation (LLPS) behavior to be over-represented (blue). Slower in-puncta fixation causes LLPS behavior to be under-represented (red). (D) Simulation of the fixation artifact as a function of the starting punctate percentage and the relative overall fixation rate (k3+k4):(k1+k2), assuming individual fixation rates are constant (k3=1, k4=2). Fast overall fixation rate compared with protein–protein interaction dynamics decreases the fixation artifact. (C) and (D) were simulated over starting punctate percentages ranging from 0% (k1=0, k2=1) to 100% (k1=1, k2=0). Level curves are marked on (C) and (D). We hypothesized that the balance between interaction and fixation dynamics in a LLPS system causes the fixation artifact and tested the hypothesis by calculating Δ Punctate Percentage as a function of various kinetic and equilibrium parameters. It is well-established that the dilute and concentrated phases of an LLPS system have different protein composition and concentrations (Currie and Rosen, 2022; Koga et al., 2011; Nott et al., 2015; Yewdall et al., 2021). The rate of fixation is known to vary with both factors by orders of magnitude, with the timescale of fixation ranging from seconds to hours (Hoffman et al., 2015; Kamps et al., 2019; Metz et al., 2006; Metz et al., 2004). Because protein–protein interactions that drive LLPS are highly dynamic with binding residence times in the range of seconds to tens of seconds (Chong et al., 2018), fixation likely happens with either lower or comparable rates than protein binding and dissociation. We thus first examined whether different fixation rates of POI in and out of puncta can cause a fixation artifact, assuming the overall fixation rates (k3+k4) are slower than protein binding and dissociation, and how the fixation artifact may depend on intrinsic protein–protein interaction equilibrium. Specifically, we calculated ΔPunctate Percentage as a function of the starting punctate percentage and the relative in-puncta fixation rate (k3:k4) when the relative overall fixation rate is constant ((k3+k4):(k1+k2) = 1:5) (Figure 5C). In the scenario where the rate of fixation is the same in and out of the puncta (k3=k4), the live-cell equilibrium is perfectly preserved in fixed cells regardless of the starting punctate percentage (ΔPunctate Percentage~0). However, when one fixation rate is faster than the other, we observe a bifurcating effect. When the fixation rate inside the puncta is greater than outside the puncta (k3 > k4), the fixed cell will have a higher punctate percentage than the live cell, that is, fixation enhances the apparent LLPS behaviors. When the balance is reversed (k4>k3), the fixed cells will have diminished apparent LLPS behaviors than in the live cell. For cases where the starting punctate percentage is near 0% or 100% due to significantly different POI binding and the dissociation rates (k2≫k1 or k1≫k2), no significant change to LLPS appearance happens after fixation (ΔPunctate Percentage~0). In short, our simulation suggests that having unequal fixation rates in and out of puncta is necessary to cause a fixation artifact of LLPS systems a

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