Engineering synthetic cells with intramembrane domains possessing distinct bilayer asymmetries

Domain: biology
Published: 2026-03-19
Editorial outcome: major_revision

Abstract
Our understanding of how membrane asymmetry governs biological function is limited by the lack of techniques to produce model membranes which can reliably and accurately mimic cellular membrane asymmetry. Not only in terms of asymmetric lipid distribution, but also how that asymmetry can be confined to specific lateral locations across the membrane. Here we present an inverted emulsion method that can be used to produce synthetic cells with symmetric and asymmetric bilayers, as well as phase separation where the intermembrane domains possess distinct bilayer asymmetries. We assess the degree of lipid asymmetry using protein-lipid interaction and quenching assays. Surprisingly, the synthetic cells with asymmetric and phase separated membranes displayed pronounced curvature of the domains and resulted in membrane budding and division. Overall, this work develops biomimetic membranes with lipid compositions akin to natural biomembranes – an essential element in the development of functional synthetic cells.

Introduction
The asymmetric nature of biomembranes is observed for all membranes present in eukaryotic cells1–4. With distinct lipid compositions in its leaflets, the plasma membrane is selective with specific functionalities5,6. For example, its inner leaflet is negatively charged, caused by a majority of charged lipids like phosphatidylserine (PS) and phosphatidylinositol phosphates. The presence of these lipids in the inner leaflet allow important cellular activities like signaling cascades, membrane protein interactions and assemblies such as ESCRTs and even viral proteins7,8. In contrast, the outer leaflet is devoid of such negatively charged lipids and instead containing mostly sphingolipids and glycolipids, which play a significant role in the formation of lipid rafts/domains and in cell-to-cell communication9,10. In fact, it is understood that having charged lipids like PS in the outer leaflet of the plasma membrane is a sign of cellular apoptosis11–13. While PS is indeed absent from the outer leaflet of healthy cell membranes, the outer leaflet can still possess a net negative charge due to the presence of alternative negatively charged components, such as gangliosides or sialylated glycolipids. These lipids contribute to the overall negative surface potential of the outer membrane, which is critical for biological processes such as cell-cell interactions and the efficacy of drug delivery systems utilizing positively charged polymers or lipids14. Recently, molecular dynamic simulations of a fully asymmetric plasma membrane were performed, excluding protein asymmetry, to understand lipid organization15. While computers simulations can reveal interesting insights into the lipid organization and their diffusion, it is still necessary to produce experimental asymmetric model membranes (including the associated membrane proteins) to not only understand various biophysical phenomena (e.g., membrane re-modeling) but also to mimic features of eukaryotic cells in the context of bottom-up synthetic biology.

Despite our knowledge of the complex and extensive lipid composition of the cellular membrane, as obtained from lipidomic studies, building asymmetric model membranes in the lab is still challenging16. Methods such as droplet interface bilayers (DIBs)17, vesicle (hemi) fusion to a supported lipid bilayer (SLB)18, or the use of methyl-β-cyclodextrin19 offer ways to produce asymmetric bilayers but the resulting membrane structures are limited. In the case of DIBs, the presence of large amounts of oil can be an issue for specific proteins or lipids, and subsequent manipulation (e.g., to add biomolecules from outside) after the formation of the bilayers is not possible. In the case of vesicle fusion-based asymmetry generation, usage of divalent cations/oppositely charged lipids could add to the complexity. Moreover, the resulting SLB or giant unilamellar vesicle (GUV) has an inaccessible inner leaflet or requires lipid flip-flop to alter its composition. The methyl-β-cyclodextrin method can produce asymmetric membranes but is limited to specific lipid types with limited access to the vesicle lumen and is process intensive. For these reasons, cell-sized free-standing GUVs with accessible outer and inner leaflets are desired. The latter point is an important one as not all GUV production methods provide high encapsulation. Being able to visualize them and encapsulate larger biomolecules within makes them a strong candidate for building synthetic cells from the bottom-up20. Techniques like electroformation and hydration methods only yield symmetric bilayers and are further limited by difficulties with using physiological buffers or low encapsulation efficiencies. Encapsulation is essential when modeling cells, therefore, researchers require not only asymmetric membranes but also high encapsulation of various biomolecules21. More sophisticated techniques such as microfluidics have been shown to create GUVs with a variety of lipid asymmetries with excellent encapsulation efficiencies22–25. For example, pulsed-jetting technique was used to create cell-size lipid vesicles with asymmetric lipid bilayers26, and double emulsion-based templating is adopted to create lipid vesicles with biotinylated lipid asymmetry27 as well as bacteria-like vesicles with lipopolysaccharides in the outer leaflet28. Microfluidic platforms have the advantage of precision in size, encapsulation efficiency, along with a high degree of asymmetry. However, the experimental complexities of microfluidic setups have curtailed their widespread applicability, leading to the requirement of a simple yet effective method to assemble asymmetric model membranes.

Previously, our lab and others have shown that the inverted emulsion method can be optimized to efficiently produce GUVs that can also encapsulate large biomolecules, while still being accessible and time effective (<20min)29–33. Using this technique, researchers have developed thermoresponsive symmetric vesicles and light activated nanopore assemblies for cargo release applications34,35. Functional applications in the form of artificial cell-based therapeutic protein delivery for tumor treatment and tissue angiogenesis was achieved36,37. Furthermore, complex multi-layered membrane systems via layer-by-layer assembly was proposed through the inverted emulsion method38. Despite the recent applications of the inverted emulsion method, the adoption of this technique remains limited amongst the biophysical community for asymmetric membrane applications. To engineer realistic synthetic cells with asymmetric membranes akin to the plasma membrane, it is necessary to improve the technique for reproducible production of vesicles with various lipid types, functional proteins, and even to include lipid domains.

Previous research in this direction has shown that it is possible to produce phase-separated GUVs using the inverted emulsion method but with limited success in cholesterol incorporation or reproducibility35,39,40. Presently, to our knowledge, there is no methodology that has been shown to produce asymmetric membranes, along with the complexity of phase separation that can also sustain protein functionality. Here, we address this shortfall and present a methodology to achieve asymmetric membranes with lipid domains and functional protein interactions.

To achieve this, we developed a triple-layer inverted emulsion method with a layer-by-layer addition of the solutions which allows production of asymmetric and/or phase-separated GUVs. First, the outer aqueous solution is layered with an oil solution containing an outer leaflet lipid composition. This is followed by layering of a ‘spacer’ oil (without lipids) and finally by a pre-formed water-in-oil (W/O) emulsion containing the inner leaflet lipid composition in the oil phase. After a quick centrifugation step, asymmetric GUVs are produced when the W/O droplets with the inner leaflet lipids pass through the interfacial monolayer containing the outer leaflet lipids. We measured the degree of asymmetry of the formed lipid bilayers via a dye quenching assay and further confirmed it through protein-lipid binding studies. Various proteins (streptavidin/GFP/Cholera toxin subunit B) either alone or in combinations were tested to asymmetrically decorate (inside and outside) GUVs tagged with specific ligand-lipid types (Biotinyl Cap PE/DGS-Ni-NTA/GM1). To demonstrate the flexibility of the method, we show the production of more complex asymmetric lipid membranes exhibiting not only phase-separation but also asymmetry in peripheral proteins. Upon formation of such complex membranes, we report synthetic cells with out-of-equilibrium morphologies leading to membrane budding and division (under osmotic deflation). By theoretical considerations, our results suggest that asymmetry in the bilayer in combination with domains can exhibit (two different values of) spontaneous curvature contributing to the formation of a closed membrane neck that undergoes fission.

Results
Triple-layer inverted emulsion method
To achieve a high degree of bilayer asymmetry in GUVs, the challenge is to prevent mixing of the two different lipid containing oil solutions. Failure to do so will result in contamination of the inner and outer leaflets with the opposite lipids and decrease the degree of asymmetry. For this reason, we have carefully selected and employed two different oils with varying densities (to avoid mixing of oils during the centrifugation step), and crucially, we use an empty oil as a spacer layer (free from lipids) in between them (Fig. 1). This avoids any lipid mixing during the production process.

After the preparation of the lipid oils containing two distinct lipid compositions for inner and outer leaflets (Fig. 1), the outer leaflet lipid composition containing squalene lipid oil is added to the microtiter plate well with outer aqueous solution (bottom left box, Fig. 1). The entire set is allowed to incubate for 30 min to form the interfacial monolayer as the lipids self-assemble along the squalene-water interface. Following this, lipid-free mineral oil is gently spread over the squalene layer, which acts as a spacer oil as described earlier. Immediately, a W/O emulsion formed from the second lipid composition containing mineral oil and the inner solution (sucrose solution with or without streptavidin, top box Fig. 1) is spread on top of the spacer oil (right, Fig. 1). Finally, centrifugation is performed to assist the movement of the inner leaflet lipid composition stabilized W/O droplets in the top-most layer towards (and through) the interface monolayer to produce vesicles with asymmetric bilayers. The same technique is used to produce symmetric bilayers by using the same lipid composition in both oil phases.

Typical examples of the resulting symmetric and asymmetric GUVs can be seen in Fig. 2a, b, respectively. Despite the change in the lipid composition for symmetric and asymmetric GUVs, there is no substantial difference in the morphology or the yield of the GUVs produced. In this method, an incubation step of 30 min is employed for the formation of interfacial monolayer to produce asymmetric GUVs. Contrary to previous methods for symmetric GUVs, we found that it is important to include an incubation step as well as an extra spacer oil to prevent any possible adulteration of the bilayers (through mixing of the oils and lipids)41. In the present work, as the W/O droplets pass through the oil-water interface (i.e., squalene-glucose), the interfacial monolayer will be depleted of lipids. Any further W/O droplets crossing the interface, in places without the interfacial monolayer, can still result in vesicles but they will only have excess oil and contain membranes that are symmetric or with low asymmetry. To avoid this, either the surface area of the interface between squalene and glucose solution has to be increased or the number of W/O droplets needs to be reduced. However, increasing the concentration of the lipids does improve monolayer formation in general, but beyond 200 μM we do not see a significant improvement33. Considering, the time implemented to form the lipid monolayer is 30 min and the centrifugation time is 3 min, the rate of formation of lipid monolayer is much longer than the rate of droplet transfer. For this reason, we reduced the number of W/O droplets by using only 3 μL of the inner aqueous solution (compared to 5 μL from our previous work33).

The asymmetry can also be compromised by contamination of inner monolayer lipids with outer ones and vice versa. If too many excess lipids from the emulsion remain, they can also get pulled down with the W/O droplets and end up in the outer leaflets of the GUVs. Here, the presence of a oil spacer (i.e., mineral oil without lipids) dilutes the lipids from W/O droplet in excess mineral oil before passing into the squalene layer and ensures a high degree of asymmetry in the final bilayers (Fig. 2c). Our previous work, without these precautionary steps to prevent contamination, has shown a lower degree of asymmetry when compared to the results of this work32.

Fluorescence quenching assay to assess bilayer asymmetry
Fluorescence quenching is an established method to assess the degree of asymmetry in lipid bilayers. In our experiments, we performed a quenching assay using NBD-tagged lipids and sodium dithionate as a quencher - a commonly used combination42. The quenching occurs when sodium dithionite, a reducing agent, interacts with the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group, reducing it to a non-fluorescent form. This reaction is highly selective and depends on the accessibility of the NBD fluorophore to the aqueous dithionite, providing a measure of its exposure in membrane systems43. In the first set of experiments, we compared the quenching percentage in symmetric GUVs produced from the previously reported double-layer method to our triple-layer method. The lipid composition for this study was 99.5 mol% POPC and 0.5 mol% NBD-DOPE. As displayed in Fig. 2c, the fluorescence intensity of the symmetric GUVs produced through the double-layer method is reduced to 62±13% from the unquenched membrane and a similar percentage of 53±11% is observed for the GUVs produced using the triple-layer method, after the addition of sodium dithionate. This result is anticipated as the NBD-DOPE lipid gets quenched on the outer leaflet of the symmetric bilayer only, and the intensity should reduce to half. From this data, statistically, it can be concluded that no significant difference in the percentage of quenching is observed amongst the symmetric GUVs produced from both the methods and quenching assay can be reliably performed on the asymmetric GUVs.

