Selective membrane wetting of phase-separated giant unilamellar vesicles by coacervate droplets

Domain: neuroscience
Published: 2026-04-11
Editorial outcome: major_revision

Abstract
Liquid-liquid phase separation (LLPS) has emerged as a key biological paradigm, playing a critical role in cellular compartmentalization and potentially contributing to the origins of life. Lipid membranes are equally important in these processes, serving as selective barriers that define and protect cellular environments. Recent studies have revealed a dynamic interaction between LLPS and lipid membranes, both in cellulo and in biomimetic systems. In vitro studies typically use simplified model membranes, while biological membranes are far more complex, often exhibiting phase-separated domains. Despite growing interest, the relationship between lipid phase separation within membranes and LLPS remains largely unexplored. Here, we explore how coacervates interact with membranes composed of liquid-ordered or liquid-disordered lipid phases using giant unilamellar vesicles (GUVs). We show that coacervate-membrane interactions are modulated by coacervate composition and lipid domain properties. Notably, we report the selective wetting of lipid domains in phase separated GUVs. Different coacervates exhibit distinct wetting behaviors, with variations in their composition influencing whether they preferentially wet liquid-disordered or liquid-ordered lipid domains, highlighting the coacervate-specific nature of these interactions. These findings highlight how lipid phase separation can mediate selective membrane wetting, offering insights into condensate-membrane interactions in cells and in simplified cell-like model systems.

Introduction
Liquid-liquid phase separation (LLPS) is emerging as a central paradigm in biology,1 and is hypothesized to have played a role in the emergence of protocells.2 In living cells, phase-separated droplets, known as biomolecular condensates, perform critical biological functions, such as metabolic regulation, signal transduction and stress management.3 In vitro, droplets produced by associative LLPS between polymers or surfactants, known as coacervates, capture key physicochemical properties of biomolecular condensates,4 5 including increased viscosity and reduced surface tension, but also serve as plausible protocell models by concentrating chemical species,6 7 enabling reactions that would otherwise be unfavorable in aqueous environments.8-11

Lipid membranes are also fundamental to both cellular and protocellular compartmentalization and evolution.1 Modern cells, but also a number of intracellular organelles, are delineated by complex proteolipidic membranes that form selectively permeable barriers capable of regulating the exchange of solutes. This structural role is equally important when considering the origins of life, as simple membranes must have been essential to define and separate the internal protocell environment from the surrounding medium.14

These two forms of compartmentalization – phase-separated droplets and membrane-bounded structures – are not mutually exclusive. Recent studies have highlighted the interactions between liquid-liquid phase separation and lipid membranes, revealing a dynamic interplay that influences both cellular function and protocell models. In living cells, biomolecular condensates often interact closely with lipid membranes.15 These interactions can drive processes such as membrane remodeling16 and protein recruitment,17 playing a key role in cellular dynamics and signaling pathways. In vitro, early experimental studies using PEG/dextran aqueous two-phase systems (ATPS) encapsulated within giant unilamellar vesicles (GUVs) provided foundational insights into membrane wetting and the role of interfacial tension balance in determining wetting morphology.18 19 More recent studies have delved deeper into the mechanisms by which coacervates interact with lipid membranes.19-21 On the one hand, the interfacial assembly of lipids around coacervates creates hybrid protocells, combining a crowded interior able to accumulate chemicals with a physical barrier that prevents droplet coalescence and limits material exchange.22-25 On the other hand, pioneering studies have started investigating interactions between biomolecular condensates or coacervates and model lipid membranes using GUVs. In a seminal example, electrostatic interactions and hydrophobic lipid bilayer anchoring were shown to promote coacervate binding to membranes.26 More recently, charge-dependent wetting of GUVs by coacervates was reported to modulate lipid packing27 and induce membrane remodeling,28 with examples showing that strong coacervate-vesicle interactions favored partial or complete endocytosis of the droplets within the GUVs.29 30

These observations represent the first steps towards elucidating coacervate-membrane interactions based on non-specific interactions. However, cellular membranes are composed of complex lipid mixtures, where lipid phase separation can give rise to domains enriched in specific lipid species. This complexity, driven by the dynamic and heterogeneous nature of biological membranes, raises fundamental questions about the role of lipid composition, membrane fluidity and domain formation in modulating coacervate-membrane interactions. In this context, the interplay between lipid phase separation within bilayers and liquid-liquid phase separation remains largely unexplored. Recent work has shown a dynamic interplay between domain formation in ternary lipid membranes and LLPS using PEG/dextran systems.31 In parallel, a study revealed that condensates preferentially interact with liquid-disordered domains in phase-separated membranes, highlighting the need to understand how both lipid composition and the chemical nature of the condensate control this selectivity.32

Here, we assemble GUVs using high- and low-melting temperature lipids, forming either liquid-disordered or liquid-ordered bilayers. We investigate their interactions with polyelectrolyte-based coacervates prepared at varying positive-to-negative charge ratios. We find that electrostatic interactions are not the primary driver of coacervate-vesicle interactions; rather, the lipid composition dictates the preferential affinity of coacervates for more fluid membranes. We identify conditions where coacervates selectively wet one type of GUVs in a binary vesicle population. Additionally, using phase-separated vesicles with micron-sized lipid domains, we demonstrate that coacervates selectively wet fluid domains, irrespective of net surface charges. We further demonstrate that such wetting is reversibly modulated by temperature, reflecting changes in lipid mixing. Importantly, we find that selective domain wetting can be inverted depending on the chemical nature of the coacervate, revealing that coacervate chemistry provides an additional lever, complementary to membrane composition, for tuning membrane affinity. Altogether, these insights underscore the potential of lipid phase separation to mediate selective membrane wetting by coacervates, offering a compelling framework for understanding the role of condensate-membrane interactions in cells and in simplified cell-like model systems.

Results
Surface charge does not dictate selective coacervate-membrane interactions
Our model coacervate droplets were assembled by mixing poly(diallyldimethylammonium chloride) (PDDA, M = 8,300 g mol-1, 5-20 mM expressed in charge concentration) and poly(sodium acrylate) (PAA, M = 5,100 g mol-1) close to charge stoichiometry in water at pH 8 (Figure 1a). Optical microscopy images of the obtained turbid suspension confirmed the formation of liquid-like micrometer-sized coacervate droplets (Figure 1b). Turbidity screening revealed that optimal coacervation conditions were met at charge neutrality, although some turbidity persisted for a PDDA/PAA molar charge ratio comprised between 0.6 and 2 (Supplementary Figure 1). The coacervate surface charge changed from negative to positive by tuning the polyelectrolyte charge ratio within this range (Figure 2a). Coacervate droplets were fluorescently labeled by spontaneous sequestration of Cyanine 5-tagged single-stranded DNA (Cy5-ssDNA, 10 µM, Supplementary Table 1) (Figure 1b, inset).

Model giant unilamellar vesicles (GUVs) were prepared using the lipid film hydration method in a 200 mM sucrose buffer, first employing either DOPC (T = -20 °C) or DPPC (T = +41.3 °C). Due to their low melting temperature, DOPC phospholipids formed membranes in a liquid disordered phase at room temperature33 34 (ℓ , Figure 1c). In contrast, the high rigidity of DPPC membranes at room temperature (gel phase33) hindered proper GUV formation, even when lipid film hydration was performed at 60°C (Supplementary Figure 2). To address this, 30 mol% cholesterol was added during DPPC lipid film formation to increase membrane fluidity and produce a liquid-ordered lipid phase35 36 (ℓ , Figure 1d), allowing for a higher GUV production yield. The DOPC- and DPPC-based GUVs were labelled with red fluorescent (Rho-PE, 0.1 mol%, Figure 1e, coloring to magenta was used) and green fluorescent (NBD-PE, 1 mol%, Figure 1f) lipids, respectively. Fluorescence recovery after photobleaching confirmed the higher lateral mobility of the lipids within DOPC membranes compared to DPPC ones (Figure 2c). Both types of vesicles exhibited a negative net charge (Figure 2b), in line with previous reports,37 38 with a slightly less negative ζ-potential observed in DPPC GUVs likely due to the addition of the fluorescently labelled NBD-PE lipid (Supplementary Figure 3).

We next mixed GUVs and coacervates in an imaging chamber to investigate their interactions using confocal fluorescence microscopy. We noted that a small fraction (approx. 20%) of the GUVs broke during this mixing step, possibly due to osmotic stress or shearing by pipetting. Nevertheless, a sufficient fraction of GUVs remained intact and was used to characterize interactions with coacervates. As an example, coacervates prepared at a PDDA/PAA molar charge ratio of 0.7 (negative surface charge) appeared to partially wet DOPC vesicles (Figure 1g) but did not interact with DPPC/cholesterol vesicles (Figure 1h). Since both types of GUVs carried a net negative ζ-potential (Figure 2b), these preliminary observations suggested that differences in electrostatic attraction could not account for the selective interaction of coacervates with DOPC vesicles.

To test this hypothesis further, we then systematically varied the PDDA/PAA molar charge ratio, thereby altering the net surface charge of the coacervates (Figure 2a), and monitored their interactions with GUVs. We found that coacervates prepared with a large excess of PAA (8 mM; ζ < 0 mV) did not interact with any of the GUVs (ζ < 0 mV), as indicated by the coexistence of non-adhering and non-deformed droplets and vesicles (Figure 2d(i,iii)). This suggested that electrostatic repulsion between same-charge coacervates and GUVs suppressed adhesion. In contrast, coacervates prepared at equimolar or lower amounts of PAA (≤ 5 mM; ζ > 0 mV) interacted with both types of GUVs (ζ < 0 mV), consistent with electrostatic attraction between oppositely charged compartments. Under these conditions, we only observed partial wetting, which primarily caused deformations to the lipid membranes while maintaining the overall spherical shape of the droplets. Most importantly, selective wetting of DOPC vesicles occurred only for a specific polyelectrolyte ratio (PDDA/PAA = 0.7) where coacervates exhibited a net negative surface charge (same as for GUVs). This observation suggests that interaction selectivity is driven by non-electrostatic factors, such as short-range interactions or differences in membrane physical properties.

Taken together, these results show that electrostatic interactions influence coacervate-GUV adhesion at the extremes of coacervate charge (highly negative coacervates repel GUVs, while positive coacervates adhere to both types of vesicles). However, the selective wetting of DOPC vesicles observed only within a narrow range of mildly negative coacervates cannot be explained by electrostatics. This indicates that additional factors govern the selective interaction between coacervates and GUVs.