Two different populations of asymmetric GUVs were then produced using both methods. One comprised of GUVs produced with NBD-DOPE in the inner leaflet of the GUVs and the other had NBD-DOPE in the outer leaflet. Like the previous condition, sodium dithionate was added to the outer solution for both populations of asymmetric GUVs. In the case of NBD-DOPE in the inner leaflet, no or very few of the GUVs should be quenched for successful asymmetricity. However, for the case when NBD-DOPE is in the outer leaflet (and therefore accessible to the quencher), all the fluorescence intensity should be quenched. A time-lapse video showing the quenching of asymmetric GUVs (with NBD-DOPE in the outer leaflet) in the presence of sodium dithionate can be seen in the supplementary information (Supplementary Video 1). In Fig. 2c, for the asymmetric NBD inside condition, both methods exhibit low quenching: double-layer (14±11%) and triple-layer (7±3%). While in the asymmetric NBD outside condition, quenching is high for both methods with 77±6% for double layer method and 97±1.3% for triple layer method. Interestingly, the triple-layer methodology shows lower variability in the degree of asymmetry. These results in conjunction with statistical analysis suggest that the two production methods lead to distinct quenching profiles under the tested conditions, with the triple-layer method demonstrating more consistent results (less variability) and improved asymmetry compared to the double-layer method. To evaluate the hypothesis that the presence of spacer oil prevents lipid mixing and improves the asymmetry, we added labels to each layer. Observations are made of the mixing of the labels after the centrifugation step. The Supplementary Fig. 1 shows that the double-layer method results in the homogeneous mixing of upper mineral oil layer with the squalene layer, and a uniform color. In the case where the triple-layer method, but without spacer, some mixing is observed. Only in the case of the triple-layer with spacer, no oil mixing was observed. This confirms that a reduced lipid mixing in the methodology contributed to the improved asymmetry, ~95% compared to ~80% or lower that is typically observed amongst works using the double-layer method32,43,44. In addition to the density differences which maintain the separation of mineral oil and squalene, this test emphasizes the role of spacer oil in further reducing the mixing of lipids, ensuring high lipid asymmetry across the membrane.

Protein-lipid bilayer asymmetry
Protein-lipid interactions are universal and play an important role in cellular activities. To demonstrate the functionality of the produced asymmetric synthetic cells, we utilized specific proteins with a high affinity towards ligand-tagged lipids that are present only in one bilayer leaflet. First, the high affinity of streptavidin towards biotin was exploited to not only utilize the asymmetry of the membranes, but also to demonstrate the assembly of a biomimetic GUV using this technique. In Fig. 3a, streptavidin (green channel) is encapsulated within the lumen of the vesicles while biotin-tagged lipids are only present in the outer leaflet of the membrane. Note that this configuration is only made possible because the inverted emulsion method can easily encapsulate large biomolecules within the GUVs along with the advantage of producing asymmetric lipid membranes. Importantly, the streptavidin protein does not bind to the GUVs as the biotin-tagged lipids are only in the outer leaflet (see also Supplementary Video 2). Indeed, the line segment analysis proves the clear demarcation between the green and red signals of the protein and lipid bilayer (Supplementary Fig. 2a). However, it is possible that the vesicle bilayers were contaminated with oil which could potentially prevent the binding of streptavidin. To investigate this possibility, streptavidin was added externally to the same set of GUVs (see Fig. 3b and Supplementary Video 3). Streptavidin can indeed bind to the biotin-tagged lipids present in the outer leaflet based on the intense green fluorescence observed along the lipid bilayer resulting from protein enrichment. This is further confirmed by line segment analysis revealing clear overlap of the green and red regions indicating binding (Supplementary Fig. 2b). This observation not only confirms that the membranes produced using the triple-layer method are highly asymmetric, but also biomimetic in nature (i.e., lipid bilayers without oil contamination) and can be functionalized.

Further experiments were performed with a different protein-ligand-tagged lipid system to establish the universal applicability of the technique. It is well understood that streptavidin-biotin interaction is one of the strongest non-covalent interactions in nature, while Ni-NTA-histidine tag interactions typically have a disassociation constant in μM range. Thus, comparing weaker and reversible coordination interactions of Ni-NTA presents a compelling proof of the biomimetic and functional asymmetry of the bilayer vesicles. Therefore, we employed His-tagged GFP and Ni-NTA-tagged lipids. The lack of signal at the membrane shown in Fig. 3c demonstrates that the GUVs with Ni-NTA lipids in the inner leaflet are successfully separated from the His-tagged GFP present in the outer solution due to the asymmetric nature of the vesicles (see also Supplementary Video 4). On the other hand, binding is observed when both Ni-NTA-tagged lipids and His-tagged GFP are located on the same side (here, outside) of the membrane, which can be seen in Fig. 3d (see also Supplementary Video 5). The line segment analysis clearly indicates the localization of the protein along the lipid bilayer when DGS Ni-NTA lipid is presented on the outer leaflet of the GUVs (Supplementary Fig. 2d) and no co-localization when the lipid is in the inner leaflet (Supplementary Fig. 2c). These results not only verify the asymmetric nature of the resulting membranes but also demonstrate that it is possible to produce functional asymmetric synthetic cells using different ligand-tagged lipid–protein pairs.

To further demonstrate the flexibility of the method, a more complex configuration of asymmetric GUVs is produced with two different ligand-tagged lipids residing in the opposite bilayer leaflets, and as above, both binding and non-binding experiments are performed. First, vesicles are prepared with biotin-tagged lipids in the inner leaflet and GM1 in the outer leaflet (see Fig. 3e). During the production process, and taking advantage of the inverted emulsion method, streptavidin (green channel) is encapsulated within the vesicles while cholera toxin subunit-B (red channel) is added to the external solution. Consequently, both proteins are observed to bind successfully to their respective ligand-tagged lipids, i.e., streptavidin binding to the biotinyl cap PE in inner leaflet and CT-BAL-594 to the GM1 in outer leaflet of the vesicles. A negative control is also performed whereby the leaflet positions of the ligand-tagged lipids are reversed while keeping both the proteins in their respective configurations, i.e., streptavidin inside the vesicles and CT-BAL-594 outside while biotin-tagged lipids are in the outer leaflet (away from streptavidin) and GM1 in the inner leaflet (away from CT-BAL-594). The data show that neither of the proteins bound to the vesicle membrane (Fig. 3f). Again, the line segment analysis further proves our observation that the produced GUVs are indeed asymmetric and allow for specific interactions in the presence of the two proteins (Supplementary Fig. 2e, f).

Finally, we quantified the results for all three distinct molecular recognition systems in Fig. 3. For each system, the positive control was when the protein in the external solution could access its target lipid on the outer leaflet, and the negative control was when the target lipid was situated on the inaccessible inner leaflet (Supplementary Fig. 3). The results demonstrate a high degree of binding specificity. For the streptavidin-biotin system, we observed binding in 89.5% of the positive control GUVs, whereas only 6.6% of negative control vesicles showed a signal. The specificity was even more pronounced for the His-tag GFP-NTA system, which resulted in 100% binding for the positive control and 0% for the negative control. Finally, we tested the binding of the CT-B to its target ganglioside, GM1. A robust binding signal was detected in 97.0% of GUVs when GM1 was on the outer leaflet, compared to only 9.1% when it was on the inner leaflet. Not only does this demonstrate the method’s flexibility to produce asymmetric GUVs with two different leaflet specific lipids able to bind different proteins, but this experiment showcases its ability to combine two asymmetries: both within the membrane (i.e., inner and outer leaflets), and across the membrane (i.e., inner and outer solutions).

Production of asymmetric phase-separated membranes
Lipid partitioning in membranes is the major contributor to raft formation in cell membranes. In association with various proteins that can influence the clustering of lipids, transbilayer coupling has been shown to influence domain formation in membranes9,45. In this context, synthetic cells also require complex lipid mixtures to produce phase-separated membranes (i.e., liquid-ordered (Lo) and liquid-disordered (Ld) domains) as well as being able to encapsulate biomolecules in the lumen to study transmembrane signaling pathways, transbilayer molecular interactions and protein assemblies. While electroformation based production of phase separated GUVs is a common strategy, it is challenging to produce them using inverted emulsion method. Previous studies demonstrated very poor incorporation of cholesterol when mineral oil was used39,46 except for one study35 (see Supplementary Table 4). However, we are unable to reproduce the phase separation using double layer method involving mineral oil alone (Supplementary Fig. 4). This inconsistency motivated us to develop an alternative method. Using the triple-layer method, we are successful in producing phase-separated GUVs from a lipid mixture containing POPC, cholesterol (chol) and sphingomyelin (SPM) (see Fig. 4).

The usage of squalene, instead of the commonly used mineral oil, paved the way for successful incorporation of cholesterol into the lipid membranes. This is because the solubility of lipids (saturated lipids such as sphingomyelin) and cholesterol in mineral oil is high, thanks to the presence of long-chain saturated hydrocarbons. The extent of phase separation and confocal images of the phase-separated GUVs are presented in Fig. 4b, c. GUVs were formed with varying concentrations of cholesterol (in both the squalene and mineral oil phases) while keeping the sphingomyelin to DOPC ratio constant (i.e., 1:1). The data is presented as a phase diagram in Fig. 4a and confocal images of the GUVs with varying percentages of cholesterol content can be seen in Fig. 4c. Beyond 60% of cholesterol, we observed no further phase separation (i.e., the majority had no visible domains). The membranes are predominantly with single-phase for GUVs containing 70%, 80% and 90% cholesterol content (Fig. 4a) as observed elsewhere35. Additionally, we compared these results to those made by the most used method to produce phase-separated GUVs, the electroformation method (Fig. 4b). Unlike similar studies, our method provides improved agreement with the results obtained by the electroformation method. Due to high solubility of cholesterol in mineral oil, studies using mineral oil have shown poor cholesterol membrane incorporation resulting in predominantly gel-phased lipid domains35,46. This signifies the importance of modifying the inverted emulsion method to replicate the gold standard, i.e., electroformation. The triple-layer method presented in this study achieves this while still being able to produce asymmetric GUVs, both membrane and solutions. Note that the electroformation method cannot produce asymmetric GUVs (neither bilayer nor solution asymmetry). Within the tested concentrations, we are also able to produce phase-separated GUVs with liquid–liquid co-existing phases by varying sphingomyelin concentration (while fixing the DOPC:Chol ratio at 1:1) (Supplementary Fig. 5). Taken together, these results provide strong evidence for the formation of phase-separated vesicles using the triple-layer method. Unlike previous work using the inverted emulsion method, the study here is exclusively focused on sphingomyelin as a major contributor for the domain partitioning and not DPPC35,39. Sphingomyelin is one of the major biomembrane lipids and exists in high concentrations in the outer leaflet of the plasma membrane. Moreover, along with cholesterol and various proteins, sphingomyelin has been proposed to play a significant role in the formation of rafts or lipid domains in the plasma membrane9. Our results present a step forward in the direction of modeling and studying rafts/domain creation via protein assemblies and transbilayer interactions.