Membrane fluidity governs the strength of coacervate wetting
To gain deeper insight into these interactions, we quantified the extent of coacervate/vesicle contact by extracting two complementary parameters: the geometric factor, Φ, used in previous studies to assess membrane curvature.28 The geometric factor, ranging between +1 (no wetting) and -1 (complete wetting, i.e., total engulfment of vesicles within coacervates), was calculated from the macroscopic contact angles on the coacervate/membrane/outer phase triple line (Figure 3a and Supplementary Figure 4). Additionally, we determined the intrinsic contact angle, θ , derived from the macroscopic contact angles, which reflects the local wetting preference of coacervates for the vesicle membrane based on interfacial tension balance at the contact line39 (Figure 3a, inset). This angle varies between 0° (non-wetting) and 180° (complete wetting). Contact angles were extracted from 2D images obtained by confocal microscopy, typically in the central plane of the compartments. Thus, the reported Φ and θ are apparent value that in do not account for three-dimensional variations of curvature outside the imaging plane. However, Φ and θ are largely independent of the relative size of coacervates and vesicles;28 nevertheless, to ensure that in both the coacervate and GUV were captured near their equatorial planes, we focused on coacervates and GUVs of similar size. Results revealed that for both DOPC and DPPC/cholesterol vesicles the geometric factor decreased while the intrinsic contact angle increased as the PDDA/PAA molar charge ratio increased, confirming the observed increased propensity of coacervates to interact with GUVs (Figures 3b,c). Specifically, Φ for DOPC vesicles decreased toward 0 and θ increased toward ~90° as the PAA in concentration decreased, reflecting an enhanced wetting tendency, whereas Φ for DPPC/cholesterol vesicles plateaued at ~0.4-0.5 and θ at ~60°, indicating that wetting remained limited even at the most in favorable coacervate compositions. Importantly, these measurements also confirmed that for a PDDA/PAA ratio of 0.7, the coacervates interacted selectively with DOPC vesicles (Φ = 0.85 ± 0.12 < 1; θ in = 17 ± 10 ° > 0) but not with DPPC/cholesterol vesicles (Φ = 1; θ = 0). Altogether, our findings indicated in that coacervates showed a higher wetting propensity of fluid DOPC membranes compared to more rigid DPPC/cholesterol bilayers.

To investigate further the role of membrane fluidity on coacervate wetting, we prepared DOPC GUVs containing 30 mol% cholesterol, which produced membranes with a slightly reduced fluidity compared to pure DOPC vesicles, as confirmed by FRAP (Figure 2c and Supplementary Table 2), while still exhibiting a net negative surface charge (Figure 2b). Similar to all other GUVs, confocal fluorescence imaging revealed that coacervates did not interact with these vesicles at low PDDA/PAA molar charge ratio but partially wetted them above a ratio of 0.8 (Figure 2d(ii)). Across this range of PDDA/PAA ratios, the geometric factor Φ plateaued at ~0.5-0.6 and θ at ~50-60°, indicating an intermediate wetting propensity that more in closely resembled DPPC/cholesterol vesicles than pure DOPC. Notably, unlike pure DOPC vesicles, DOPC/cholesterol GUVs did not interact with coacervates prepared at a PDDA/PAA ratio of 0.7 (Figures 3b,c). These findings indicated that addition of cholesterol to DOPC membranes, which produced more ordered lipid bilayers, reduced the propensity of coacervates to interact with GUVs.

Coacervates selectively wet fluid lipid domains
We then sought to exploit the preferential affinity of coacervates for fluid membranes to demonstrate selective wetting capability. Initially, we simply mixed a binary population of DOPC and DPPC/cholesterol vesicles with coacervate droplets prepared at a PDDA/PAA molar charge ratio of 0.7. In these conditions, we observed that coacervates interacted selectively with DOPC GUVs (Figure 4a), mirroring results in individual GUV populations. In comparison, the same experiment performed at a PDDA/PAA molar charge ratio of 1 showed that coacervates interacted with both types of GUVs, leading to the formation of larger clusters (Supplementary Figure 5).

Building upon the well-established ternary DOPC/DPPC/cholesterol phase diagram,33 40 we then moved to preparing phase-separated GUVs exhibiting DOPC- and DPPC-rich domains at room temperature (Supplementary Figure 6). We mixed these phase-separated GUVs with coacervate droplets prepared at a PDDA/PAA molar charge ratio of 0.7. Coacervates appeared to selectively wet the DOPC-rich domains on GUVs (Figure 4b). We repeated these observations at varying PDDA/PAA molar charge ratio (Figure 4c). Similar to DOPC, DOPC/cholesterol and DPPC/cholesterol, excess PAA (8 mM) resulted in non-wetting of GUVs. Partial wetting, associated with a geometric factor lower than 1 and an intrinsic contact angle of ca. 60° (Figures 3b,c), was observed for PDDA/PAA molar charge ratios higher than 0.7, similar to pure DOPC vesicles. Specifically, for lower PAA concentrations, Φ decreased toward ~0.2 and θ increased to in ~70-80° on DOPC-rich domains, indicating a higher wetting propensity than for DOPC/cholesterol domains but lower than for pure DOPC vesicles. This likely reflects the partitioning of cholesterol into DPPC-rich domains,40 making DOPC-rich domains more fluid than DOPC/cholesterol but less fluid than pure DOPC. Importantly, for all these ratios, we observed that coacervates selectively wetted DOPC-rich domains (Figure 4c), despite the affinity of coacervates for both individual DOPC and DPPC/cholesterol vesicles. Statistical analyses confirmed these observations, showing a > 95% preference for DOPC domains at a PDDA/PAA ratio of 0.7 (Figure 4d). When this ratio was increased, the selective wetting of DOPC domains persisted, although with a slightly decreased preference (~75%). Time-lapse imaging at a PDDA/PAA ratio of 1 showed that coacervate wetting reached a stable configuration within 30 seconds-3 minutes after initial contact and remained unchanged for at least 20 minutes (Supplementary Figure 7 and Supplementary Movies 1-2). During this brief transient period, some coacervates initially adhered at the boundary between the two lipid phases, followed by lipid rearrangement that relocated the droplets onto the DOPC-rich regions, indicating that selective wetting was dynamically established through short-lived membrane reorganization.

We further explored the effect of temperature on selective wetting using PDDA/PAA coacervates at a fixed molar charge ratio of 1. Phase-separated GUVs were heated up to 50°C and subsequently cooled down to room temperature while monitoring coacervate-membrane interactions by confocal microscopy (Figure 4e and Supplementary Movie 3). Upon heating, mixing of the two lipid domains was detected at 34-35°C in the coacervate-free membrane segment (Supplementary Figure 8). This lipid mixing was accompanied by a gradual decrease in coacervate wetting propensity, reflected by a smooth two-fold increase in the geometric factor Φ (Figure 4f) and ca. 1.2-fold decrease in the intrinsic contact angle (Figure 4g). Upon cooling, lipid domains re-emerged at around 34-35°C (Supplementary Figure 8), concomitant with the restoration of the initial wetting configuration and a sharp decrease and increase in Φ and θ , respectively, toward their initial value below 34°C. We note a hysteresis between heating and cooling, which may reflect the coupling between lipid phase behavior and droplet adhesion; however, additional experiments would be required to establish the origin of this effect. Importantly, reversibility indicates that selective wetting of DOPC domains is an equilibrium property and not a kinetically trapped state. Statistical analyses on 6 different GUVs confirmed a heterogeneous yet significant two-fold increase of Φ (Figure 4h) and ca. 1.2-fold decrease of θ (Figure 4i) at 50°C compared to room temperature. In comparison, control experiments using homogeneous DOPC and DPPC/cholesterol vesicles showed no significant temperature dependence of wetting (Supplementary Figure 9 and Figures 4h,i). These observations indicate that temperature modulates wetting primarily through changes in membrane organization due to lipid mixing rather than through membrane fluidization or an effect on coacervate properties. In particular, the reduced wetting observed at elevated temperature for Janus-like GUVs is consistent with lipid mixing diminishing the contrast between fluid and ordered membrane environments that underlies selective wetting.

Overall, these observations indicate that PDDA/PAA coacervates preferentially wetted more fluid lipid domains on phase-separated GUVs. This behavior aligns with a recent study showing that lipid packing primarily determines protein- and peptide-based coacervates affinity for fluid membranes.32 Our results further show that this preference persists across PDDA/PAA ratios, although the extent of wetting increases at lower PAA concentrations. Temperature-controlled experiments reveal that mixing of lipid domains above the miscibility transition reversibly decreased coacervate wetting. Taken together, these findings highlight that coacervate-membrane interactions are strongly influenced by membrane fluidity, with the strength of wetting modulated by coacervate composition and temperature, while the preference for fluid domains remains consistent.

Chemical composition of coacervates modulates wetting selectivity
Last, to assess the generality of this selective wetting behavior, we tested several other coacervates which ζ-potential was also assessed (Supplementary Figure 10). First, we replaced PAA with carboxymethyl-dextran (CM-dex, M = 20 kg mol-1) to produce PDDA/CM-dextran coacervates. These droplets exhibited a wetting behavior similar to that of PDDA/PAA coacervates (Figure 5a, e), namely, no wetting of GUVs at high polyanion concentrations (ζ < 0 mV) and selective wetting of DOPC domains at lower concentrations (ζ > 0 mV, Figures 5b, f). Next, we tested coacervates assembled from PDDA and a low molecular weight anion, adenosine triphosphate (ATP), which ζ-potential remained slightly negative across ratios (Supplementary Figure 10). At low ATP concentrations, the coacervates selectively wetted the DOPC domain, similar to PDDA/PAA droplets. However, when ATP was used in excess, we observed that coacervates wetted preferentially DPPC domains (Figure 5c), with a selectivity higher than 90% (Figure 5g). Finally, we substituted PDDA with diethylaminoethyl-dextran (DEAE-dex M = 500 kg mol-1) to generate DEAE-dex/PAA coacervates. These droplets displayed a markedly different wetting behavior: while no wetting of GUVs occurred at low polycation concentration (ζ < 0 mV), higher concentrations (ζ > 0 mV) led to the preferential wetting of less fluid DPPC domain (Figures 5d, h), together with complete wetting at the highest DEAE-dex concentration (Supplementary Figure 11). Notably, in complete-wetting configurations, the coacervate wrapped the DPPC-rich domain, while the DOPC-rich domain tended to face the surrounding continuous phase.

These results highlight that the chemical nature of the coacervating species influences selectivity towards specific lipid domains. Three distinct behaviors emerged: (i) no wetting at low polycation/polyanion ratios and selective wetting of DOPC domains at higher ratios (PDDA/PAA and PDDA/CM-dextran); (ii) selective wetting of DPPC domains at low polycation/polyanion ratios and selective wetting of DOPC domains at higher ratios (PDDA/ATP); (iii) no wetting at low polycation/polyanion ratios and selective wetting of DPPC domains at higher ratios (DEAE-dextran/PAA).

Control experiments on pure DOPC and DPPC/cholesterol vesicles were performed for all conditions to assess whether the domain selectivity observed on phase-separated GUVs reflected an intrinsic preference for one membrane composition (Figure 5a-d). PDDA/CM-dex coacervates behaved identically to PDDA/PAA: selective wetting of the DOPC-rich domain on phase-separated GUVs was accompanied by wetting of both pure DOPC and DPPC/cholesterol vesicles, whereas conditions showing no wetting on Janus-like GUVs also showed no wetting of either pure vesicle type. PDDA/ATP coacervates exhibited two regimes: under conditions where they selectively wetted the DOPC-rich domain on phase-separated GUVs, they wetted both pure vesicle types, similar to PDDA/PAA and PDDA/CM-dex; in contrast, when they selectively wetted the DPPC-rich domain on Janus-like GUVs, they wetted DPPC/cholesterol but not DOPC vesicles, revealing a genuine preference for DPPC-rich membranes. DEAE-dex/PAA coacervates showed a similarly strong DPPC selectivity, wetting DPPC/cholesterol but not DOPC vesicles whenever wetting occurred on Janus-like GUVs.

These interactions were quantified via the geometric factor and intrinsic contact angle analyses for one select polyion ratio (Supplementary Figure 12). Specifically, we fixed the polycation/polyanion ratio to explore different wetting scenarios, including selective wetting of DOPC domains (PDDA/PAA: 5 mM/5 mM; PDDA/CM-dex: 5 mM/2.5 mg mL-1; and PDDA/ATP: 5 mM/2.5 mM), and selective wetting of DPPC domains (DEAE-dex/PAA: 2.5 mg mL-1/5 mM). Interestingly, even when no clear preference for either pure DOPC or DPPC/cholesterol vesicles was apparent from confocal images, geometric factor and intrinsic contact angle analyses revealed a consistent trend: the domain selectively wetted on phase-separated GUVs corresponded to the membrane composition that exhibited slightly lower Φ and highest θ on individual vesicles. While these differences were modest, they suggest that the domain selected on phase-separated membranes is always the more-wetted of the two individual membrane types. Together, these controls demonstrate that, depending on the chemical nature of the coacervating species, selective wetting on phase-separated GUVs can reflect either a weak relative bias between two otherwise wettable membranes or, in some cases, an exclusive affinity for one membrane composition.

To rationalize these observations further, we characterized different coacervate properties, including their critical salt concentration (CSC) – conventionally used as an indicator of the coacervate robustness –, interfacial tension, and inverse capillary velocity – the ratio of viscosity to surface tension (see Methods, Supplementary Figure 13 and Supplementary Tables 3-5).

CSC values showed no clear correlation with wetting behavior (Figure 5i). For instance, PDDA/PAA (CSC = 356 ± 42 mM), PDDA/CM-dex (CSC = 118 ± 6 mM) and PDDA/ATP (CSC = 48 ± 2 mM) had significantly different CSC values, despite both selectively wetting DOPC domains. In contrast, interfacial tension displayed a pronounced difference between coacervates with distinct wetting selectivity. DEAE-dex/PAA coacervates, which wetted DPPC/cholesterol-rich domains, exhibited an ultra-low interfacial tension (~2.5 μN m-1), markedly lower than that of coacervates selectively wetting DOPC-rich domains (~35-90 μN m-1) (Figure 5j). In parallel, inverse capillary velocity measurements showed that DEAE-dex/PAA also exhibited an order-of-magnitude higher η/γ compared to PDDA/PAA, PDDA/CM-dex and PDDA/ATP (Figure 5k), resulting in a viscosity intermediate between that of PDDA/ATP or PDDA/CM-dextran and PDDA/PAA systems (Figure 5l and Supplementary Table 6). Hence, coacervate viscosity did not show any correlation with wetting selectivity. Viscosity is indeed expected to primarily affect wetting dynamics rather than equilibrium wetting configurations.

Last, using the measured coacervate interfacial tensions, we calculated the coacervate-membrane (Σ ) and membrane-dilute phase (Σ ) segment tensions associated with wetting on pure DOPC and DPPC/cholesterol vesicles (see Methods and Supplementary Figure 14). For PDDA/PAA and PDDA/ATP coacervates, which selectively wet DOPC-rich domains on phase-separated GUVs, Σ (resp. Σ ) was slightly lower (resp. higher) on DOPC compared to DPPC/cholesterol vesicles, suggesting a slight preference for coacervates to wet DOPC membranes, although the differences were modest. For PDDA/CM-dex coacervates, both Σ and Σ were slightly lower on DPPC/cholesterol vesicles than on DOPC vesicles, despite preferential wetting of DOPC-rich domains on phase-separated membranes. In contrast, DEAE-dex/PAA coacervates, which selectively wet DPPC-rich domains, displayed very small absolute values of both Σ and Σ . Wetting by DEAE-dex/PAA coacervates was observed on DPPC/cholesterol vesicles but not on DOPC vesicles, precluding a direct quantitative comparison of segment tensions between the two membrane compositions in this case. Overall, across the different coacervate systems investigated, segment tensions measured on pure DOPC and DPPC/cholesterol vesicles differed only modestly and did not exhibit a consistent correspondence with the domain selectivity observed on phase-separated GUVs when considered independently of other membrane or coacervate properties.

Taken together, these results reveal a clear empirical distinction between coacervates displaying different wetting behaviors. DEAE-dex/PAA coacervates, which are characterized by an ultra-low coacervate interfacial tension, are the only system observed (i) to selectively wet DPPC-rich domains and to fail to wet DOPC domains in phase-separated GUVs, (ii) to completely wet GUVs at a given polyion ratio. In contrast, coacervates with substantially higher interfacial tensions systematically wet preferentially DOPC-rich domains in phase-separated GUVs. Segment tension differences measured on individual DOPC and DPPC/cholesterol vesicles are small and do not systematically correlate with domain selectivity, indicating that they do not, by themselves, account for the observed selective wetting.

Methods
Materials
The phospholipids, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rho-PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD-PE) were purchased from Avanti polar lipids (IL, USA). Hydrochloric acid (CAS: 7647-01-0), sodium hydroxide (CAS: 1310-73-2), polyvinyl alcohol (PVA) (CAS: 9002-89-5), cholesterol (CAS: 57-88-5), adenosine 5′-triphosphate (ATP) disodium salt hydrate (CAS: 34369-07-8) and poly(acrylic acid sodium salt) [PAA, M = 5,100 g mol-1] (CAS: 9003-04-7), DEAE-dextran hydrochloride [DEAE-dex, M = 500,000 g mol-1] (CAS:9064-91-9) and CM-dextran sodium salt [CM-dex, M = 10,000-20,000 g mol-1] (CAS:39422-83-8) were purchased from Sigma Aldrich. PDMS (Sylgard 184) was purchased from Dow Corning. Poly(diallyldimethylammonium chloride) [PDDA, M = 8,500 g mol-1, 28 wt. % in H O] (CAS: 26062-79-3) was purchased from Polysciences. Cyanine 5-labelled single stranded DNA (Cy5-ssDNA) was purchased from Integrated DNA Technologies (the sequence is provided in Supplementary Table 1).

Giant vesicle preparation
Giant Unilamellar Vesicles (GUVs) were prepared by the modified lipid film hydration method as reported elsewhere.43 Briefly, 50 μL of the desired lipid or lipid mixture solution at 4 mM in chloroform were spin coated onto a glass slide at 1,000 rpm for 120 seconds at room temperature. The lipid compositions were as follow: 99.9 mol% DOPC + 0.1 mol% Rho-PE (DOPC), 69.9 mol% DOPC + 30 mol% cholesterol + 0.1 mol% Rho-PE (DOPC/cholesterol), 99 mol% DPPC + 1 mol% NBD-PE (DPPC), 69 mol% DPPC + 30 mol% cholesterol + 1 mol% NBD-PE (DPPC/cholesterol), 34.9 mol% DOPC + 34 mol% DPPC + 30 mol% cholesterol + 0.1 mol% Rho-PE + 1 mol% NBD-PE (DOPC/DPPC/cholesterol). The spin coated lipid film on the glass slide was dried under vacuum for 1h to remove any traces of chloroform. A rectangular chamber was assembled using a PTFE spacer (25 mm x 35 mm x 5 mm), filled with 500 μL of a 200 mM sucrose solution (swelling solution), and closed with a second glass slide held with two binder clips. The dried lipid film was hydrated with this swelling solution for 30 minutes at 25°C for DOPC and DOPC/cholesterol lipid films and 60°C for DPPC/cholesterol and DOPC/DPPC/cholesterol lipid films using a heating plate equipped with a temperature controller (IKA RCT standard 2.0). The resulting suspension of GUVs was transferred to Eppendorf tubes shielded from light to avoid photobleaching and stored at room temperature for a maximum duration of one week.

Preparation of stock solutions for coacervate formulation
Stock solutions of PDDA, PAA and ATP were prepared at 100 mM or 1 M (expressed in monomer concentrations) by diluting the 28 wt% PDDA solution or dissolving the appropriate amount of PAA or ATP powder using Milli-Q water and adjusting the pH to 8.0 with NaOH (1M) or HCl (1M). The concentration of ATP was checked via UV-Vis spectroscopy (PerkinElmer) at 260 nm using an excitation co-efficient of 15.4 × 103 L mol-1 cm-1. Carboxymethyl (CM)-dextran and diethylaminoethyl (DEAE)-dextran stock solutions were made at 50-100 mg mL-1 in Milli-Q water and the pH adjusted to 8.0. Given that the fraction of charge units on these polysaccharides is not defined, we do not provide a charge concentration for these two polymers but keep the concentration as mg mL-1. All stock solutions were stored at 4°C, expect for ATP where aliquots were stored at -20°C.

Coacervate characterization
Turbidity screening: The optimal polymer ratio for PDDA/PAA coacervate formation was determined by measuring the turbidity at 700 nm of 20 µL solutions prepared at fixed (5 mM) PDDA and variable PAA concentrations (0.5-50 mM) in sucrose/glucose medium (90 mM each, final concentration) using a 384-well plate (Corning, flat bottom) and a microplate spectrophotometer (Molecular Devices). For critical salt concentration (CSC) determination, 20 µL coacervates suspensions were prepared at 10 mM PDDA and 10 mM, 5 mM or 5 mg mL-1 PAA, ATP, CM-dextran, respectively; or 10 mM PAA and 4 mg mL-1 DEAE-dextran in sucrose/glucose medium (90 mM each, final concentration) and varying NaCl concentrations and the turbidity determined at 700 nm. The data was fitted with a sigmoidal logistic function. The CSC was determined by extrapolating the first-order derivative at the inflection point to zero turbidity.

Interfacial tension measurements: The interfacial tension of the different coacervates were estimated by the interfacial profile between the dense coacervate phase and dilute supernatant after macroscopic phase separation in a cell with parallel walls at room temperature, as described in ref. 44. Coacervate suspensions were here prepared at high concentration to produce sufficient amount of bulk coacervate phase, specifically, for PDDA/PAA: 200 mM PDDA and 200 mM PAA; for PDDA/ATP: 60 mM PDDA and 30 mM ATP; for PDDA/CM-dextran: 50 mM PDDA and 25 mg mL-1 CM-dextran; for DEAE-dextran/PAA: 40 mg mL-1 DEAE-dextran and 100 mM PAA. All systems except PDDA/ATP were formulated in sucrose/glucose medium (90 mM each, final concentration). These suspensions were placed into a 1 mm thick quartz cell (Hellma), then centrifuged at 1,400 ×g for 5 minutes to favor complete macroscopic phase separation, resulting in a net interface between a denser polymer-rich (coacervate) phase and a polymer-dilute (supernatant) upper phase. The static interfacial profile near the vertical wall of the cell was recorded using an optical microscope rotated by 90° with a digital camera using a ×5 objective, and computed using ImageJ.