The triple-layer method also allows combining the above to produce complex asymmetric phase-separated vesicles. The schematic shown in Fig. 5a depict the trans-bilayer asymmetry of lipids confined to the Ld and Lo domains of the GUV. In the Ld domain, asymmetry is achieved through selective addition of biotinyl cap PE lipid to the inner leaflet of the Ld domain only. In the case of Lo domain, GM1 lipid is confined to the outer leaflet of the Lo domain only. This is confirmed through the binding of streptavidin and CT-B Al-594 proteins that have high specificity towards biotin and ganglioside (Fig. 5a and Supplementary Fig. 6). However, the addition of both streptavidin (inside) and CT-B Al-594 (outside) proteins further add to the asymmetry of the system. Such a design mimics the natural asymmetry present within biological membranes (distinct lipid types and peripheral proteins on either side of the membrane), creates distinct internal and external functionalities, and broadens the system’s use for modeling of biomembranes. For example, this setup could be used to study the selective binding of signaling proteins to one side of the membrane while simultaneously investigating receptor-ligand interactions or membrane curvature on the external membrane surface. In general, we believe that more complex asymmetric membranes will be crucial for increasing the functionality of synthetic cells, either within the field of bottom-up synthetic biology or for applications in biotechnology and biomedicine47. Indeed, upon addition of CT-B Al-594 two distinct spontaneous curvatures are observed, here. Figure 5b shows confocal images of asymmetric phase-separated GUVs incubated with CTB subunit (tagged with Alexa-594 dye, red). The asymmetric phase-separated GUVs have lipid composition that induces phase-separation (DOPC:SPM:Chol - 1:1:2, with 0.2 mol% biotinyl cap PE in the inner leaflet, and 1 mol% GM1 in the outer leaflet). The lipid asymmetry is achieved through doping of the inner leaflet with 0.2 mol% biotinyl cap PE which partitions into the Ld phase and the outer leaflet with 1 mol% of GM1 which partitions into the Lo phase48. This domain-specific asymmetry resulted in unexpected membrane dynamics within the asymmetric phase-separated GUVs that have not been reported in literature. Unlike the vesicles with symmetric bilayers, the asymmetric phase-separated GUVs exhibited a noticeable outward protrusion after the binding of CT-B Al-594 to the GM1 within the Lo domain (Fig. 5b, c and Supplementary Video 6). Typically, Lo domains are tightly packed and are less flexible compared to Ld domains49,50.

Here, the Lo domain protrudes toward the GM1-containing outer leaflet after the addition of CT-B Al-594 (Fig. 5b, c, and Supplementary Video 6). The outward-budding of the smaller Lo domain acts to lower the line free energy of the domain boundary between the two domains51,52. In contrast, when adding CT-B Al-594 to symmetric phase-separated GUVs, no such protrusion was observed (Supplementary Fig. 6). The same can be said of other reports in which membrane nanotubes were pulled from GUVs53. Previously, it has been observed that GM1 asymmetry in non-phase-separated GUVs (10% GM1) resulted in high membrane positive curvature which generated membrane nanotubes54,55. Indeed, we also observed outward curvature, but with only 1% GM1 and with a phase-separating membrane composition (Fig. 5c and Supplementary Video 6). This pronounced curvature with less GM1 compared to previous work could be due to the enrichment of GM1 to the Lo domain. Furthermore, the protrusions remain for over 6 h (Supplementary Video 7), suggesting that there is no flipping of GM1 or biotinylated lipids for long time periods. This opens the possibly of using such morphologies as polarized synthetic cells or anisotropic novel drug carrier systems. Thus, the outward-budding of the Lo domain is driven by two synergetic mechanisms. First, the GM1-enriched outer leaflet leads to a positive spontaneous curvature of this domain, arising from the large headgroups of the GM1 glycolipids. Second, the domain boundary between the Lo- and the Ld-domain has a positive line tension. Therefore, the free energy of the Lo-domain is reduced when the budding decreases the length of the domain boundary48.

To establish control of synthetic cell morphology, we modulated the protein concentration in one domain whilst keeping the other one constant. To do this, we systematically increased the concentration of streptavidin (10 nM, 20 nM, 40 nM, and 80 nM) inside the vesicle while maintaining a constant CT-B Al-594 concentration outside. As shown in Fig. 6, at equilibrium (after an hour), the pronounced membrane curvature in the Lo domain (red) was progressively reduced as the concentration of internal bound streptavidin in the Ld domain (green) increased (n≥15). This modulation of the protein concentration at the inner Ld leaflet only, counteracted the Lo domain curvature induced by CT-B Al-594 binding. At streptavidin concentrations of 40 nM and above, the vesicle’s morphology is restored to a state close to its original spherical configuration (i.e., without CT-B).

This phenomenon mirrors the mechanism by which the outward budding of the Lo domain reduces the line free energy at the domain boundary when CT-B Al-594 is added. Therefore, we demonstrate the ability to control membrane curvature in one domain by modulating protein binding in another within a complex asymmetric phase-separated system. These findings suggest that such asymmetric systems offer a novel approach for studying the dynamic interplay between membrane domains within the same vesicle, enabling unprecedented control over domain-specific membrane curvature.

Shape analysis and budding dynamics of asymmetric phase-separated GUVs
Next, we applied shape analysis to better understand the dynamic morphologies observed in our asymmetric phase-separated synthetic cells. This was done by minimizing the elastic energy of the two-domain vesicles in Fig. 5 as given by Eq. (S6) (see detailed discussion in the SI). Taking into account the constraints on the vesicle volume and surface areas, one has to minimize the shape functional given by Eq. (S7) for fixed volume V and fixed surface areas Ao and Ad of the Lo and Ld domains. The elastic energy of the two-domain vesicle includes the bending energies of the two membrane domains which depend on the bending rigidities and the spontaneous curvatures of these domains. The bending rigidity of the Lo domain is larger than the bending rigidity of the Ld domain, which can be understood by the increased bilayer thickness of the Lo domain56–58. In addition to the difference in bending rigidity, protein asymmetry in our systems changes the spontaneous curvatures of the two domains. In general, the spontaneous curvature arising from protein asymmetry is unable to induce the formation of a closed neck for fixed vesicle volume but such a neck can be formed during osmotic deflation52. Therefore, we used deflation to achieve neck closure as in Fig. 5d and Supplementary Fig. 7a, similar to previous studies52,59–61. Together with the Supplementary Video 8, we conclude that osmotic deflation can also promote domain-induced budding in asymmetric GUVs, leading to the formation of two spherical membrane domains connected by a closed neck. In general, domain-induced budding is facilitated by the line tension of the domain boundary and by a large spontaneous curvature of the domain that forms the bud51,52,59. Furthermore, we also observed neck fission and, thus, vesicle division (see Fig. 5e and Supplementary Video 9). In the Supplementary Fig. 7b, the Lo buds have a bright red ring (due to CT-B Al-594 binding) and are devoid of streptavidin (green) binding. The boundaries between the Ld and Lo membrane domains contribute the term λLdb to the free energy of the phase-separated membrane, which is proportional to the line tension λ and to the total length Ldb of the boundaries. The line tension λ is always positive, which implies that the membrane’s free energy can be reduced by domain coarsening, i.e., by fusing two or more domains into one larger domain. This coarsening process eventually leads to only two domains and one domain boundary (as observed here). The additional asymmetric binding of proteins to specific membrane domains, changes their mean curvatures, with a more pronounced change of the Lo domain as displayed in Fig. 5b–d.

The observed two-domain vesicles are axisymmetric which implies that these shapes can be determined by their contours as illustrated in Fig. 7. The axis of rotational symmetry corresponds to the dotted line, which intersects the Lo domain at the north pole and the Ld domain at the south pole of the vesicle. The mean curvature of the vesicle membrane at the two poles is obtained by fitting two osculating circles to the shape contour, one at the north pole and another one at the south pole (see Fig. 7b). In general, an osculating circle is tangent to the curve, and has the same curvature at the point of contact. The radii of these two circles, denoted by Rnpol and Rspol, determine the mean curvatures Mo,npol and Md,spol of the vesicle at the north (Lo) and south (Ld) pole according to Mo,npol = 1/Rnpol and Md,spol = 1/Rspol. Inspection of Fig. 7b reveals that the osculating circle at the south pole provides a good fit to the whole shape contour of the Ld domain. As a consequence, the Ld domain forms a spherical cap with constant mean curvature Md = 1/Rspol, has several interesting consequences. First, these necks can be characterized by their effective mean curvatures Meffij as defined in Eq. (S10). Second, the formation of the closed necks in Fig. 5d, e implies that the spontaneous curvature md of the Ld domain exceeds the effective neck curvature Meffij as in Eq. (S11). Shape analysis leads to the values Meffij = 0.336 μm−1 for Fig. 5d and Meffij = 0.308 μm−1 for Fig. 5e. The same analysis has been applied to other vesicles with closed necks, leading to the numerical values in the Supplementary Table 6. Third, because the closed neck of the asymmetric two-domain vesicle in Fig. 5d, e undergoes fission we can also conclude that, in this case, the constriction force f acting against this neck exceeds 20 pN as follows from Eq. (S13) and previous experiments60.

Methods
Materials
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), Sphingomyelin (SPM), Cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) (DGS-Ni-NTA), GM1 Ganglioside (Brain, Ovine-Sodium Salt) (GM1), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (biotinyl Cap PE), and L-α-Phosphatidylethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (Ammonium Salt) (NBD-DOPE) are purchased from Avanti polar lipids. Cholera Toxin Subunit B (Recombinant), Alexa Fluor ™ 594 Conjugate (CT-B Al-594), 1,1’-Dioctadecyl-3,3,3 ‘, 3’-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DiD), Streptavidin Alexa-488, and Corning™ Glass Bottom 96-well microplates for fluorescence-based assays were all purchased from Thermo Fisher scientific. Chloroform, Beta-casein, mineral oil, squalene, glucose, sucrose, Tris-(hydroxymethyl)-amino methane (Tris), and sodium chloride (NaCl) were purchased from Sigma Aldrich. All chemicals were used as purchased unless otherwise stated. MilliQ® water is used for making all solutions.

Inverted emulsion method
A desired lipid mixture (see Supplementary Tables 2 and 3, for various lipid compositions) in chloroform (400 μM) is dried in a glass vial using argon gas and further left in vacuum for 60 min. Following this, mineral oil is added to the same glass vial in the case of double layer method and mineral oil (for the inner leaflet lipid mixture) and squalene (for the outer leaflet lipid mixture), in the case of triple layer method. The vials are subjected to bath sonication in degassing mode for 15 min at room temperature. The final lipid-oil mixture, with a concentration of 400 μM, is stored at 4°C and used within a week. First, 30 μL of mineral oil lipid-oil mixture (for double layer method) and squalene lipid-oil mixture (containing the outer leaflet composition) (for triple layer method) is added to β-casein coated (2 mg/mL for 30 min) microtiter plate (96-well) prefilled with 50 μL of desired outer solution (glucose or glucose + 10 mM Tris and 100 mM NaCl for experiments with proteins, ~900 mM). At this stage, the formation of a lipid monolayer is allowed to take place for 30 min. Following this, 30 μL lipid-free mineral oil is added on top of the squalene lipid-oil mixture, in the case of triple layer method. Immediately, 50 μL of pre-fabricated W/O emulsion formed by rubbing an Eppendorf® tube containing inner aqueous solution (3 μL of sucrose or sucrose + 10 mM Tris and 100 mM NaCl buffer for experiments with proteins, ~900 mM) in 200 μL of mineral oil on an Eppendorf® rack, three times, is added on top of the spacer lipid-free mineral oil (for triple layer method) and mineral oil (for double layer method). Finally, a centrifugation step is performed in swinging buckets at 200 × g for 3 min. In the case of lipid mixtures for phase separation (to generate lipid domains), the solutions are pre-heated, and the inverted emulsion method is performed at 60°C. All the lipid compositions are presented in Supplementary Table 1 and Supplementary Table 2 in the supplementary information. At all instances, equi-osmolar solutions are used, unless otherwise stated.

Electroformation
Electroformation experiments for the generation of phase separated GUVs were performed to compare with the population of phase separated GUVs produced using inverted emulsion methods. For this purpose, two indium tin oxide (ITO) coated plates are preheated to 90°C and cooled to room temperature before applying 10 μL of lipid mixture (POPC:SPM – 1:1, Chol 10/20/30/40/50/60/70/80) in chloroform on the ITO coated side. The lipid-coated ITO plates were desiccated under vacuum for 45 min. A chamber was formed with the lipid coatings sides facing toward each other using a 2 mm thick hollow rectangular Teflon spacer (20 mm × 40 mm). Pre-heated 900 mM sucrose solution was injected into the chamber and sealed with metal clips. Using copper tapes, the ITO plates are connected to a function generator and placed inside a hot oven set at 60°C. A sine wave function at 10 Hz and 2 Vpp was applied to the chamber for 3 h. Finally, the GUV suspension was collected by tapping and pipetting, and imaging was performed at room temperature. Measurements were taken from distinct samples where N ≥ 3.