This profile can be described by the generalized Young–Laplace equation connecting capillary pressure with curvature and surface tension for the case of a flat wall45 46 as:
x(y) 2ℓ 2ℓ y2 h2
c c
= arccosh( )−arccosh( )−√4− +√4− (1)
ℓ h y ℓ 2 ℓ 2
c c c
where x is the distance to the vertical wall, y the height of the profile above the flat level far away from the wall, h is the meniscus contact height at x = 0, and ℓ is the capillary length, defined as:
c
γ
ℓ = √ (2)
c Δρg
where Δρ is the mass density difference between the dense and dilute phases, g the gravitational acceleration and γ the interfacial tension between the coacervate and supernatant phases.

The interfacial profile was fitted using OriginLab to extract the capillary length ℓ . To determine Δρ, 1-2 mL of coacervate suspensions at the concentrations listed above in sucrose/glucose medium (except for PDDA/ATP) were centrifuged at 10,000 ×g for 5 min. 20-50 μL of the dense and dilute phases were accurately weighed in order to quantify their densities, either by pipetting for sufficiently fluid coacervates (PDDA/PAA, PDDA/CM-dextran, and PDDA/ATP) or by filling a square glass capillary (800 × 800 µm, VitroCom Inc.) via capillarity with the supernatant or bulk coacervate phase (DEAE-dextran/PAA), and normalizing the measured weight by that of water in the same capillary. These measurements were repeated 3 times for each phase and the average difference used as Δρ to determine the surface tension using the relationship between ℓ and γ above. The left and right profiles on both sides of the cell were analyzed and the average value and standard deviation of the surface tension for these 4 profiles reported.

Inverse capillary velocity: The inverse capillary velocity, defined as the ratio of viscosity over surface tension, was calculated for the different coacervates by studying the kinetics of two droplets fusing together. Coacervates were prepared at 20 mM PDDA and the following polyanion concentrations: 20 mM PAA, 10 mM ATP and 10 mg mL-1 CM-dextran, and 4 mg mL-1 DEAE-dextran and 10 mM PAA, to be at the same PDDA:polyanion ratio as for surface tension measurements, in sucrose/glucose medium (90 mM each, final concentration). The suspension was rapidly loaded into a passivated chamber (see below) and several fusion events recorded on an inverted Olympus microscope (IX71) equipped with a ×40 objective. A high-speed camera (Phantom Miro M-310 12-28VDC) was used for PDDA-based coacervates due to their fast fusion speed.

The aspect ratio, namely, the ratio between the long and short axis, L/l, during droplet fusion, decreased exponentially with time as the fusing droplets relax to a spherical shape, with a characteristic time τ:
L L
= +βe -t⁄τ (3)
l l
0
In the case of a Newtonian liquid, we can relate the relaxation time τ to the viscosity η and surface tension γ. By repeating this experiment with droplets of different sizes, we extracted the inverse capillary velocity, η/ γ, using the relationship:
η
τ = d (4)
γ
where d is the diameter of the two droplets after fusion. All the image and data treatment was performed with ImageJ.

Zeta potential measurements
The ζ-potential of coacervate suspensions and of lipid vesicles (with and without dyes) were quantified at room temperature using a Malvern Zetasizer (Nano ZS) device. Dispersions of coacervates were prepared immediately prior to the experiments at 5 mM PDDA final concentration and varying PAA (2.5, 4, 5, 6, 7, 8 mM), CM-dextran (2.5, 5, 10 mg mL-1) and ATP (2.5, 5, 8 mM) concentrations, or at 5 mM PAA final concentration and varying DEAE-dextran concentrations (1.5, 2, 2.5 mg mL-1), in sucrose/glucose medium (90 mM each, final concentration). Five measurements were performed for each sample, and the average value and standard deviation reported. For GUVs, measurements were conducted following the approach of Dimova et al.47 adapted from Carvalho et al.48 Briefly, the voltage for electrophoretic movement was set to 150 V in the instrument settings to limit vesicle disruption, and only measurements on populations fitting to the instruments quality criteria were considered. Five measurements were performed for each sample, and the average value and standard deviation reported.

Preparation and surface functionalization of imaging chambers
Custom-made PDMS-based chambers with a glass coverslip at the bottom were prepared for imaging, and glass coverslips were functionalized using polyvinylalcohol (PVA) to prevent coacervate spreading onto the glass surface. Briefly, PDMS was prepared by thoroughly mixing Part A and Part B in 10:1 ratio using a Sylgard 184 silicone elastomer kit, followed by crosslinking at 55° C for 12 h. The cross-linked PDMS was cut into rectangular blocks (40 × 20 mm), in which a 8 mm-diameter hole was punched. These blocks were subjected to oxygen activation using a plasma cleaner (Pico, Diener Electronic) at 80% power for 2 min. Glass coverslips (24 × 36 mm) were also plasma activated using oxygen. The plasma treated PDMS blocks and glass coverslips were sealed and cross-linked at 55°C for 1h, resulting in homemade imaging chambers (Supplementary Figure 15).

A 2 wt% PVA solution was prepared by dissolving the desired amount of powder in Milli-Q water and stirred for 6h using a magnetic stirrer. The assembled imaging chambers underwent plasma treatment for 2 minutes at 80% power, then 200 µL of the PVA solution were added to the chambers for 15 minutes to create a thin PVA coating. The solution was removed, then the chambers were dried at 110°C for 15 minutes. This PVA coating process was repeated 3 times to achieve stable functionalization.

Imaging of coacervate-vesicle interactions
Vesicles suspensions prepared in 200 mM sucrose were diluted with an isotonic 200 mM glucose solution (1:1 v/v) to facilitate density mismatch-base sedimentation of GUVs on the glass coverslip in the imaging chamber. PDDA/polyanion coacervates were prepared in an Eppendorf tube by adding varying volumes of polyanion stock solution (100 mM PAA or ATP, and 50 mg mL-1 CM-dextran) to 10 µL of PDDA stock solution (100 mM), then added to the vesicle suspension at a 10% v/v (final total volume of 200 µL) to reach a final PDDA concentration of 5 mM within the chamber and varying polyanion concentration. Similarly, DEAE-dextran/PAA coacervates were prepared in a test tube by adding varying volumes of DEAE-dextran stock solution (50 mg mL-1) to 10 µL of PAA stock solution (100 mM), then added to the vesicle suspension at a 10% v/v (final total volume of 200 µL) to reach a final PAA concentration of 5 mM within the chamber and varying DEAE-dextran concentration. In all conditions, the final sucrose and glucose concentrations were 90 mM each. Coacervate droplets were fluorescently labeled by adding Cy5-ssDNA (10 μM final concentration) to the droplet suspension. After gentle mixing the vesicle–coacervate suspension by pipetting, the sample was allowed to sediment at the bottom of the passivated imaging chamber for 10 minutes before imaging.

Epifluorescence and confocal fluorescence microscopy images were acquired on a Zeiss LSM 980 microscope using a ×63 oil immersion lens (HCX PL APO, 1.4 NA). Rho-PE, NBD-PE, and Cy5-ssDNA were excited using the 561 nm, 488 nm and 633 nm laser lines respectively.

Fluorescence recovery after photobleaching (FRAP)
FRAP measurements were conducted on GUVs to determine the mobility of labelled lipids within the bilayers. The photo-bleached area was chosen as a rectangular region with a size of 5×2 µm on the GUV membrane on the equatorial plane. Measurements were performed on three different GUVs prepared from the same sample. A pre-bleach sequence of minimum 10 frames was followed by the bleaching sequence, using the 488 nm laser (for NBD-PE) or 561 nm laser (for Rho-PE). Fluorescence recovery was monitored over the post-bleach sequence on ImageJ. The fluorescent intensity was normalized using a double normalization approach:49
[P(t)−B(t)] [R(t = 0)−B(t = 0)]
Fn (t) = . (5)
[R(t)−B(t)] [P(t = 0)−B(t = 0)]
where Fn (t) refers to the normalized fluorescent intensity, P is the average fluorescence intensity in the photo-bleached area, R is the average fluorescence intensity in a reference area on the GUV membrane outside of the bleached area, and B is the average fluorescence intensity in a randomly selected background area. The fluorescence recovery curve was fitted with a mono-exponential function using OriginLab to give the fluorescence recovery time constant τf.

Wetting geometries quantification
All wetting geometries involve three aqueous solutions: the interior solution of the GUV, the exterior solution of the GUV and coacervates droplets. At the contact point, the apparent contact angles α, β, and γ sum up to 360° (Supplementary Figure 4).

Geometric factor: A detailed explanation of the contact analysis and geometric parameters used in this study is reported elsewhere.28 50 51 Briefly, the contact angle between the surface segments from the microscopy images requires the rotational axis of symmetry of the vesicle-coacervate system to lie on the 2D projected plane of the image. Any improper orientation can cause artifacts in the contact angle measurements. Because of this, we focused our analysis on vesicle-coacervate pairs that preserve the rotational axis of symmetry of the system by choosing systems where coacervates and GUVs have the same size (and therefore whose equatorial plane lie in the same z-plane).

The contact angles α, β and γ shown in Supplementary Figure 4 were estimated as detailed in the Supplementary Methods, and used to extract geometric factor, Φ, which is a purely geometric quantity related to the adhesion free energies of the membrane segments:28
sinγ−sinβ
Φ = (6)
sinα
The geometric factor varies from +1, if the vesicle and coacervates and not interacting, to -1, if the coacervate droplet completely wets the vesicle.

Intrinsic contact angle: We also extracted the intrinsic contact angle θin, which represents the contact angle between the vesicle and coacervate at the nanoscale, where the membrane is smoothly curved, and is defined as:39
sinγ−sinβ
cosθin = (7)
sinα
The value of θin varies between 0° and 180° depending on the affinity of the coacervate for the membrane, with 0° meaning non-wetting/dewetting and 180° indicating complete wetting.

Segment tensions: Thanks to the measured coacervate interfacial tension γcoacervate, we calculated the coacervate-membrane tension Σcm and membrane-dilute phase tension Σmd using the following equations:39
sinγ
Σcm = γcoacervate (8)
sinα
sinβ
Σmd = γcoacervate (9)
sinα

Selective wetting quantification
Selective wetting behavior was assessed using confocal fluorescence imaging at the central plane of interacting coacervate–GUV systems. To ensure adequate sampling of binding events, image tiles of 20×20 confocal fields were acquired.

Selective wetting was defined as the preferential adhesion of a coacervate droplet to either the DOPC-rich or DPPC/cholesterol-rich membrane domain. Events in which coacervates exhibited complete wetting of the vesicle were not assigned to a specific membrane domain, as domain identity cannot be resolved in this configuration. These complete-wetting events are reported separately where relevant (e.g., for DEAE-dex/PAA coacervates).

Temperature-controlled wetting experiments
Rectangular glass capillaries (200 µm × 2 mm × 5 cm; VitroCom) were PEGylated to prevent nonspecific adhesion via incubation in a 5 vol% PEG-silane (3-[methoxy(polyethyleneoxy)propyl]tri-methoxysilane, 90%, 6-9 PE units, abcr GmbH) solution in toluene for 48h, then rinsing with ethanol and Milli-Q water. Vesicles-coacervate mixtures were prepared as described above for imaging directly into a PDMS-based imaging chamber, where samples were allowed to settle for 15 min. A PEGylated glass capillary was then gently inserted into the chamber, positioned at the bottom, and filled by capillary action. The capillary was transferred onto a glass coverslip, and both ends were sealed using UV-curing adhesive (NOA63, Norland) to prevent evaporation and sample drift.