Lipid mixing analysis
Using tracer lipids in the oil layers, we tried to visualize the extent of lipid mixing in both double-layer and triple-layer methods as well as triple-layer without spacer oil. For this purpose, initially 600 μL of 900 mM glucose solution is added to the Eppendorf® tubes, followed by pipetting 200 μL of NBD-DOPE loaded mineral oil or squalene over the glucose solution. In one of the conditions, spacer oil, mineral oil without lipids, is added on top of the squalene layer. In the next step, W/O droplets (900 mM sucrose solution) in DID containing mineral oil is added. Following this, the tubes are centrifuged at 1000 × g for 3 min in an Eppendorf® mini centrifuge (Centrifuge 5424 R). After the centrifugation step, the bottom-most oil layer, interfacing the glucose solution, was observed for any change in color (from yellow to bluish yellow).

Quenching assay
After the production of NBD-tagged PE (0.5 mol%) containing vesicles using the above described double and triple layer inverted emulsion methods, a 10 mM final concentration of freshly prepared sodium dithionate is injected in the outer solution. This injection can be either done after removing the top oil layers or without it based on the requirements of the experiment. After the successful injection of the quencher, end-point fluorescence intensity measurements or time-lapse experiments were performed using confocal microscopy. Fluorescence intensity percentage is calculated via Eq. 1 using Fiji (ImageJ) software for vesicles with uniform membrane intensity before and after the addition of the quencher. Statistical significance between groups was assessed using a two-sided Mann–Whitney U test (OriginPro, OriginLabs software). The null hypothesis was that the median quenching percentages between the two groups compared (double-layer vs. triple-layer) were equal. The significance level was set at p < 0.01. For the ‘Asymmetric NBD Outside’ condition, the difference between the double-layer and triple-layer methods was statistically significant (p < 0.0001), leading to the rejection of the null hypothesis, lack of a significant difference (p > 0.01) is denoted with n.s.

Protein-lipid asymmetry experiments
For experiments with proteins encapsulated within the lumen of the vesicle, a streptavidin concentration of 10 nM, 20 nM, 40 nM or 80 nM was used to bind biotinylated lipids present in the membrane. In this case, the protein solution is mixed with an inner solution that is used to make W/O emulsions. In all other cases, where the proteins are in the outer solution, the desired concentration of protein solution (final concentrations, GFP 100 nM, streptavidin 20 nM, and cholera toxin subunit B (CT-B Al-647) 20 nM) with matching osmolarity is injected into the outer solution after the formation of the GUVs. For osmolarity induced bud division experiments, 2.5 μL of 2 M glucose solution was injected into the outer solution creating a 50 mOsm hypertonic solution to deflate the GUVs, after cholera toxin binding. Time-lapse and end-point experiments were performed using confocal microscopy. Measurements were taken from distinct samples where N ≥ 3.

Microscopy
All the experimental results were obtained using a confocal fluorescence microscope (SP8, Leica). Vesicles with NBD-tagged lipids/streptavidin-alexa 488/GFP were excited with a laser excitation of 488 nm and the emission was detected between 498 and 540 nm. In the case of cholera toxin subunit-B tagged with Alexa Fluor-594, an excitation wavelength of 551 nm is used, and emission is collected between 565 and 620 nm. Finally, for DiD labeled vesicles, excitations are set at 638 nm and emissions were captured in the range of 648–710 nm. Sequential channel acquisition is performed whenever multiple fluorophores are employed. Acquired confocal images and videos are treated using LASX (Leica) or Fiji (ImageJ) software. Measurements were taken from distinct samples where N ≥ 3.

Vesicle shape contour analysis
For shape analysis, Autodesk® AutoCAD® was used to fit the vesicles. An exemplary CAD file for the GUV fitting method is included in the supplementary information. To perform the contour analysis, the GUV image file with scale bar is imported into the AutoCAD® file and the image is adjusted to match the dimensions of the GUVs to that of the CAD file via scaling (in the example file, the scale bar is 10 μm). To determine the axis of rotational symmetry for a two-domain GUV (Fig. 7a), two osculating circles fitting the Ld (green) and Lo (red) domains at the south and north pole are drawn with radii, Rspol and Rnpol, respectively (Fig. 7b). The line connecting the centers of the two circles represent the axis of rotational symmetry. The diameter 2Rdb of the domain boundary is then obtained by drawing a line perpendicular to the rotational symmetry axis and connecting the two cross-sectional points of the domain boundary with the shape contour (where the Ld domain (green) meet the Lo domain (red)), shown as a dashed line in Fig. 7b. A semicircle is drawn with its corner at the domain boundary (left or right side of the GUV, in Fig. 7b, it is drawn on the right side) having a radius of Ro,semi, with the arc of the semicircle fitting the Lo domain contour (red) as shown in Fig. 7b and Supplementary Fig. 8. The base line of this semicircle is parallel to the membrane’s normal vector at the domain boundary, which forms the tilt angle ψ with the rotational symmetry axis (Fig. 7b). These geometric constructions are used to calculate the parameters for the equations mentioned in the main text as well as in the SI and presented in Supplementary Table 5.

Similarly, the effective mean curvatures (Meffij) for vesicles with closed neck configurations are calculated by measuring the radii of the green region (Ri) and red region (Rj). The radii were measured by fitting a circle using circle function in AutoCAD® (an example fit can be found in the supplementary information). Measurements were taken from distinct samples where N ≥ 3.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Yandrapalli et al. focuses on the production of asymmetric, phase separated vesicles prepared by emulsion phase transfer, where an alteration of the conventional method, employing a lipid-free oil spacer, is used to more controllably form asymmetric giant unilamellar vesicles, as determined by protein binding and fluorescence quenching of the vesicle membrane. The authors then use this method to produce phase separated vesicles, successfully segregating protein-binding lipids to the liquid disordered (Ld) and liquid ordered (Lo) phases respectively, and show that protein binding can be proteins can be controllably localized to either face (and domain) of the phase separated vesicle. Finally, the budding off of the protein-bound Lo phase is monitored, and analysis of the shape and budding dynamics is evaluated. This work represents an advance in the fabrication of lipid vesicle-based cell models by combining asymmetry and phase separation control to localize proteins to the membrane in a user-defined manner. This builds on work in the field that have shown (i) the production of asymmetric vesicles, including the author’s prior work (10.1016/j.bpj.2022.12.017) and others which combine membrane asymmetry and protein binding to localize proteins to different faces of a vesicle membrane (https://doi.org/10.1021/acssynbio.2c00564), as well as (ii) the production of phase separated vesicles by emulsion phase transfer (10.1039/c7sc04309k). As such, whilst the work is interesting, my main concern is in its novelty. The main focus of this paper is on vesicle characterisation, including their budding behavior, and for such work to be considered in Nature Communications (as opposed to a field-specific journal) I think the novelty of this manuscript needs to be strengthened. To do so I suggest the following additions; 1) Demonstrating novel functionality (beyond simply binding fluorescent proteins) of the asymmetric phase separated vesicles. Could a proof-of-concept experiment be performed which shows how such control of protein localization results in improved performance of the vesicles as synthetic cells, for example through adhesion, biosynthesis or other improved functionality? 2) Further investigation into the effects of protein localization and stability of such vesicles. The budding, whilst interesting, could be seen as a disadvantage with respect to synthetic cell stability, and it would be interesting to further investigate this. If proteins are localized to both faces of the Lo domain, can this budding be prevented during vesicle deflation? And how long does the protrusion of the asymmetric, phase-separated GUVs persist? This is very interesting from the perspective of lipid flip-flop kinetics, where the presence of the domain boundary may potentially affect flip-flop rates compared to a single phase asymmetric membrane. I also have some suggestions and questions regarding the experimental approach, which should be explored to better validate the new method presented in the manuscript; 3) The authors present the triple-layer inverted emulsion method as an improvement on prior work. The use of a spacer oil is interesting, and I can see how the use of oils of differing density could be used to better separate the lipid-containing squalene layer (outer leaflet) and W/O mineral oil emulsion (inner leaflet). The authors state that there is no lipid mixing during the production process, but there is no data that studies this. It would be interesting to observe whether lipid mixing occurs (perhaps through the use of fluorescent tracer lipids in the squalene and mineral oil emulsion phases), with and without the mineral oil spacer oil. This would facilitate a better understanding of the role of the mineral oil spacer, and how necessary it is in the formation of the asymmetric vesicles. 4) In Figure 1C, the authors show fluorescence quenching data for double-layer and triple-layer emulsion phase separated-vesicles. The authors include convincing data demonstrating the asymmetry of GUVs produced via the triple-layer method, but only produce symmetric vesicles using the double-layer method. The authors cite their prior work (10.1016/j.bpj.2022.12.017) and say that the triple-layer method gave better results in this study, than the double-layer method in the previous study. However, I believe the membrane asymmetry is different, with the focus of the prior work the asymmetric incorporation of NBD-labelled bacterial lipopolysaccharides. The authors should perform fluorescence quenching on GUVs prepared via the double-layer method using the NBD-DOPE lipid used in this work, and the fluorescent quenching compared for double- and triple-layered. This, combined with 3) above will enable a better evaluation for the use of the triple-layer phase transfer method as compared to the simpler double-layer method. 5) The authors report improved cholesterol integration within phase-separated GUVs due to poorer solubility of the molecule in squalene. Has this been reported previously using the double-layer method? Is the cholesterol present in the squalene layer of the assembled phase column only, or also in the mineral oil emulsion that is layered on top? 6) Could the authors provide more data for the curvature of the Lo phase for i) asymmetric phase separated vesicles and ii) the symmetric phase separated vesicles after Ct-B AL 594 binding? In each case only one GUV is presented (asymmetric Fig 4C, symmetric Fig S3). For Fig S3, in the overlay of the image the domains do not align (probably due to timelapse between taking measurements in different fluorescence channels). Providing images of more GUVs would enable the reader to better evaluate the range of curvatures shown for asymmetric/symmetric phase separated GUVs respectively. 7) The shape analysis of vesicle curvature is interesting, but is only applied to a small number of asymmetric GUVs (for curvature discontinuity, 6 GUVs are evaluated and for the closed membrane necks 10 GUVs are evaluated). Were these GUVs prepared from a single phase transfer experiment? It would be good to apply this method to a larger number of GUVs, to better understand the range of curvatures observed the frequency with which the protrusion of the Lo domain occurred across a vesicle population. Finally, the methods section needs to be improved to enable reproducibility of the work; 8) For osmolarity-induced bud division experiments, the final solution osmolarity is not described (only that 2.5 ul of 2M glucose solution is added). Please amend this so it is clear to the reader the final external solution osmolarity (and the osmotic gradient across the membrane). 9) The description of shape analysis in the method is minimally described - the only detail is that Autodesk AutoCAD was used to fit the vesicles. Please give sufficient information for a reader to reproduce the shape analysis. 10) How are the mathematical calculations performed? Please give details.", "reviewer_2": "This is a very interesting manuscript. It’s well written, details are clear, data presentation is very logical, and conclusions are generally well justified. The curvature formalism capable of considering the lipid mixture and effects of lipid ordered and lipid disordered phase is appreciably rigorous, and I haven't seen that applied in the field of synthetic cell engineering before. Authors used more biologically relevant lipids, which increases the value of this work. Authors also interestingly claim better incorporation of cholesterol. Addressing few issues would improve this manuscript: The authors mention that others have proffered methods for preparing asymmetric membranes, and the manuscript compares double layer emulsion to triple layer emulsion in figure 1. However, the authors did not compare the ability to form asymmetric bilayers between their method (triple layer emulsion) and double layer emulsion. Moreover, authors significantly modify the lipid compositions and emulsion formulation between published double layer emulsions and what they test as double layer here. That they didn't overtly compare asymmetry between the double and triple layer methods is odd too because they validate that their assay would work with both methods. Authors were not adequately specific about how successful they were with cholesterol incorporation beyond saying that they were able to see changes in phase separation dynamics with a range of working concentrations during the preparation. Interesting methods: The spacer oil that separates the two lipid-in-oil solutions that contain the intended inner and outer leaflet lipid compositions, Strictly speaking Kruskal-Wallis should never be conflated with ANOVA, also I want more explanation as to why the authors used something really exotic here. They chose a nonparametric multi category test, if nonparametric due to dynamics of liposome formation, why not just Mann- Whitney with Bonferroni correction. There should be justification for nonparametric tests (even though they are always more stringent than parametric tests) and explanation for the choice of Kruskal Wallis over the more common Mann-Whitney. The authors say that cholesterol is more soluble in mineral oil than in squalene, which I am inclined to believe, but the manuscript don't give citation or explanation as to why (the explanation to me would be that mineral oil has cyclic hydrocarbons and aromatics). Likewise there are many small molecule quenchers used, I would like a citation or quick mechanistic comment about the sodium dithionate. Overall, it’s a very interesting paper. I believe after fixing and clarifying the above described issues, it will be a valuable publication of interest to our community.", "reviewer_3": "The manuscript by Yandrapalli et al. describes the development of an emulsion phase transfer method to generate giant unilamellar vesicles with an asymmetric lipid bilayer. This is an interesting concept but I have a number of concerns that need to be address, especially regarding the controls used to quantify if the method is an improvement over existing procedures. 1) Page 3. In the introduction, the authors state that “In contrast, the outer leaflet is devoid of such negatively charged lipids”. While they are correct PS is not present in the outer membrane of healthy cells, the outer membrane is still negatively charged using alternative lipids. If this were not the case, drug delivery with positively charged polymers/lipids would not work. 2) Page 4. The authors have not included droplet interface bilayers as other methods to create asymmetric bilayers. 3) Page 4/5. There needs to be more discussion of previous methods to produce asymmetric bilayers and their drawbacks/differences to what the authors have developed. For instance, the sentence “Presently, to our knowledge, there is no methodology that can produce asymmetric membranes, along with the complexity of phase separation that can also sustain protein functionality.” needs to be more thoroughly evidences and explained. 4) Page 7. The authors state “To achieve a high degree of bilayer asymmetry in GUVs, the challenge is to prevent mixing of the two different lipid containing oil solutions. Failure to do so will result in contamination of inner leaflet and outer leaflets with the opposite lipids and decrease the degree of asymmetry.” This is not evidenced. Furthermore, these are the critical control experiments that are not performed throughout the paper. The control vesicles are prepared symmetrically, but this is not what the authors are claiming they are improving on. There needs to be controls for alternative state of the art asymmetric vesicle preps, for instance those generated by the authors themselves (doi: 10.1016/j.bpj.2022.12.017) and/or just adding an emulsion formed with the inner lipid to a phase transfer column prepared with the outer lipid. These controls are required throughout the work (especially Fig 1, 2, and 4) to actually back up the claims that they are improving on previous methods. 5) Page 9. Authors state “To avoid this, either the surface area of the interface between squalene and glucose solution has to be increased or the number of W/O droplets needs to be reduced.” Can the lipid concentration be increased? 6) Page 10. Authors state “The lipid composition for this study was 99.5 mol% POPC and 0.5 mol% NBD-DOPE.” Did the asymmetric quantification change when different % of the asymmetric lipid was used. I.e. only 0.5% is put on one side. This is the case in later experiments too, when only a small % is made asymmetric. 7) Page 11. Authors state “This suggests that a significant amount of the GUVs, 93 %, remained asymmetric for the duration of the experiment (approximately 1 hour) with no observable permeation or morphological changes.” Did the authors measure past 1 hour? This seems like a very short time to quantify. 8) Fig 2. Why did the authors only test the single asymmetric membrane by adding the proteins from the outside? Looking at 2e/f the streptavidin signal is much weaker at the membrane this way (albeit with the other lipid on the outside too), is there a reason for this? 9) Page 20. Authors state “Both streptavidin (inside) and CT-B AL-594 (outside) proteins were used to further enhance the asymmetry and applicability of the system.” Please explain this in more detail. 10) Page 20 and throughout. There needs to be a better way of explaining the lipid mix and which are in the outer/inner. For instance “DOPC:SPM:Chol - 1:1:2, 0.2 mol% biotinyl cap PE, 1 mol% GM1 and 0.5 mol% DiD” does not explain the system well enough. 11) Page 22. Authors state “In contrast, when adding CT-B AL-594 to symmetric phase-separated GUVs, no such protrusion was observed (Figure S3).” Can they explain why this might be? I.e. the presence of GM1 in the inner phase stops the protein from protruding the phase, but this is not obvious. Also on the same page there is another sentence that is not explained and difficult to understand “Note that there is no binding of streptavidin in the Lo domain (red) suggesting that there are no biotinylated lipids in the Lo phase, and that there is no large head group bearing lipids on the inner leaflet to compensate for the asymmetry of the GM1 in the outer leaflet unlike in symmetric phase-separated GUVs”. 12) With regards to the curvatures quantified. There is mathematical modelling of individual vesicles, but no discussion or analysis of how they compare or how consistent they are. Or how these values change with altered composition/concentrations. 13) Page 29, authors state “These unique synthetic cells with complex membranes were able to exhibit cell-like behavior, with budding and division events, from a purely biophysical origin with no additional energy input.” Is this fair if the authors are adding protein and putting the vesicles under osmotic pressure to attain these features? 14) Methods. The authors use the phase ‘briefly’ multiple times. The materials and methods should be exhaustive for someone to repeat. Regarding density of squalene vs mineral oil, this depends on the mineral you purchase, which is not given. There are multiple points where concentrations are not obvious or amounts added at steps are not given. 15) As is good practice, source data should be made available on a repository."