The sealed capillary was mounted on a custom-built temperature-controlled microscope stage. Samples were heated from 25 °C to 50 °C at a rate of 1 °C min-1, held at 50 °C for 10 min, and subsequently cooled back to 25 °C at the same rate. Confocal fluorescence microscopy images were acquired continuously during the thermal cycle using a Zeiss LSM980 microscope equipped with the Definite Focus system (Zeiss) to minimize focal drift during temperature changes, and using a long-working distance 63× air objective (Plan-Neofluor, 0.75 NA).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript investigates how coacervate droplets interact with lipid membranes of differing fluidity by tuning the\npolyelectrolyte charge ratio of PDDA and PAA. The authors demonstrate that coacervates preferentially wet more fluid\n(liquid-disordered) membranes and that this wetting behavior is modulated not only by the PDDA/PAA ratio but also by the\nchemical nature of the coacervate components. By comparing several types of coacervates, the study highlights the\nselective wetting phenomenon as a generalizable feature, potentially governed by the viscosity of the\ncoacervate and the properties of the membrane phase. This manuscript presents potentially publishable results; however, the current version\nlacks sufficient statistical robustness, mechanistic depth, and accurate contextual framing (e.g., in terms of relevance to\norigins-of-life). Substantial additional discussion, quantitative analysis, and clarification are needed before the manuscript\ncan be reconsidered for publication.\n1) Although the study conceptually addresses the origin of life in the Introduction, the use of synthetic polymers (PDDA and\nPAA), which lack direct biological relevance, may weaken the argument. The manuscript appears to overstate the\nsignificance of the system to prebiotic chemistry, and I suggest the authors moderate their claims regarding the implications\nfor origins-of-life research.\n2) The observation that zeta potential becomes significantly negative upon the addition of only 0.1–1 mol% of fluorescent\nlipids is puzzling, especially considering the use of a neutral sucrose buffer. The authors should clarify the mechanism\nunderlying this drastic shift and explain whether such small additions can indeed induce meaningful surface charge\nchanges.\n3) The dataset for quantifying wetting interactions (n = 5) appears limited. Additionally, the authors themselves acknowledge\nthat deformation length ratio (L*) is size-dependent. It would strengthen the study to include the vesicle/coacervate size\nratios for each condition and present trends of φ and L* as a function of this size ratio to ensure meaningful comparison.\n4) In the main text (page 5), what is referred to as \"Figure 2a(ii)\" appears to be a mislabeling and should be \"Figure 2d(ii)\".\n5) The finding that membrane fluidity (rather than charge) dominates wetting behavior is highly interesting. However, the\ndiscussion of the underlying physicochemical mechanism is insufficient. Since membrane adhesion typically results from a\nbalance between attractive and repulsive forces, I suggest the authors discuss—preferably quantitatively—how vesicle\nmembrane fluidity (i.e., deformability) and coacervate viscosity contribute to the overall adhesion energy. Why does\ncomplete fusion not occur among the tested conditions? Is there an inherent incompatibility in material properties between\nthe coacervates and vesicles?\n6) It would be helpful to include representative images used to calculate the interfacial tension and to indicate which\nparameters (x, y, h, and so on) correspond to the length in the image.", "reviewer_2": "The manuscript by Joseph et al. describes the interaction between coacervates and GUV membranes composed of either\nordered or disordered liquid lipid phases. This significant manuscript is, in my opinion, highly relevant to a broad audience,\nincluding researchers in origin-of-life studies and materials science. However, I have several concerns that should be\naddressed through discussion or additional experiments:\nDoes temperature influence the wetting behavior? Investigating temperature’s role in wetting behavior is crucial, as it often\ninfluences molecular interactions and phase behavior in lipid systems.\nThe manuscript lacks mechanistic discussion, particularly regarding the molecular interactions driving selective wetting. The\nlack of mechanistic discussion is a significant gap in many studies of complex systems like coacervates and GUVs.\nAlthough computational modelling is out-of-scope, I would encourage a thorough discussion to clarify this.\nIs the wetting behavior permanent or reversible over time? Does it evolve or change in morphology? Time-lapse imaging\ncould clarify this.\nThe authors draw parallels to living cells, but these comparisons are vague, as the systems are synthetic and engineered.\nCould these systems be adapted for use with living systems? How might one engineer systems exhibiting similar behavior\nin biological contexts? By using biologically relevant electrolytes for instance?This is, in my opinion, critical for broader\napplications. For example, could biologically relevant polyelectrolytes be used?", "reviewer_3": "This manuscript describes how coacervate droplets contact lipid membranes as a function of the lipid ordering in the\nmembrane (liquid disordered DOPC and DOPC/cholesterol vs liquid ordered DPPC and DPPC/cholesterol membranes)\nand shows preferential wetting of lipid membrane domains by coacervates that depend on coacervate composition. The\nauthors have examined multiple coacervate compositions and demonstrated that the most obvious explanations based on\ncharge-charge interactions alone cannot explain their results for these systems, in which the membranes are based on\nzwitterionic lipids. They have quantified wetting behavior from image analysis to compare multiple\ncoacervate-membrane combinations and provide measurements on the coacervate physical properties such as surface charge,\ninterfacial tension and viscosity. Although a compelling overall explanation for the findings is not clear, the authors have shown that a variety of\noutcomes can be achieved based on the combination of coacervate composition and membrane phase (Lo vs Ld), and that\ncoacervates can preferentially interact with only one membrane phase domain in mixed-phase membranes. The work will\ninterest the large and growing community of researchers interested in coacervate-lipid membrane contact and wetting, which\nis increasingly understood to play crucial roles in cell biology and is further of interest for design of artificial cells and\npotential relevance to the origins of life.\nI do have some comments and suggestions for improving the manuscript:\n1. The discussion is somewhat lacking in explanations for the observed results and would benefit from some additional\ncontent. For example, the authors point to coacervate viscosity as a key determinant of the coacervate-membrane\ninteractions, but do not provide a mechanism by which coacervate viscosity would have this effect. Additionally, the authors\nfound that coacervate surface tensions were not predictive, and I wonder if considering all relevant surface tensions\n(coacervate-dilute phase, coacervate-membrane, and membrane-dilute phase) would be more instructive, particularly since\nfree polyelectrolytes from the coacervate suspension are likely to be interacting with the membranes (see also next\ncomment). The discussion of ATP-membrane interactions and their potential role is interesting and raises the broader\nquestion of how all the coacervate molecules interact with the membranes to potentially change their properties.\n2. The coacervate suspensions added to the vesicle samples also bring with them molecules in the dilute phase, that could\ninteract with lipid membranes even if the coacervates do not contact the membranes. This possibility is not discussed in the\nmanuscript but could in principle play an important role in the outcomes, particularly for any experiments where the relative\namounts of coacervate components are varied to alter the droplet charge, which presumably also changes the amount of\nfree polyelectrolyte outside of the coacervates, and/or where one coacervate component is a small molecule and the other a\npolymer (as for PDDA/ATP).\n3. Characterization of the physical properties of the coacervates do not appear to have been performed in the same medium\nas the membrane interaction experiments. Coacervate surface tension and viscosity is likely different in their initial\nsuspensions, which contain the coacervate and dilute phases, than after addition to the vesicle samples, which contain a\nsubstantial concentration of sugars (sucrose/glucose) from the vesicle preparation. What impact does this have on\ncoacervate properties?\n4. Why are the membranes charged (Figure 2b)? DOPC and DPPC are both zwitterionic, and both have the same\nheadgroup. Is the relatively large negative zeta potential for DOPC membranes, and the positive zeta potential for DPPC\nmembranes expected from prior literature?\n5. The manuscript does not mention earlier LLPS-membrane studies that used PEG/dextran LLPS. This is a missed\nopportunity to compare/contrast these new findings with the prior work (see, for example, an overview in Annu. Rev.\nBiophys. 2024. 53:319–41, and specific examples that also incorporate Lo/Ld domains in the phase-contacting lipid\nmembranes: Nature Chem. 2024. 16: 54–62, Am. Chem. Soc. 2008. 130(23): 7400–7406). The current manuscript’s work is\nnonetheless novel; it differs substantially from these previous studies due to its focus on coacervates, which are a better\nmodel system for most aspects of biocondensates than the PEG/dextran ATPS and which can be expected to have more\nvaried membrane wetting properties than PEG/dextran systems.\n6. The SI notes that 10 minutes elapsed after mixing before imaging. Did the wetting behavior change further over time or did\nit remain constant after 10 minutes?\n7. In the SI, the authors state how the “Selective wetting quantification” was performed. I found the explanation confusing as\nit seemed that several types of potentially important wetting scenarios were excluded from analysis (“wetting on both\nmembrane domains” and “complete wetting” were excluded). This requires further explanation, since it would seem\nimportant to include these events in datasets meant to elucidate selective wetting behavior."}, "author_response": "Point-by-point response\nWe sincerely thank the reviewers for their constructive comments, which have significantly helped\nus strengthen our manuscript. In response, we performed several new experiments and\nsubstantially revised the analysis and discussion. Specifically, we (i) added temperature-dependent\nexperiments demonstrating the reversible coupling between lipid phase separation and coacervate\nwetting, (ii) carried out systematic control wetting experiments on homogeneous DOPC and\nDPPC/cholesterol vesicles for all four coacervate systems across three polyion ratios, and\nquantitatively determined all membrane segment tensions; and (iii) re-measured coacervate\nproperties, including interfacial tension and viscosity, in the sucrose/glucose medium used for\nvesicle experiments.\nThese new data prompted an extensive revision of the ‘Discussion’, leading to a more constrained\nand physically grounded interpretation of domain selectivity. In particular, we identify a clear\nempirical distinction between coacervates with higher interfacial tensions, which preferentially wet\nmore fluid DOPC-rich membranes, and the DEAE-dex/PAA system with ultra-low interfacial\ntension, which uniquely exhibits DPPC-rich domain selectivity and complete wetting under specific\nconditions. Overall, the revised manuscript emphasizes the combined roles of interfacial energetics\nand membrane mechanical properties in governing selective wetting, and we believe these\nadditions significantly improve the rigor and clarity of the work.\nReviewer #1 (Remarks to the Author):\nThis manuscript investigates how coacervate droplets interact with lipid membranes of differing\nfluidity by tuning the polyelectrolyte charge ratio of PDDA and PAA. The authors demonstrate that\ncoacervates preferentially wet more fluid (liquid-disordered) membranes and that this wetting\nbehavior is modulated not only by the PDDA/PAA ratio but also by the chemical nature of the\ncoacervate components. By comparing several types of coacervates, the study highlights the\nselective wetting phenomenon as a generalizable feature, potentially governed by the viscosity of the\ncoacervate and the properties of the membrane phase. This manuscript presents potentially\npublishable results; however, the current version lacks sufficient statistical robustness, mechanistic\ndepth, and accurate contextual framing (e.g., in terms of relevance to origins-of-life). Substantial\nadditional discussion, quantitative analysis, and clarification are needed before the manuscript can be\nreconsidered for publication.