}, "author_response": "We appreciate the reviewer’s thoughtful feedback and suggestions. While we recognize the value of demonstrating additional functionality such as adhesion or biosynthesis, we believe the current focus on controlling protein localization (not only to a domain but also to a leaflet) and analysing the resulting budding dynamics already offers significant novelty as this has never been done before. Our work uniquely integrates lipid membrane asymmetry and liquid-liquid membrane phase separation to precisely localize proteins to specific lipid domains, a combination not previously achieved. This platform mimics key aspects of cellular membranes, particularly the asymmetric distribution of lipids and proteins, and allows for the controlled study of protein-induced membrane budding and shape changes. Relevant for bottom-up synthetic cell formation and a process relevant to biological vesicle formation and trafficking. While future studies can explore additional functionalities, the primary novelty here lies in our ability to controllably localize proteins and induce budding in phase-separated vesicles, which represents a substantial advancement in synthetic cell design and provides a robust foundation for future applications. We believe this contribution is sufficiently novel for Nature Communications. We would like to highlight that we have not just shown binding of proteins, we have also shown controllable morphological (curvature) changes which were a direct result of the asymmetry binding. Furthermore, the curvature changes resulted in budding of smaller “daughter” GUVs. In the context of bottom-up synthetic cells, this is important to demonstrate a new mechanism for synthetic cell division. Note that both of these functionalities (morphological changes and budding off) were not seen in simple symmetry systems (i.e. controls). To emphasize the points above, the following text has added to the discussion section: Furthermore, existing techniques that are able produce complex combinations of lipid membranes such as hemifusion-based18 and methyl-cyclodextrin-based19 are limited to outer leaflet modifications, are restricted to specific lipids, require external agents such as fusogens, and require long preparation times The latter point being unsuitable for membrane dynamic studies. Our method, on the other hand, not only addresses these drawbacks but also provides three important advantages over existing techniques for synthetic cell production: (i) generating lipid membranes with a high degree of asymmetry and no restriction on lipid type, (ii) reproducible production of phase separated GUVs using the inverted emulsion method, and (iii) the combination of lipid asymmetry and phase separation in the same GUV. These unique advantages allowed us to assemble and study the consequences of asymmetric protein binding on asymmetric membranes, i.e., stable and controllable spontaneous membrane curvatures resulting in membrane budding. In addition, these budded regions could be further increased to induce membrane neck closure and complete fission of our synthetic cells. 2) Further investigation into the effects of protein localization and stability of such vesicles. The budding, whilst interesting, could be seen as a disadvantage with respect to synthetic cell stability, and it would be interesting to further investigate this. If proteins are localized to both faces of the Lo domain, can this budding be prevented during vesicle deflation? And how long does the protrusion of the asymmetric, phase-separated GUVs persist? This is very interesting from the perspective of lipid flip-flop kinetics, where the presence of the domain boundary may potentially affect flip-flop rates compared to a single-phase asymmetric membrane. Thank you for your insightful suggestions. We understand the importance of further investigating the stability of the asymmetric, phase-separated vesicles, particularly in relation to protein localization and its impact on budding. To address your first point, investigating whether protein localization to both faces of the Lo domain can prevent budding during vesicle deflation is the logical next step. Such experiments could reveal how symmetric protein distribution across both leaflets impacts membrane stability, potentially counteracting forces that drive budding. Multiple works using proteins, and symmetric membranes have already been shown, cited in the manuscript as well (https://doi.org/10.1021/acssynbio.2c00564). However, your proposed experiment, i.e. investigating whether protein localization to both faces of the Lo domain can prevent budding during vesicle deflation, is counter intuitive. This is because, there will not be a bud to undergo fission in the first place as the membrane will be saturated with proteins on both sides of the membrane (as seen in Figure 2e). In our system, membrane curvature is induced by asymmetry alone, and not by symmetry (i.e. proteins localised to both Lo faces as suggested). We can also observe that in our systems where we have large head group lipids on both sides of the membrane bound to proteins, we see no protrusions – a condition similar to what you are suggesting. Furthermore, we have included a general explanation on why vesicles remain spherical and without any protrusions when the area is conserved, for more clarity. We have included the following text in the SI: Geometric constraint for shape transformations The volume-to-area ratio 𝑣 is restricted to the range 0 < 𝑣 ≤ 1, where the maximal value 𝑣 = 1 corresponds to a spherical vesicle shape. Thus, for a given surface area A of the vesicle membrane, the vesicle volume V cannot exceed its maximal value V = 𝐴!/#/(6√𝜋 ). In general, we should distinguish the apparent area of the membrane from its true area because some excess area can be stored in the membrane’s shape fluctuations. However, the vesicles analyzed here did not exhibit such fluctuations on the scales above 300 nm as resolvable by confocal light microscopy. As a consequence, the excess area stored in the shape fluctuations did not exceed a few percent of its apparent area and can therefore be ignored. If a tense vesicle with 𝑣 = 1 is further inflated, the vesicle membrane will rupture and lose its structural integrity. This geometric constraint applies to both vesicle membranes with a uniform composition (both lipid and bound protein) and for vesicle membranes with two intramembrane domains as studied here. However, you have raised an interesting point about preventing budding. Therefore, based on your suggestion, we tried a different approach to counteract the budding via increasing the concentration of streptavidin at the Ld domain. Figure S6: Exemplary GUVs when increasing the initial concentration of Streptavidin protein (from left to right) in the lumen of the vesicles (prepared from four separate experiments) results in reduced to no membrane protrusion of the Lo domain after Cholera toxin addition. Scale bar corresponds to 10 µm. In the new Figure S6 above, we have varied the final streptavidin concentration inside the vesicles while keeping the concentration of cholera toxin-B constant outside. We found that at higher concentrations, streptavidin is effective in preventing the protrusions. The below text has been added to the main text, with the new Figure S6 in SI. In order to establish control of synthetic cell morphology, we modulated the protein concentration in one domain whilst keeping the other one constant. To do this, we systematically increased the concentration of streptavidin (10 nM, 20 nM, 40 nM, and 80 nM) inside the vesicle while maintaining a constant CT-B Al-594 concentration outside. As shown in Supplementary Figure S6, at equilibrium (after an hour), the pronounced membrane curvature in the Lo domain (red) was progressively reduced as the concentration of internal bound streptavidin in the Ld domain (green) increased. This modulation of the protein concentration at the inner Ld leaflet only, counteracted the Lo domain curvature induced by CT-B Al-594 binding. At streptavidin concentrations of 40 nM and above, the vesicle's morphology is restored to a state close to its original spherical configuration (i.e. without CT-B). This phenomenon mirrors the mechanism by which the outward budding of the Lo domain reduces the line free energy at the domain boundary when CT-B Al-594 is added. Therefore, we demonstrate the ability to control membrane curvature in one domain by modulating protein binding in another within a complex asymmetric phase-separated system. These findings suggest that such asymmetric systems offer a novel approach for studying the dynamic interplay between membrane domains within the same vesicle, enabling unprecedented control over domain-specific membrane curvature. Note that we could also add more protein (CT-B) outside after the above in order to try and bring back the protrusion. However, this will be the focus of further studies, where we also modulate the protein binding more systematically and with different proteins. We believe that these new experiments are more relevant and in-line with the ideas that have already presented in this manuscript. Moreover, controlling budded domain curvature by altering the concentration of proteins (and therefore spontaneous curvature) has not been seen in such a system previously. This further strengthens the novelty of our work. We thank the reviewer for this idea. Regarding the stability of the protrusions. They remain stable. To demonstrate this, we have added a video (new video 7 in SI) with a vesicle having stable protrusion over a duration of >6 hours. We also want to reiterate that we don’t see protrusion as a disadvantage or a lack of stability. On the contrary, we think it is a result of membrane asymmetry that is stable at equilibrium. To be able to produce these interesting shapes and control the morphology merely via asymmetric membrane construction is very interesting for us as well as to the community. In particular, for synthetic cells that become polarised and for novel drug carrier systems (i.e. anisotropic particles). The below text has been added to reflect the above: This pronounced curvature with less GM1 compared to previous work could be due to the enrichment of GM1 to the Lo domain. Furthermore, the protrusions remain stable for over 6 hours (Supplementary Video 7), suggests that there is no flipping of GM1 or biotinylated lipids for long time periods. This opens the possibly of using such morphologies as polarised synthetic cells or anisotropic novel drug carrier systems. I also have some suggestions and questions regarding the experimental approach, which should be explored to better validate the new method presented in the manuscript. 3) The authors present the triple-layer inverted emulsion method as an improvement on prior work. The use of a spacer oil is interesting, and I can see how the use of oils of differing density could be used to better separate the lipid-containing squalene layer (outer leaflet) and W/O mineral oil emulsion (inner leaflet). The authors state that there is no lipid mixing during the production process, but there is no data that studies this. It would be interesting to observe whether lipid mixing occurs (perhaps through the use of fluorescent tracer lipids in the squalene and mineral oil emulsion phases), with and without the mineral oil spacer oil. This would facilitate a better understanding of the role of the mineral oil spacer, and how necessary it is in the formation of the asymmetric vesicles. Thank you for your valuable suggestion. We appreciate your interest in the role of the mineral oil spacer in our triple-layer inverted emulsion method and the concern regarding potential lipid mixing during the production process. While we have hypothesized that the use of a lipid-free oil spacer can effectively prevent lipid mixing between the inner (mineral oil) and outer (squalene) leaflets, as we see improvements in the asymmetry, we agree that providing direct experimental evidence would strengthen our claims. Therefore, we performed the below experiment. With three conditions, i) double layer ii) triple layer without spacer oil, and iii) triple layer with spacer oil (from left to right). The figure below shows photographic images of inverted emulsion method performed in Eppendorf® tubes after the centrifugation step. In all conditions, PE-NBD (yellow) is added as a tracer lipid in the oil interfacing the bottom aqueous solution, while the top layered oil containing w/o droplets is with DID (blue). After performing the centrifugation, only in the condition where there is spacer oil (right), we observe is a distinct yellow colour of unmixed yellow NDB (bottom). In the condition with triple layer without spacer (centre), a blue layer can be seen above the yellow layer resulting from a partial mixing of lipids. For the double layer method (left), a homogeneous blue layer is seen resulting from significant mixing of the lipid layers. This demonstrates that there is clear mixing of lipids in the order double layer, less in the triple layer without spacer, and none in triple layer with spacer. Note, the topmost orange part (seen in all three) is a reflection of the Eppendorf rack. This figure now added to the SI, with the following text to the main manuscript. Figure S1: Observing the mixing of lipid dyes after the transfer of W/O droplets. The fluorophores DID (blue) and NBD-DOPE (yellow) are added to the upper and lower most oil layers respectively for (i) the double-layer (ii) triple-layer without spacer and (iii) triple-layer with spacer. The lipid dye mixing is more prominent in the case of double-layer method compared to that of triple layer method without spacer oil and with spacer oil. To evaluate the hypothesis that the presence of spacer oil prevents lipid mixing and improves the asymmetry, we added labels to each layer. Observations are made of the mixing of the labels after the centrifugation step. Figure S1 shows that the double-layer method results in the homogeneous mixing of upper mineral oil layer with the squalene layer, and a uniform colour. In the case where the triple-layer method, but without spacer, some mixing is observed. Only in the case of the triple-layer with spacer, no oil mixing was observed. This confirms that a reduced lipid mixing in the new methodology contributed to the improved asymmetry, ~95% compared to ~80% or lower that is typically observed amongst works using the double-layer method32,43,44. In addition to the density differences which maintain the separation of mineral oil and squalene, this test emphasizes the role of spacer oil in further reducing the mixing of lipids, ensuring high lipid asymmetry across the membrane. 4) In Figure 1C, the authors show fluorescence quenching data for double-layer and triple-layer emulsion phase separated-vesicles. The authors include convincing data demonstrating the asymmetry of GUVs produced via the triple-layer method, but only produce symmetric vesicles using the double-layer method. The authors cite their prior work (10.1016/j.bpj.2022.12.017) and say that the triple-layer method gave better results in this study, than the double-layer method in the previous study. However, I believe the membrane asymmetry is different, with the focus of the prior work the asymmetric incorporation of NBD-labelled bacterial lipopolysaccharides. The authors should perform fluorescence quenching on GUVs prepared via the double-layer method using the NBD-DOPE lipid used in this work, and the fluorescent quenching compared for double- and triple-layered. This, combined with 3) above will enable a better evaluation for the use of the triple-layer phase transfer method as compared to the simpler double-layer method. We thank the reviewer for pointing out the differences. Multiple groups have already performed the double layer method with mineral oil and NBD lipids-based quenching. We assumed this data could be used to justify the differences. After recommendation by all the reviewers, we have now performed the corresponding asymmetry experiments with double layer method as well. The plot below suggests that double layer method can achieve asymmetry albeit with large error bars (77 ± 6%) when compared to that of the results from our new triple layer method (97 ± 1.3%). A statistical analysis resulted in no significant variation between both the methods for the condition where NBD lipids are inside, but the quenching distribution for double layer method is broad compared to the triple layer method. For the condition where NBD lipids are outside, the difference is significant. Thus, solidifying our claim on high efficiency of asymmetricity through triple layer method. The below plot and text are now included in the manuscript under the “Fluorescence quenching assay to assess bilayer asymmetry” section. Below caption for Figure 1(c) is included for clarity., The change in fluorescence intensity for symmetric GUVs and asymmetric GUVs induced by quenching from the addition of 10 mM sodium dithionate to the outer solution. Inserts show confocal images of exemplary GUVs before and after quenching produced via the new triple-layer method. Scale bars correspond to 5 µm. Statistical significance was determined using Mann Whitney method. Fluorescence quenching is an established method to