\n1) Although the study conceptually addresses the origin of life in the Introduction, the use of synthetic\npolymers (PDDA and PAA), which lack direct biological relevance, may weaken the argument. The\nmanuscript appears to overstate the significance of the system to prebiotic chemistry, and I suggest\nthe authors moderate their claims regarding the implications for origins-of-life research.\nWe thank the reviewer for pointing this out. We agree that our system is not intended to model\nprebiotic chemistry directly. To avoid overstating origins-of-life implications, we have softened the\nlanguage in both the ‘Introduction’ and ‘Conclusion’ by removing or rephrasing statements referring\nto the emergence of life and prebiotic systems. The revised text now focuses on general physical\nprinciples and on cell-like model systems rather than prebiotic scenarios.\n2) The observation that zeta potential becomes significantly negative upon the addition of only 0.1–1\nmol% of fluorescent lipids is puzzling, especially considering the use of a neutral sucrose buffer. The\nauthors should clarify the mechanism underlying this drastic shift and explain whether such small\nadditions can indeed induce meaningful surface charge changes.\nWe thank the reviewer for pointing out this issue. Following the reviewer’s comment, we re-\nexamined our ζ-potential measurements using the protocol recommended by Dimova et al. (Sci.\nRep. 8, 11838 (2018)), adapted from Carvalho et al. (Biophys. J. 95, 4348–4360 (2008)). As\nemphasized in these references, ζ-potential measurements on GUVs require special caution\nbecause vesicles are polydisperse and may destabilize under electric fields. This included\ncontrolling the applied voltage (150 V), performing no more than 5 measurements per sample, and\nrejecting measurements that did not pass the instrument’s “quality report” filter.\nAfter re-measuring all conditions using this stricter procedure, we found that DOPC and\nDOPC/cholesterol GUVs exhibited essentially identical ζ-potential values with and without Rh-PE\ndye (~ -30 mV); and DPPC/cholesterol GUVs showed a shift from -31 mV to -11 mV when 1 mol%\nNBD-PE dye was included. These revised measurements therefore show that the previously\nreported large shifts with labelled lipids were largely due to limitations of our initial measurement\nconditions; yet, the shift towards less negative ζ-potential observed for DPPC/cholesterol GUVs\nwhen NBD-PE is added likely reflects a dye-dependent effect specific to that membrane\ncomposition.\nWe have now updated Figure 2b and Supplementary Figure 3 with the corrected values and\nclarified these points in the ‘Methods’.\nPlease also see our reply to Reviewer 3 (point 4) regarding the net negative charge of PC-type lipid\nvesicles.\n3) The dataset for quantifying wetting interactions (n = 5) appears limited. Additionally, the authors\nthemselves acknowledge that deformation length ratio (L*) is size-dependent. It would strengthen the\nstudy to include the vesicle/coacervate size ratios for each condition and present trends of φ and L*\nas a function of this size ratio to ensure meaningful comparison.\nWe thank the reviewer for these constructive suggestions. We now report n = 10 vesicle-coacervate\npairs for each condition shown in Figure 3. The expanded dataset yields the same trends as those\noriginally reported.\nRegarding the deformation length ratio L*, we agree with the reviewer that it is size-dependent and\ntherefore not ideal for comparing across systems where vesicle and coacervate sizes may vary.\nHowever, as discussed in the text, we targeted vesicles and coacervates of comparable sizes (to\nmake sure both were imaged near their equatorial plane), so with a size ratio of close to 1 for all\nconditions. Nevertheless, L* still intrinsically carries a geometric dependence, making cross-\ncondition comparisons potentially misleading.\nTo provide a more meaningful and size-independent measure of wetting, we now replaced L* by\nthe intrinsic contact angle (θ ) of the coacervate-membrane interface in Figure 3c, following the\nin\ntheoretical framework of Kusumaatmaja & Lipowsky, Phys. Rev. Lett. 103, 238103 (2009). The\nintrinsic contact angle is defined by the local force balance at the contact line and is largely\nindependent of droplet/vesicle size. It is a material parameter that reflects the underlying balance\nof interfacial tensions, and therefore offers a more robust descriptor of wetting affinity than L*.\nHence, the calculated θ values enables more direct comparison of wetting across conditions. It\nin\nhas been used as a relevant wetting parameter by Dimova et al. in her recent studies of protein\ncondensate/GUVs interactions (refs. 27, 32).\nThis new analysis confirms that the PDDA/PAA ratio tunes wetting of GUVs in a membrane-\ndependent manner. At the lowest PDDA/PAA ratio, θ remains 0° for all membrane compositions,\nin\nindicating a non-wetting regime. Increasing the PDDA/PAA ratio leads to a progressive increase in\nθ , reflecting a transition toward stronger membrane wetting. The magnitude and evolution of this\nin\nincrease depend on membrane composition: fluid DOPC membranes exhibit the largest change in\nθ , cholesterol-containing membranes show a more limited response, and Janus GUVs display\nin\nintermediate behavior. These trends indicate that modifying the coacervate composition alters the\nbalance of interfacial tensions at the membrane-coacervate contact line, while the membrane\nphysical state sets the extent to which this tuning can translate into enhanced wetting.\nWe have added this intrinsic contact angle analysis to the ‘Results’ section, and provided\nmethodological details in the ‘Methods’.\n4) In the main text (page 5), what is referred to as \"Figure 2a(ii)\" appears to be a mislabeling and should\nbe \"Figure 2d(ii)\".\nDone, thank you for spotting out this typo.\n5) The finding that membrane fluidity (rather than charge) dominates wetting behavior is highly\ninteresting. However, the discussion of the underlying physicochemical mechanism is insufficient.\nSince membrane adhesion typically results from a balance between attractive and repulsive forces, I\nsuggest the authors discuss—preferably quantitatively—how vesicle membrane fluidity (i.e.,\ndeformability) and coacervate viscosity contribute to the overall adhesion energy. Why does\ncomplete fusion not occur among the tested conditions? Is there an inherent incompatibility in material\nproperties between the coacervates and vesicles?\nWe agree with the reviewer that understanding the physicochemical origin of membrane-\ncoacervate adhesion is an important issue. In the revised manuscript, we have substantially\nexpanded both the experimental controls, analysis and discussion to address this point\nquantitatively.\nFirst, we now quantified coacervate interfacial tensions and viscosities in the same sucrose/glucose\nmedium used for vesicle experiments (see comment from Reviewer 3, point 3). The new values\nsuggest that coacervate viscosity no longer correlates with wetting selectivity (see Figure 5).\nViscosity is indeed expected to primarily affect wetting dynamics rather than the equilibrium\nconfigurations. This now excludes coacervate viscosity as a dominant control parameter for\nselective wetting under our conditions. Nevertheless, these new measurements confirm that DEAE-\ndex/PAA coacervates, which are the only ones to preferentially wet DPPC-rich domains and show\ncomplete wetting, exhibit one order of magnitude lower interfacial tension compared to the other\nthree coacervates.\nSecond, we performed systematic control experiments on homogeneous DOPC and\nDPPC/cholesterol vesicles for all coacervate systems and the 3 polyion ratios (see Figure 5). We\nfurther quantified geometric factors, intrinsic contact angles (Supplementary Figure 12) and\ncoacervate-membrane and membrane-dilute phase segment tensions (Supplementary Figure 14)\non these homogeneous vesicles for one select polyion ratio. We fixed this ratio to explore different\nselective wetting scenarios of either DOPC-rich domain (for PDDA/PAA, PDDA/ATP and\nPDDA/CM-dextran coacervates) or DPPC-rich domain (for DEAE-dextran/PAA coacervates) in\nJanus-like GUVs. These measurements show that, for most coacervates, both individual\nmembranes are wetted under the same conditions, and that differences in geometric factors,\nintrinsic contact angles, and segment tensions between DOPC and DPPC/cholesterol membranes\nare modest. This demonstrates that domain selectivity observed on phase-separated GUVs cannot\nbe explained by a simple intrinsic preference for one membrane composition or by local interfacial\ntensions alone.\nWe suggest that membrane mechanics provides an additional contribution that influences wetting\nselectivity by modulating the energetic cost of membrane deformation during droplet adhesion.\nWetting requires membrane deformation, which incurs a bending energy cost governed by the\nmembrane bending rigidity and the curvature imposed by the droplet. For coacervates with higher\ninterfacial tension (in the tens of µN/m), selective wetting is consistent with an elastocapillary\nbalance in which wetting is favored on more deformable DOPC-rich domains, where the bending\nenergy penalty associated with membrane deformation is lower compared to stiffer\nDPPC/cholesterol-rich domains, which resist deformation.\nDEAE-dex/PAA coacervates constitute a distinct case due to their ultra-low interfacial tension.\nIn this regime, the free-energy difference between distinct wetting configurations is small, rendering\nwetting behavior particularly sensitive to variations in local membrane properties. Under these\nconditions, membrane elasticity may no longer simply penalize deformation but instead bias which\nwetting geometries are mechanically stable. One preliminary hypothesis is that the higher bending\nrigidity of DPPC/cholesterol-rich domains may constrain membrane deformation in a way that\nstabilizes specific droplet-membrane contact geometries, leading to an apparent preference for\nthese domains.\nIn this framework, domain selectivity arises from the interplay between coacervate interfacial\nenergetics and membrane mechanical properties, with their relative importance depending on the\nmagnitude of the coacervate interfacial tension.\nRegarding complete wetting, we note that it is only observed for DEAE-dex/PAA at specific\ncompositions, consistent with its ultra-low interfacial tension, as discussed theoretically in a recent\nreference (Lipowsky, Soft Matter, 2025, 21, 7370-7392). Under all other conditions, partial wetting\nis expected when the balance of interfacial and membrane elastic energies does not favor complete\nspreading.\nWe have expanded our ‘Discussion’ accordingly. While our data do not allow a full quantitative\ndecomposition of adhesion energy into capillary and elastic contributions, they establish clear\nexperimental constraints and identify membrane mechanics as a key factor that must be\nincorporated in future theoretical and experimental treatments.\n6) It would be helpful to include representative images used to calculate the interfacial tension and to\nindicate which parameters (x, y, h, and so on) correspond to the length in the image.\nThank you for this suggestion. We have added in Supplementary Figure 13 a scheme of the profile,\ntogether with a representative image of interfacial profiles for all four coacervates, with the x,y axis\nshown and the parameter ‘h’ represented.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Joseph et al. describes the interaction between coacervates and GUV membranes\ncomposed of either ordered or disordered liquid lipid phases. This significant manuscript is, in my\nopinion, highly relevant to a broad audience, including researchers in origin-of-life studies and\nmaterials science. However, I have several concerns that should be addressed through discussion or\nadditional experiments:\nDoes temperature influence the wetting behavior? Investigating temperature’s role in wetting\nbehavior is crucial, as it often influences molecular interactions and phase behavior in lipid systems.\nWe thank the reviewer for highlighting the importance of temperature in wetting phenomena.