assess the degree of asymmetry in lipid bilayers. In our experiments, we performed a quenching assay using NBD-tagged lipids and sodium dithionate as a quencher - a commonly used combination.42 The quenching occurs when sodium dithionite, a reducing agent, interacts with the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group, reducing it to a non-fluorescent form. This reaction is highly selective and depends on the accessibility of the NBD fluorophore to the aqueous dithionite, providing a measure of its exposure in membrane systems.43 In the first set of experiments, we compared the quenching percentage in symmetric GUVs produced from the previously reported double-layer method to our new triple-layer method. The lipid composition for this study was 99.5 mol% POPC and 0.5 mol% NBD-DOPE. As displayed in Figure 1(c), the fluorescence intensity of the symmetric GUVs produced through the double-layer method is reduced to 62 ± 13 % from the unquenched membrane and a similar percentage of 53 ± 11% is observed for the GUVs produced using the triple-layer method, after the addition of sodium dithionate. This result is anticipated as the NBD-DOPE lipid gets quenched on the outer leaflet of the symmetric bilayer only, and the intensity should reduce to half. From this data, statistically, it can be concluded that no significant difference in the percentage of quenching is observed amongst the symmetric GUVs produced from both the methods and quenching assay can be reliably performed on the asymmetric GUVs. Two different populations of asymmetric GUVs were then produced using both methods. One comprised of GUVs produced with NBD-DOPE in the inner leaflet of the GUVs and the other had NBD-DOPE in the outer leaflet. Like the previous condition, sodium dithionate was added to the outer solution for both populations of asymmetric GUVs. In the case of NBD-DOPE in the inner leaflet, no or very few of the GUVs should be quenched for successful asymmetricity. However, for the case when NBD-DOPE is in the outer leaflet (and therefore accessible to the quencher), all the fluorescence intensity should be quenched. A time-lapse video showing the quenching of asymmetric GUVs (with NBD-DOPE in the outer leaflet) in the presence of sodium dithionate can be seen in the supplementary information (Supplementary Video S1). In Figure 1(c), for the asymmetric NBD inside condition, both methods exhibit low quenching: double-layer (14 ± 11%) and triple-layer (7 ± 3%). While in the asymmetric NBD outside condition, quenching is high for both methods with 77 ± 6% for double layer method and 97 ± 1.3% for triple layer method. Interestingly, the triple-layer methodology shows lower variability in the degree of asymmetry. These results in conjunction with statistical analysis suggest that the two production methods lead to distinct quenching profiles under the tested conditions, with the triple-layer method demonstrating more consistent results (less variability) and improved asymmetry compared the double-layer method. 5) The authors report improved cholesterol integration within phase-separated GUVs due to poorer solubility of the molecule in squalene. Has this been reported previously using the double-layer method? Indeed, the cholesterol incorporation is improved. Previous works have not attempted to produce phase separation with squalene. To better explain this, we have now added a new table to the SI (Table S4) where previous attempts were made with mineral oil alone (double-layer method) to achieve phase separated GUVs and have shown contrasting results. Moreover, our own experiments using the double layer method (mineral oil alone), resulted in lower levels of cholesterol incorporation leading to <1% of the GUVs showing liquid-liquid phase separation. We have now included a new Figure S3 below. Table S4: Various phase transfer methods using mineral oil and ≥50% cholesterol to induce membrane phase separation in lipid-based vesicles. % GUVs with Method Lipid composition Oil used liquid-liquid phase Ref. separation 1/1 DOPC/DPPC Blosser et. al. cDICE and 100% Mineral oil <1% 2016 cholesterol 1/1 DOPC/DPPC and 40 to 80% cholesterol Inverted Karamdad et. or 1/1 Mineral oil 95% emulsion al. 2018 DOPC/EggSPM and 40 to 80% cholesterol 1/1 DOPC/DPPC and 50% cholesterol Inverted or Mineral oil <1% This study emulsion 1/1 DOPC/EggSPM and 50% cholesterol Triple layer 1/1 DOPC/EggSPM Mineral oil & inverted and 30% to 60% >60% This study emulsion cholesterol In the below Figure we used the double layer method, and we were unable to reproduce the work performed by Karamdad et al. (2018) for both DPPC and Sphingomyelin conditions. Figure S3: Symmetric GUVs with phase-separation compositions (50% Chol, DOPC:SPM – 1:1 & 50% Chol, DOPC:SPM – 1:1) produced using the double-layer method and showing no phase separation. Scale bar corresponds to 10 µm. Hence, we developed this new method to overcome this issue. It is clear from the literature that varying results exist. Hence, we aimed (and succeeded) to present a new method that we hope the community will use. Or at least provide more method options. Moreover, compared to the findings of Karamdad et al. (2018), our triple-layer method achieves results that are well-aligned with those obtained using the electroformation (i.e. no oil) technique. Below text is added to the main: While electroformation based production of phase separated GUVs is a common strategy, it is challenging to produce them using inverted emulsion method. Previous studies demonstrated very poor incorporation of cholesterol when mineral oil was used.46,47 except for one study48 (see Table S4). However, we are unable to reproduce the phase separation using double layer method involving mineral oil alone (Supplementary Figure S3). This inconsistency motivated us to develop a new method. Together with this data, we can confirm that cholesterol incorporation is better using our new triple layer method with squalene. Is the cholesterol present in the squalene layer of the assembled phase column only, or also in the mineral oil emulsion that is layered on top? Cholesterol is present in both of the layers (but not the spacer). We have added the following text to the manuscript to make this point clearer. GUVs were formed with varying concentrations of cholesterol (in both the squalene and mineral oil phases) while keeping the sphingomyelin to DOPC ratio constant (i.e., 1:1). Note that lipid compositional information (per leaflet) is also provided in Tables S2 and S3, but now we have amended the tables to explicitly say which oils phases they are in. 6) Could the authors provide more data for the curvature of the Lo phase for i) asymmetric phase separated vesicles and ii) the symmetric phase separated vesicles after Ct-B AL 594 binding? In each case only one GUV is presented (asymmetric Fig 4C, symmetric Fig S3). For Fig S3, in the overlay of the image the domains do not align (probably due to timelapse between taking measurements in different fluorescence channels). Providing images of more GUVs would enable the reader to better evaluate the range of curvatures shown for asymmetric/symmetric phase separated GUVs respectively. Thank you for noticing the issue. Figure S5 is now updated to better represent the lack of curvature compared to the GUVs with asymmetric phase separation. More images are uploaded in the supplementary material now under Figure S8. Figure S5: Symmetric GUVs with domain phase-separation (50% Chol, DOPC:SPM – 1:1 1% GM1 in the 0.5% DiD) showing no protrusions. CT-B binding to the liquid-ordered region (red channel) and DiD to the liquid disordered region (green channel). Scale bar corresponds to 10 µm. 7) The shape analysis of vesicle curvature is interesting but is only applied to a small number of asymmetric GUVs (for curvature discontinuity, 6 GUVs are evaluated and for the closed membrane necks 10 GUVs are evaluated). Were these GUVs prepared from a single-phase transfer experiment? It would be good to apply this method to a larger number of GUVs, to better understand the range of curvatures observed the frequency with which the protrusion of the Lo domain occurred across a vesicle population. We have now analysed and added 4 more GUVs for the curvature discontinuity data to strengthen findings. These analyses are taken from N = between 3 or 4 number of different phase transfer preparations. We all did not see a large variability in the resulting analysis for each condition which also strengthens the data (and method). We have now updated Table S5 (below) to reflect this, as well as including additional figures to Figure S8 (below). We thank the reviewer for this and hope this is now sufficient. Similar extensive analysis (16 GUVs, previously 10) also performed with vesicles undergoing neck closure – associated data is presented in Table S6 and a new Figure S9. Table S5: Membrane curvature discontinuity analysis across the domain boundary of two-domain asymmetric GUVs. G Ro, Rd, C2 R C2= R R M M U se se d ψ C C =sin( M M M s 1/R o, d, M 1o 1d o d np sp Vs mi mi b ψ)/r pol db db ol ol - - 1.9 0.23 0.0 1.0 1.0 3. 0.12 1.0 0.9 4.9 0.5 7 8. 0.0 1 4 0.2 230 7714 184 803 988 9 3456 232 845 8 2 2 1 386 008 77 865 56 96 52 5 79 67 94 7 - 3. - 0.25 0.0 1.6 1.6 3. 0.15 1.6 1.5 0.3 6 3.1 6. 0.0 2 4.2 41 0.2 3516 077 892 969 1 2439 387 958 2 0 25 56 428 5 381 79 11 58 69 2 024 2 91 3 - 4 0.1 0.12 0.0 0.1 0.1 5. 10 0.09 0.0 0.1 0.1 61. 9.4 5. 0.0 3 0. 058 1796 527 138 665 5 .1 8135 408 019 428 1 5 33 163 5 2 532 15 08 23 2 9 427 84 78 62 7 - 0.0 0.14 0.0 0.1 0.1 5. 0.11 0.0 0.0 0.1 17. 16. 5 0.0 8. 4 6 602 2816 428 015 443 2 5340 290 877 168 48 61 9 572 67 05 386 04 11 15 7 254 66 72 38 1 - - - 0.5 0.13 0.3 0.3 3. 13 0.07 0.3 0.2 4.3 1.6 4. 4 0.2 0.0 0.0 5 917 5838 637 162 3 .1 5872 337 562 3 9 73 0 309 475 775 16 573 77 23 2 8 534 94 57 5 5 4 - - - 0.7 0.22 0.4 0.4 3. 0.14 0.4 0.3 4.4 1.3 2. 3 0.2 0.0 6. 0.0 6 299 3112 765 754 3 7275 386 996 4 7 44 3 252 010 79 389 27 349 2 63 8 405 01 26 3 6 7 - - 6. 1.0 1.0 8.7 0.4 6. 7 0.1 2.0 0.14 0.0 1.1 1.1 10 0.09 0.0 7 1 902 813 1 8 68 0 148 83 06 129 119 249 .3 708 088 4 1 49 1 6 - - 11. 0.0 5.7 3. 5.6 5.6 5.5 0.0 2. 8 0.1 0.37 5.8 7. 0.13 0.0 8 111 985 442 2 244 032 5 9 63 3 801 735 428 26 7741 212 1 9 35 1 26 07 8 2 - - 0.4 0.24 0.0 0.3 0.3 4. 0.14 0.2 0.2 5.0 3. 5 6. 0.0 9 2.4 0.1 166 5842 231 312 543 9 5985 813 545 1 16 1 85 268 996 7 3 21 55 75 8 4 26 18 1 - - 0.5 0.4 0.3 3. 0.18 - 0.3 0.2 2.4 1.9 2. 4 0.4 0.31 0.0 5. 10 050 090 614 5 1159 0.1 431 296 5 8 41 9 081 3039 475 52 51 45 83 1 42 135 05 03 6 6 Figure S8: Confocal images of six GUVs along with the analysis of their shape contours as in Figure 5, with annotations using Autodesk AutoCAD®. Scale bar corresponds to 10 µm. Figure S9: Confocal images of 16 GUVs with neck closure, fitted with a spherical circle for both green segment (Ld phase) and red segment (Lo phase), with annotations using Autodesk AutoCAD®. Scale bar corresponds to 10 µm. Table S6: Effective Membrane curvature of two domain asymmetric GUVs with neck closure. No. Ri Rj 1/Mi 1/Mj Mij 1 3.45 6.5 0.289855 0.153846 0.221851 2 4.34 4.48 0.230415 0.223214 0.226815 3 3.1 11.4 0.322581 0.087719 0.20515 4 4.66 7.8 0.214592 0.128205 0.171399 5 7.45 28.48 0.134228 0.035112 0.08467 6 3 19.82 0.333333 0.050454 0.191894 7 1.4 4.15 0.714286 0.240964 0.477625 8 2.1 8.4 0.47619 0.119048 0.297619 9 2.8 6.35 0.357143 0.15748 0.257312 10 1.85 6.6 0.540541 0.151515 0.346028 11 1.57 5.4 0.636943 0.185185 0.411064 12 1.5 6.12 0.666667 0.163399 0.415033 13 2.4 4.65 0.416667 0.215054 0.31586 14 2.7 16.25 0.37037 0.061538 0.215954 15 2.3 8.01 0.434783 0.124844 0.279813 16 1.89 6.11 0.529101 0.163666 0.346383 Finally, the methods section needs to be improved to enable reproducibility of the work; We thank the reviewer for pointing out these discrepancies. 8) For osmolarity-induced bud division experiments, the final solution osmolarity is not described (only that 2.5 ul of 2M glucose solution is added). Please amend this so it is clear to the reader the final external solution osmolarity (and the osmotic gradient across the membrane). Methods section is updated, and the new information is added for clarity and reproducibility. For osmolarity induced bud division experiments, 2.5µL of 2 M glucose solution was injected into the outer solution creating a 50 mOsm hypertonic solution to deflate the GUVs, after cholera toxin binding. 9) The description of shape analysis in the method is minimally described - the only detail is that Autodesk AutoCAD was used to fit the vesicles. Please give sufficient information for a reader to reproduce the shape analysis. A new methods section describing the shape analysis and fitting is added. In addition, an exemplary CAD file with a fitted vesicle is now included with the manuscript as a supplementary file. Vesicle shape contour analysis For shape analysis, Autodesk® AutoCAD® was used to fit the vesicles. An exemplary CAD file for the GUV fitting method is included in the supplementary information. To perform the contour analysis, the GUV image file with scale bar is imported into the AutoCAD® file and the image is adjusted to match the dimensions of the GUVs to that of the CAD file via scaling (in the example file, the scale bar is 10 µm). In the next step to draw axi-symmetric line passing along the GUVs centre (Figure 5(a)), two osculating circles fitting the Ld (green) and Lo (red) domains are drawn with radii, Rspol and Rnpol, respectively (Figure 5(b)). Next, connecting the centres of the two circles, a line is drawn, representing the symmetrical axis. This is followed by drawing a line perpendicular to the axi-symmetric line to the domain boundary (where Ld domain (green) meets the Lo domain (red)), shown as a dashed line in Figure 5(b). A semicircle is drawn with its corner at the domain boundary (left or right side of the GUV, in Figure 5(b), it is drawn on the right side) having a radius of Ro,semi, until the arc of the semicricle fits the Lo domain (red) as shown in Figure 5(b) and Figure S8. A line is drawn along and parallel to the diameter of the semicircle until it meets the axi-symmetric line giving an angle, tilt angle (ψ) (Figure 5(b)). Thus, obtained measurements are used to calculate various parameters using the equations mentioned in the main text as well as in SI and presented in Table S5. &'' Similarly, the effective mean curvatures (𝑀 ) for vesicles with closed neck configurations are $% calculated by measuring the radii of the green region (R) and red region (R). The radii were measured i j by fitting a circle using circle function in AutoCAD® (an example fit can be found in the CAD file uploaded as in the supplementary information). 10) How are the mathematical calculations performed? Please give details. Mathematical calculations are presented as a table in the SI, along with all the relevant equations. Another reviewer has requested the source data which we will upload to the SI as well. The following has been added to make this location clearer in the main text. The corresponding data obtained after fitting the vesicles (refer to Methods section, Vesicle shape contour analysis and Supplementary CAD file) are provided in Table S5 of the Supplementary Information."