\nAcquiring confocal fluorescence microscopy images at elevated temperatures is technically\nchallenging, as conventional objectives cannot be used without heat dissipation, and specialized\nsetups are required. Despite these constraints, we successfully managed to perform temperature-\ncontrolled wetting studies on an example, using a custom-built temperature-controlled stage and a\nlong-working distance, lower resolution ×63 air objective (0.75 NA).\nThese experiments were acquired for a PDDA/PAA ratio of 1 in which Janus-like GUVs were\nheated up to 50°C (i.e., above the lipid mixing temperature, ~ 34°) at 1°C/min, held at 50°C for 10\nminutes, and subsequently cooled down to room temperature at the same rate. Control\nexperiments using pure DOPC and DPPC/cholesterol vesicles were also conducted using the same\nprotocol.\nFor Janus GUVs, heating induced mixing of the two lipid domains, resulting in a measurable\nincrease and decrease in the geometric factor and intrinsic contact angle, respectively, namely a\nlower wetting propensity. This is also visible on confocal images through a change in the\nmembrane-coacervate segment curvature. Importantly, this behavior is reversible upon cooling\ndown to room temperature, indicating that selective wetting of DOPC domains is an equilibrium\nproperty and not a kinetically trapped state. Additionally, control experiments show that pure DOPC\nand DPPC/cholesterol vesicles exhibit temperature-independent wetting.\nBased on these observations, we interpret the decreased wetting propensity of Janus-like GUVs at\nhigh temperature to lipid mixing : initially, coacervates wet the more fluid DOPC domain, then upon\nlipid mixing above their lipid mixing temperature, coacervates experience a modified local lipid\nenvironment now containing mixed DOPC and DPPC lipids, resulting in a lower interaction strength.\nWe also note a modest hysteresis between heating and cooling, which may reflect the coupling\nbetween lipid phase behavior and droplet adhesion; however, additional experiments would be\nrequired to establish the origin of this effect.\nWe have added a corresponding Movie of the behavior (Supplementary Movie 3) new panels e-i in\nFigure 4 showing (i) representative images of PDDA/PAA coacervates wetting Janus-like GUVs at\nroom temperature, at 50 °C, and after cooling back to room temperature, (ii) a representative\nevolution of the geometric factor and intrinsic contact angle during a heating-cooling cycle, and (iii)\nnormalized changes in geometric factor and intrinsic contact angle at different temperature. We\nhave added how we detected lipid mixing in Supplementary Figure 8. We have also added\nrepresentative images of temperature-independent wetting on pure DOPC and DPPC/cholesterol\nas Supplementary Figure 9.\nWe discuss these results in the main text, and have added the corresponding experimental details\nin the ‘Methods’.\nThe manuscript lacks mechanistic discussion, particularly regarding the molecular interactions driving\nselective wetting. The lack of mechanistic discussion is a significant gap in many studies of complex\nsystems like coacervates and GUVs. Although computational modelling is out-of-scope, I would\nencourage a thorough discussion to clarify this.\nWe thank the reviewer for emphasizing the need to discuss mechanisms underlying selective\nwetting. Please see our reply to Reviewer 1, point 5.\nBriefly: in the revised manuscript, we have (i) measured coacervate properties, including interfacial\ntensions, in the same sucrose/glucose medium used for vesicle experiments (see comment from\nReviewer 3, point 3), (ii) added systematic control experiments on homogeneous DOPC and\nDPPC/cholesterol vesicles for all coacervate systems and 3 polyion ratios, and (iii) quantified\ngeometric factors, intrinsic contact angles, and coacervate-membrane and membrane-dilute phase\nsegment tensions on homogeneous DOPC and DPPC/cholesterol GUVs for one select polyion\nratio resulting in different selective wetting scenarios.\nThese new data show that:\n- Coacervate viscosity no longer correlates with wetting selectivity (it is indeed expected to\nprimarily affect wetting dynamics – which we do not monitor in our study – rather than\nequilibrium behavior).\n- A qualitative correlation emerges between coacervates interfacial tension and selective\nwetting, where coacervates with higher interfacial tensions (35-90 µN/m) selectively wet\nDOPC-rich domains, while coacervates with an ultra-low interfacial tension (2.5 µN/m)\nselectively wet DPPC-rich domains.\n- Segment tensions, geometric factors, and contact angles on individual DOPC and\nDPPC/cholesterol vesicles differ only modestly between membrane types and cannot, by\nthemselves, explain the pronounced selective wetting observed on phase-separated\nGUVs.\nBuilding on these observations, we suggest that membrane mechanics provides an additional\ncontribution that influences wetting selectivity by modulating the energetic cost of membrane\ndeformation during droplet adhesion. Wetting requires membrane deformation, which incurs a\nbending energy cost governed by the membrane bending rigidity and the curvature imposed by the\ndroplet. For coacervates with higher interfacial tension (in the tens of µN/m), selective wetting is\nconsistent with an elastocapillary balance in which wetting is favored on more deformable DOPC-\nrich domains, where the bending energy penalty associated with membrane deformation is lower\ncompared to stiffer DPPC/cholesterol-rich domains, which resist deformation.\nDEAE-dex/PAA coacervates constitute a distinct case due to their ultra-low interfacial tension.\nIn this regime, the free-energy difference between distinct wetting configurations is small, rendering\nwetting behavior particularly sensitive to variations in local membrane properties. Under these\nconditions, membrane elasticity may no longer simply penalize deformation but instead bias which\nwetting geometries are mechanically stable. One preliminary hypothesis is that the higher bending\nrigidity of DPPC/cholesterol-rich domains may constrain membrane deformation in a way that\nstabilizes specific droplet-membrane contact geometries, leading to an apparent preference for\nthese domains.\nIn this framework, domain selectivity arises from the interplay between coacervate interfacial\nenergetics and membrane mechanical properties, with their relative importance depending on the\nmagnitude of the coacervate interfacial tension.\nWe have expanded our ‘Discussion’ accordingly. While our data do not allow a full quantitative\ndecomposition of adhesion energy into interfacial and elastic contributions, they establish clear\nexperimental constraints and identify membrane mechanics as a key factor that must be\nincorporated in future theoretical and experimental treatments.\nIs the wetting behavior permanent or reversible over time? Does it evolve or change in morphology?\nTime-lapse imaging could clarify this.\nWe have performed time-lapse imaging of coacervate-GUV interactions over ~20 minutes for\nJanus-like vesicles (for a PDDA/PAA ratio of 1). These new observations show that the wetting\nconfiguration stabilizes within the first 30 seconds-to-3 minutes after contact and remains\nessentially unchanged over at least 20 minutes. We typically quantify the geometric factor after ~10\nminutes, which is well within this stable regime. Please note that in the longer movie (20 min, Movie\nS1), the GUV appears smaller towards the end due to focus drift, but the coacervate continues to\nremain bound to the DOPC-rich domain with the same morphology.\nInterestingly, the time-lapse movies also reveal that during the initial approach, coacervates\nsometimes make contact at an interface between DOPC-rich and DPPC-rich regions. Over the next\n30 seconds-to-3 minutes, lipid reorganization occurs, and the coacervate relocates to a more\nfavorable position on the DOPC-rich region, indicating that selective wetting is dynamically\nestablished through short-lived membrane reorganization. After this brief rearrangement, the\nwetting morphology becomes stationary.\nWe have included two representative time-lapse sequences and movies as Supplementary Figure\n7 and Supplementary Movies 1 and 2, and added a short description in the Supporting Information.\nThese observations confirm that wetting is stable over the time window relevant to our\nmeasurements.\nThe authors draw parallels to living cells, but these comparisons are vague, as the systems are synthetic\nand engineered. Could these systems be adapted for use with living systems? How might one engineer\nsystems exhibiting similar behavior in biological contexts? By using biologically relevant electrolytes\nfor instance?This is, in my opinion, critical for broader applications. For example, could biologically\nrelevant polyelectrolytes be used?\nThank you for this point. Indeed, the biophysical principles we uncover (e.g., the role of membrane\nfluidity and coacervate interfacial tension) align well with emerging data in more biologically relevant\nsystems. We note that Dimova and colleagues recently reported that condensates formed from\nglycinin, Synapsin 1, PEG/dextran, and oppositely charged oligopeptides all display lipid-packing–\ndependent wetting behavior on membranes, resulting in selective wetting of L domains in Janus\no\nGUVs (ref. 32). This suggests that the mechanisms we describe are likely to be broadly applicable,\nincluding protein- or RNA-based condensates in cellular contexts. However, we also reveal that the\nnature of the coacervates plays a role in this selectivity, with the ultra-low-surface-tension-\ncoacervate (DEAE-dextran/PAA) preferring the L domain, and with the PDDA/ATP systems\nd\nexhibiting a polycation/polyanion-ratio-dependent selective wetting.\nWe have added comments in the ‘Discussion’ to highlight these connections and mention that\nfuture work could test membrane wetting with biologically relevant polyelectrolytes (e.g., IDPs,\nRNA) to translate these physical principles into a more cellular or physiological framework.\nReviewer #3 (Remarks to the Author):\nThis manuscript describes how coacervate droplets contact lipid membranes as a function of the lipid\nordering in the membrane (liquid disordered DOPC and DOPC/cholesterol vs liquid ordered DPPC and\nDPPC/cholesterol membranes) and shows preferential wetting of lipid membrane domains by\ncoacervates that depend on coacervate composition. The authors have examined multiple coacervate\ncompositions and demonstrated that the most obvious explanations based on charge-charge\ninteractions alone cannot explain their results for these systems, in which the membranes are based\non zwitterionic lipids. They have quantified wetting behavior from image analysis to compare multiple\ncoacervate-membrane combinations and provide measurements on the coacervate physical\nproperties such as surface charge, interfacial tension and viscosity. Although a compelling overall\nexplanation for the findings is not clear, the authors have shown that a variety of outcomes can be\nachieved based on the combination of coacervate composition and membrane phase (Lo vs Ld), and\nthat coacervates can preferentially interact with only one membrane phase domain in mixed-phase\nmembranes. The work will interest the large and growing community of researchers interested in\ncoacervate-lipid membrane contact and wetting, which is increasingly understood to play crucial roles\nin cell biology and is further of interest for design of artificial cells and potential relevance to the origins\nof life.\nI do have some comments and suggestions for improving the manuscript:\n1. The discussion is somewhat lacking in explanations for the observed results and would benefit from\nsome additional content. For example, the authors point to coacervate viscosity as a key determinant\nof the coacervate-membrane interactions, but do not provide a mechanism by which coacervate\nviscosity would have this effect. Additionally, the authors found that coacervate surface tensions were\nnot predictive, and I wonder if considering all relevant surface tensions (coacervate-dilute phase,\ncoacervate-membrane, and membrane-dilute phase) would be more instructive, particularly since\nfree polyelectrolytes from the coacervate suspension are likely to be interacting with the membranes (see\nalso next comment). The discussion of ATP-membrane interactions and their potential role is\ninteresting and raises the broader\nquestion of how all the coacervate molecules interact with the membranes to potentially change their properties.\nWe thank the reviewer for this insight. Please see our replies to Reviewer 1, point 5 and to Reviewer\n2, point 2. Briefly: in the revised manuscript, we have (i) measured coacervate properties, including\ninterfacial tensions, in the same sucrose/glucose medium used for vesicle experiments (see our\nresponse to point 3), (ii) added systematic control experiments on homogeneous DOPC and\nDPPC/cholesterol vesicles for all coacervate systems and 3 polyion ratios, and (iii) quantified\ngeometric factors, intrinsic contact angles, and coacervate-membrane and membrane-dilute phase\nsegment tensions on homogeneous DOPC and DPPC/cholesterol GUVs for one select polyion\nratio resulting in different selective wetting scenarios.\nThese new data show that:\n- Coacervate viscosity no longer correlates with wetting selectivity (it is indeed expected to\nprimarily affect wetting dynamics – which we do not monitor in our study – rather than\nequilibrium behavior).\n- A qualitative correlation emerges between coacervates interfacial tension and selective\nwetting, where coacervates with higher interfacial tensions (35-90 µN/m) selectively wet\nDOPC-rich domains, while coacervates with an ultra-low interfacial tension (2.5 µN/m)\nselectively wet DPPC-rich domains.\n- Segment tensions, geometric factors, and contact angles on individual DOPC and\nDPPC/cholesterol vesicles differ only modestly between membrane types and cannot, by\nthemselves, explain the pronounced selective wetting observed on phase-separated\nGUVs.\nBuilding on these observations, we suggest that membrane mechanics provides an additional\ncontribution that influences wetting selectivity by modulating the energetic cost of membrane\ndeformation during droplet adhesion. Wetting requires membrane deformation, which incurs a\nbending energy cost governed by the membrane bending rigidity and the curvature imposed by the\ndroplet. For coacervates with higher interfacial tension (in the tens of µN/m), selective wetting is\nconsistent with an elastocapillary balance in which wetting is favored on more deformable DOPC-\nrich domains, where the bending energy penalty associated with membrane deformation is lower\ncompared to stiffer DPPC/cholesterol-rich domains, which resist deformation.\nDEAE-dex/PAA coacervates constitute a distinct case due to their ultra-low interfacial tension.\nIn this regime, the free-energy difference between distinct wetting configurations is small, rendering\nwetting behavior particularly sensitive to variations in local membrane properties. Under these\nconditions, membrane elasticity may no longer simply penalize deformation but instead bias which\nwetting geometries are mechanically stable. One preliminary hypothesis is that the higher bending\nrigidity of DPPC/cholesterol-rich domains may constrain membrane deformation in a way that\nstabilizes specific droplet-membrane contact geometries, leading to an apparent preference for\nthese domains.\nIn this framework, domain selectivity arises from the interplay between coacervate interfacial\nenergetics and membrane mechanical properties, with their relative importance depending on the\nmagnitude of the coacervate interfacial tension.\nWe have expanded our ‘Discussion’ accordingly. While our data do not allow a full quantitative\ndecomposition of adhesion energy into interfacial and elastic contributions, they establish clear\nexperimental constraints and identify membrane mechanics as a key factor that must be\nincorporated in future theoretical and experimental treatments.\n2. The coacervate suspensions added to the vesicle samples also bring with them molecules in the\ndilute phase, that could interact with lipid membranes even if the coacervates do not contact the\nmembranes. This possibility is not discussed in the manuscript but could in principle play an important\nrole in the outcomes, particularly for any experiments where the relative amounts of coacervate\ncomponents are varied to alter the droplet charge, which presumably also changes the amount of\nfree polyelectrolyte outside of the coacervates, and/or where one coacervate component is a small\nmolecule and the other a polymer (as for PDDA/ATP).\nWe thank the reviewer for this thoughtful comment. We fully agree that coacervate suspensions\nnecessarily contain coacervate components in the surrounding dilute phase, and that these species\nmay interact with lipid membranes independently of direct droplet contact. This effect is particularly\nrelevant when varying polycation/polyanion stoichiometry, which alters both droplet properties and\nthe concentration of free components in solution, and in systems involving small-molecule\ncomponents such as ATP.\nIn the revised manuscript, we now explicitly acknowledge and discuss this possibility in the\n‘Discussion’. We emphasize that changes in wetting behavior may reflect not only properties of the\ncondensed phase, but also contributions from coacervate components present in the dilute phase\nthat can modulate membrane properties or interfacial energies. While the present experiments do\nnot allow us to disentangle the respective roles of condensed and dilute phases, we highlight this\nas an important additional layer of complexity inherent to coacervate-membrane systems and a\nkey direction for future work.\n3. Characterization of the physical properties of the coacervates do not appear to have been\nperformed in the same medium as the membrane interaction experiments. Coacervate surface tension\nand viscosity is likely different in their initial suspensions, which contain the coacervate and dilute\nphases, than after addition to the vesicle samples, which contain a substantial concentration of sugars\n(sucrose/glucose) from the vesicle preparation. What impact does this have on coacervate properties?\nWe thank the reviewer for raising this important point. We have now re-done the full set of\ncoacervate characterizations in the same sucrose/glucose (90 mM each) medium used in the\nmembrane-coacervate interaction experiments. Specifically, we re-measured ζ-potential, critical\nsalt concentration, inverse capillary velocity and interfacial tension for all coacervate systems under\nthese conditions. However, the interfacial tension for the PDDA/ATP system could not be measured\nin the sucrose/glucose medium because bulk coacervate could not be formed at sufficiently high\namount. We therefore kept the value in water, as clearly labelled.\nOverall, we find that while ζ-potentials remain essentially unchanged, some coacervate systems\ndisplay moderate shifts in critical salt concentration, inverse capillary velocity and interfacial tension\nwhen transferred to the sugar-containing medium. Importantly, these new measurements show that\ncoacervate viscosity no longer correlates with wetting selectivity (as expected since it primarily\naffects wetting dynamics rather than equilibrium behavior). However, a qualitative correlation\nemerges between coacervates interfacial tension and selective wetting, where coacervates with\nhigher interfacial tensions (35-90 µN/m) selectively wet DOPC-rich domains, while coacervates\nwith an ultra-low interfacial tension (2.5 µN/m) selectively wet DPPC-rich domains.\nWe have updated Figures 2a, 5i-l, Supplementary Figure 13) and Supplementary Tables 3-6 with\nthese correct measurements and added the corresponding experimental details in the ‘Methods’.\n4. Why are the membranes charged (Figure 2b)? DOPC and DPPC are both zwitterionic, and both have\nthe same headgroup. Is the relatively large negative zeta potential for DOPC membranes, and the positive zeta potential for DPPC\nmembranes expected from prior literature?\nWe appreciate the reviewer’s question. As noted in our response to Reviewer 1, point 2, our ζ-\npotential measurements were repeated using a more stringent protocol, which revealed that both\nDOPC and DPPC/cholesterol GUVs carry a net negative ζ-potential under our experimental\nconditions (see revised Figure 2b and detailed explanation above). Thus, the previously reported\nsign difference was due to limitations of our initial measurements rather than a true compositional\neffect.\nAlthough DOPC and DPPC have no net formal charge, the observation that zwitterionic PC\nmembranes exhibit a non-zero ζ-potential is well supported in the literature. For example, for DPPC\nGUVs prepared by electroformation, Sek et al. (Chem. Phys. Lipids, 235, 105059 (2021)) report a\nζ-potential of 4.7 mV, while Wang et al. (Curr. Issues Mol. Biol. 45, 3757-3771 (2023)) report a\nvalue of –23 mV. In the later study, the negative ζ-potential is attributed to headgroup reorientation,\nwhereby the phosphate group becomes preferentially exposed to the aqueous phase. Additional\nstudies (Sachs et al., Biophys. J., 86, 3772-3782 (2004); Seelig et al., Biochim. Biophys. Acta, 467,\n109-119 (1977)) likewise show that electrostatic and hydration forces can bias the PC headgroup\norientation, making it energetically favorable for the phosphate moiety to tilt so the negative charge\nis more exposed, and yielding a negative surface potential.\nTaken together, these findings support that the negative ζ-potentials we observe for both DOPC\nand DPPC/cholesterol GUVs are consistent with established behavior of PC lipid bilayers. We have\nclarified this point and added the relevant references in the manuscript.\n5. The manuscript does not mention earlier LLPS-membrane studies that used PEG/dextran LLPS. This\nis a missed opportunity to compare/contrast these new findings with the prior work (see, for example,\nan overview in Annu. Rev. Biophys. 2024. 53:319–41, and specific examples that also incorporate Lo/Ld\ndomains in the phase-contacting lipid membranes: Nature Chem. 2024. 16: 54–62, Am. Chem. Soc.\n2008. 130(23): 7400–7406). The current manuscript’s work is nonetheless novel; it differs substantially\nfrom these previous studies due to its focus on coacervates, which are a better model system for most\naspects of biocondensates than the PEG/dextran ATPS and which can be expected to have more\nvaried membrane wetting properties than PEG/dextran systems.\nWe thank the reviewer for highlighting the earlier work on PEG/dextran LLPS and its interactions\nwith lipid membranes. We have now included references to these pioneering studies in the\nIntroduction and added a brief contextual sentence in the Discussion noting that early ATPS\nexperiments provided foundational insights into wetting and interfacial tension effects. We\nemphasize that our study is complementary and distinct: by focusing on polyelectrolyte\ncoacervates, we reveal varied selective wetting behaviors on phase-separated membranes.\n6. The SI notes that 10 minutes elapsed after mixing before imaging. Did the wetting behavior change\nfurther over time or did it remain constant after 10 minutes?\nPlease see our reply to Reviewer 2.\nBriefly, we have now performed time-lapse imaging of coacervate-GUV interactions over ~20\nminutes for Janus vesicles (for a PDDA/PAA ratio of 1). These measurements show that the wetting\nconfiguration stabilizes within the first 1-3 minutes after contact and remains essentially unchanged\nover at least 20 minutes. We typically quantify the geometric factor after ~10 minutes, which is well\nwithin this stable regime.\n7. In the SI, the authors state how the “Selective wetting quantification” was performed. I found the\nexplanation confusing as it seemed that several types of potentially important wetting scenarios were\nexcluded from analysis (“wetting on both membrane domains” and “complete wetting” were\nexcluded). This requires further explanation, since it would seem important to include these events in\ndatasets meant to elucidate selective wetting behavior.\nWe thank the reviewer for pointing out this lack of clarity. We have revised the SI Methods to\nremove the confusing exclusion criteria and to clarify that selective wetting is quantified only when\na coacervate unambiguously wets a single membrane domain. Complete wetting events, which\ncannot be assigned to a specific domain, are treated separately and explicitly discussed in the main\ntext."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have adequately addressed all the comments and concerns raised by the reviewers. I also checked the\ncomments raised by Reviewer 1, and the authors have done a great job addressing them. I would like to congratulate the\nauthors on their work. I recommend publishing this manuscript in Nature Communications.", "reviewer_3": "The revised manuscript and author response address concerns raised by the reviewers. I recommend that the manuscript be\naccepted for publication."}, "author_response": ""}]

Decision letter