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I was pleased to re-review this manuscript by Yandrapalli et al. The authors have taken on board reviewer comments and strengthened their manuscript, particularly regarding the control experiments necessary to verify the new triple-layer inverted emulsion phase transfer method. Further experiments were also conducted that demonstrate control of vesicle domain curvature through binding of proteins to differing domains and faces of the vesicle. These additions have largely addressed my concerns, and I believe the manuscript is suitable for publication in Nature Communications after addressing the following: • I could not see information on the lipid mass and concentration in mineral oil / squalene in the inverted emulsion method section – please add to improve reproducibility. • Figure 2C lists GFP as inside the GUV and the NTA lipid on the outside. I believe the opposite is the case? Please correct. • Figure 3 – can you list the number of vesicles analysed for each chol% studied? • Figure S4: Please show exemplar images for each of the compositions studied alongside the phase diagram. • Figure S6 is a very interesting addition to the manuscript. Could you please list the number of vesicles studied for each streptavidin concentration? This is missing. • “In the Supplementary Figure S4(b), the Lo buds have a bright red ring (due to CT-B Al-594 binding) and are devoid of streptavidin (green) binding” – should be Supplementary Figure S7? • In the SI, in the “Shape parameters of two-domain vesicles” section, the final sentence ends stating there are five elastic shape parameters, but this sentence is not finished? • Figure S8: 10 vesicles are presented but the caption states 6 are shown. • Please check the naming of all equations in the manuscript and SI – I believe due to manuscript additions there are multiple equations mislabelled in the manuscript text.", "reviewer_2": "The authors addressed all my concerns and questions. I think the new data and explanations improved the manuscript. I have no more comments.", "reviewer_3": "I thank the authors for their extensive response and adding in the new data to the manuscript, although I still have a number of concerns and queries. From my reading this manuscript mentions two main findings, that they produce higher degree of lipid bilayer asymmetry (1) and better phase separated asymmetric GUVs (2), compared to previous emulsion phase transfer methods. For the first point, all reviewers asked for more comparison to previous methods, which the authors gave in the form of the double-layer method (Fig 1). Surprisingly, they only saw an improvement in the symmetry of the outside leaflet, which dramatically reduces their argument. Furthermore, the authors did not even quantify the comparison of the triple layer (with an oil free layer) to triple layer (without an oil free layer). They did this with a visual inspection (Fig S1), which did show the oil free version kept the two layers separated somewhat, even after centrifugation. They need to actually measure the asymmetry to see if the oil free layer is required. Furthermore, in my original review I said the authors should also add an emulsion formed with the inner lipid+mineral oil (with time for the monolayer to form) to a phase transfer column prepared with the outer lipid+squalene (again with time for the monolayer to form). This again is likely to keep the asymmetry to a similar level and would be much simpler. The authors even state that “the rate of formation of lipid monolayer is much longer than the rate of droplet transfer”, therefore minimal lipid/oil mixing is likely not to cause a problem if the monolayers are already formed. Furthermore, there is little quantification of protein binding in Fig 2 (just an individual GUV for each condition in Fig S2), could the other control methods provide similar results here even if their asymmetric values measured using NBD aren’t as good (is protein being less sensitive than the NBD assay)? For the second point, the improved phase separation seems to come entirely from the improved ability to add cholesterol into the membrane (from my reading). The authors state this is because of the use of squalene. However, as like the first point, the squalene is not reliant on the triple layer with an oil-free layer. Therefore, can similar results for this part also be obtained using triple layer without the oil-free layer or incubating the monolayers separately? Overall, you can see that my concern is that the system is over-engineered, and not all of the parts are required. Or that similar results can actually be obtained in a simpler way than careful layering of four layers together. Other points: For “Even with experimental observations lasting well beyond 6 h, the observed asymmetry and the resulting membrane curvature remained stable for the entire duration” the authors need to show this data. The supplier or preparation of His-GFP is missing from the text."}, "author_response": "In the Methods section, the first sentence of the ‘Inverted emulsion method’ states that lipid concentration is 400 µM. To be clearer, we will reiterate it in the next sentence as below: “The final lipid-oil mixture, with a concentration of 400 µM, is stored at 4 °C and used within a week.” • Figure 2C lists GFP as inside the GUV and the NTA lipid on the outside. I believe the opposite is the case? Please correct. You are correct. Thanks for pointing this out. We have replaced it with the correct illustration. • Figure 3 – can you list the number of vesicles analysed for each chol% studied? We have added this to the caption, now - n≥70 • Figure S4: Please show exemplar images for each of the compositions studied alongside the phase diagram. The figure is now updated with the one below. • Figure S6 is a very interesting addition to the manuscript. Could you please list the number of vesicles studied for each streptavidin concentration? This is missing. That information (n≥15) is now added into the caption of the figure as well as in the main text. • “In the Supplementary Figure S4(b), the Lo buds have a bright red ring (due to CT-B Al-594 binding) and are devoid of streptavidin (green) binding” – should be Supplementary Figure S7? It is. It is now S8 after new additions. Thanks again for pointing it out. • In the SI, in the “Shape parameters of two-domain vesicles” section, the final sentence ends stating there are five elastic shape parameters, but this sentence is not finished? It is now corrected. Using the vesicle size R again as the basic length scale and the bending rigidity κ as the basic ve o energy scale, we are then left with five elastic shape parameters: the dimensionless spontaneous curvatures m R and m R of the Lo and Ld domains; the ratio κ / κ of the two bending o ve d ve d o rigidities; as well the dimensionless Gaussian curvature difference and line tension (κ - κ )/κ Go Gd o and λR /κ . ve o • Figure S8: 10 vesicles are presented but the caption states 6 are shown. Thanks for pointing it out, this is corrected now. • Please check the naming of all equations in the manuscript and SI – I believe due to manuscript additions there are multiple equations mislabelled in the manuscript text. Checked and updated where necessary. Thank you.. Reviewer #2 (Remarks to the Author): The authors addressed all my concerns and questions. I think the new data and explanations improved the manuscript. I have no more comments. We thank the reviewer. Reviewer #3 (Remarks to the Author): I thank the authors for their extensive response and adding in the new data to the manuscript, although I still have a number of concerns and queries. From my reading this manuscript mentions two main findings, that they produce higher degree of lipid bilayer asymmetry (1) and better phase separated asymmetric GUVs (2), compared to previous emulsion phase transfer methods. For the first point, all reviewers asked for more comparison to previous methods, which the authors gave in the form of the double-layer method (Fig 1). Surprisingly, they only saw an improvement in the symmetry of the outside leaflet, which dramatically reduces their argument. While the improvement in the degree of asymmetry for the inner leaflet is not statistically significant, the error bars are dramatically reduced which significantly improves the reproducibly of the method. Furthermore, the authors did not even quantify the comparison of the triple layer (with an oil free layer) to triple layer (without an oil free layer). They did this with a visual inspection (Fig S1), which did show the oil free version kept the two layers separated somewhat, even after centrifugation. They need to actually measure the asymmetry to see if the oil free layer is required. Furthermore, in my original review I said the authors should also add an emulsion formed with the inner lipid+mineral oil (with time for the monolayer to form) to a phase transfer column prepared with the outer lipid+squalene (again with time for the monolayer to form). This again is likely to keep the asymmetry to a similar level and would be much simpler. The authors even state that “the rate of formation of lipid monolayer is much longer than the rate of droplet transfer”, therefore minimal lipid/oil mixing is likely not to cause a problem if the monolayers are already formed. We appreciate the opportunity to clarify our methodology. This new method remains straightforward and is a rational extension of established emulation phase transfer techniques. Our method involves transferring an emulsion of [inner lipid + mineral oil] droplets through a column containing the [outer lipid + squalene] phase. The only modification is the use of squalene (for domains) and the inclusion of an intermediate, lipid-free mineral oil layer that rests on top of the squalene phase. This is not a complication but a critical simplification that serves two purposes. First to act as a physical barrier, that can still be seen after the centrifugation and movement of droplets through it. The second is to perform a washing step. As the droplets pass through this layer, it washes away any loosely associated lipids from the droplet surface before entering the squalene layer, further ensuring the purity and defined composition of the final outer leaflet. We cannot increase the incubation time for the water in mineral oil emulsion because they quickly begin sediment and fuse into larger droplets. This was fundamental issue with the previous method and why our inclusion of the spacer layer works to separate the unwanted free lipids from going to the other monolayer. Furthermore, there is little quantification of protein binding in Fig 2 (just an individual GUV for each condition in Fig S2), could the other control methods provide similar results here even if their asymmetric values measured using NBD aren’t as good (is protein being less sensitive than the NBD assay)? In response to your valid criticism regarding the quantification in Figure 2, we have now updated the Supplementary Information (Figure S3) to include analysis of a larger population of GUVs for all conditions, moving beyond single representative images. This new data provides robust statistical support for our conclusions. Figure S3: Confocal images of asymmetric GUVs with varying lipid compositions and protein interactions corresponding to the data shown in Figure 2 in the main text. Plots on the right showing the percentage of GUVs with protein binding for three distinct molecular recognition systems. For each system, binding was assessed in a positive control configuration (e.g., protein and its target lipid are accessible) and a negative control configuration (e.g., protein and its target lipid are inaccessible). Data is compiled from N > 30 vesicles for all conditions. Scale bars: 20 µm. The following has been added into the main text: Finally, we quantified the results for all three distinct molecular recognition systems in Figure 2. For each system, the positive control was when the protein in the external solution could access its target lipid on the outer leaflet, and the negative control was when the target lipid was situated on the inaccessible inner leaflet (Figure S3). The results demonstrate a high degree of binding specificity. For the streptavidin-biotin system, we observed binding in 89.5% of the positive control GUVs, whereas only 6.6% of negative control vesicles showed a signal. The specificity was even more pronounced for the His-tag GFP-NTA system, which resulted in 100% binding for the positive control and 0% for the negative control. Finally, we tested the binding of the CT-B to its target ganglioside, GM1. A robust binding signal was detected in 97.0% of GUVs when GM1 was on the outer leaflet, compared to only 9.1% when it was on the inner leaflet. For the second point, the improved phase separation seems to come entirely from the improved ability to add cholesterol into the membrane (from my reading). The authors state this is because of the use of squalene. However, as like the first point, the squalene is not reliant on the triple layer with an oil-free layer. Therefore, can similar results for this part also be obtained using triple layer without the oil-free layer or incubating the monolayers separately? With respect to your second point, we have already tried squalene and other organic solvents as sole lipid mixtures. We published this information in our paper in 2019 (Moga et al. 2019). Squalene-based lipid mixture yields very few numbers of GUVs and at >400 x g, only. In our experience, such high centrifugal speeds results in excessive vesicle clustering and bursting (Moga et al. 2019). Such low GUV numbers mean that the majority of droplets fail to convert into GUVs, resulting in excessive leakage of internal components into the outer solution and thus affecting the overall asymmetry that we are aiming for. To make this clear, we have added the text below to the manuscript in the Discussion section: However, employing squalene as the sole oil phase for the entire inverted emulsion process is not a viable strategy. As we have previously demonstrated, GUVs generated from a single squalene-oil phase exhibit very low formation efficiency and suffer significant leakage of their aqueous contents due to the required at high centrifugation speeds (>400 x g) (Moga et al., 2019). This instability fundamentally undermines the goal of creating and maintaining a well-defined lipid asymmetry. To overcome these established limitations, we engineered the current oil system. Mineral oil is used for the initial droplet encapsulation, and as a physical barrier to prevent lipid mixing (also serving as a wash step to remove excess, non-incorporated dye). By reserving squalene for the destination phase, we decouple the requirements for stable emulsion formation from the final membrane composition. This design allows us to leverage the unique benefits of squalene for inducing phase separation (i.e. with cholesterol) without the associated penalties of poor yield and vesicle instability, thus enabling the reliable production of intact, asymmetric as well as phase-separated GUVs. Overall, you can see that my concern is that the system is over-engineered, and not all of the parts are required. Or that similar results can actually be obtained in a simpler way than careful layering of four layers together. We have carefully considered our options before compiling the oil layers, not over engineering. Our primary motivation is to achieve robust bilateral asymmetry - controlling the composition of both the inner and outer leaflets independently - not just one side. We validated this by demonstrating highly specific protein binding (also to domains) with three distinct protein-lipid pairs, which constitute an extensive functional evaluation of the system's specificity. Moreover, the choice of oil (i.e. squalene, which is denser than mineral) allows us to also achieve phase separation without additional oils keeping the system as simple as possible for both asymmetry and domains. Other points: For “Even with experimental observations lasting well beyond 6 h, the observed asymmetry and the resulting membrane curvature remained stable for the entire duration” the authors need to show this data. To clarify, a typical experiment starts with centrifugation in a microtiter plate, followed by imaging under the microscope. On any given day, centrifugation is performed only once (by mid-day) and the vesicles in multiple wells will be imaged for the rest of the day (up to 6pm for example). So, most of the experiments are well beyond 1h and up to 6h. The below example is shown for vesicles with Ni-NTA lipid on the inside at time 0h and again imaged after GFP addition at time 6h. If there is change in asymmetry, one should observe the binding of GFP to the Ni-NTA lipids, this is not the case even after 6h. A similar example for membrane curvature system is included and both have been added to the SI as a new Figure which is also referenced in the text. Moreover, videos S7 shows curvature as well. Figure S11: Stability of asymmetric GUVs with and without asymmetry induced membrane curvature. Top panels, confocal cross-sectional images of vesicles containing Ni-NTA lipids on the inner leaflet at the start (left) and no binding of His-tag GFP is observed even after 6h (right). Bottom panels, membrane curvature of phase separated GUVs after CT-B binding at time 30 min (bottom, left) and sustained membrane curvature after 4h (right). Scale bars: 20 µm. The supplier or preparation of His-GFP is missing from the text. Thank you for noticing, it is now included in Table S1 of the SI."}, {"round": 3, "reviewer_comments": {"reviewer_3": "All my final comments have been addressed. I support publication."}, "author_response": ""}]

Decision letter