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A fundamental feature of cellular plasma membranes (PMs) is an asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being approximately twofold more unsaturated than the exoplasmic leaflet. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in the asymmetric structures of protein transmembrane domains. These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.

Eukaryotic membranes can be partitioned into lipid-driven membrane microdomains called lipid rafts, which function to sort lipids and proteins in the plane of the membrane. As protein selectivity underlies all functions of lipid rafts, there has been significant interest in understanding the structural and molecular determinants of raft affinity. Such determinants have been described for lipids and single-spanning transmembrane proteins; however, how multi-pass transmembrane proteins (TMPs) partition between ordered and disordered phases has not been widely explored. Here we used cell-derived Giant Plasma Membrane Vesicles (GPMVs) to systematically measure multi-pass TMP partitioning to ordered membrane domains. Across a set of 24 structurally and functionally diverse multi-pass TMPs, the large majority (92%) had minimal raft affinity. The only exceptions were two myelin-associated four pass TMPs, Myelin and Lymphocyte protein (MAL) and ProteoLipid Protein (PLP). We characterized the potential mechanisms for their exceptional raft affinity and observed that PLP requires cholesterol and sphingolipids for optimal association with ordered membrane domains and that PLP and MAL appear to compete for cholesterol-mediated raft affinity. These observations suggest broad conclusions about the composition of ordered membrane domains in cells and point to previously unrecognized drivers of raft affinity for multi-pass transmembrane proteins.

Eukaryotic plasma membranes are compartmentalized into functional lateral domains, including lipid-driven membrane rafts. Rafts are involved in most plasma membrane functions by selective recruitment and retention of specific proteins. However, the structural determinants of transmembrane protein partitioning to raft domains are not fully understood. Hypothesizing that protein transmembrane domains (TMDs) determine raft association, here we directly quantify raft affinity for dozens of TMDs. We identify three physical features that independently affect raft partitioning, namely TMD surface area, length, and palmitoylation. We rationalize these findings into a mechanistic, physical model that predicts raft affinity from the protein sequence. Application of these concepts to the human proteome reveals that plasma membrane proteins have higher raft affinity than those of intracellular membranes, consistent with raft-mediated plasma membrane sorting. Overall, our experimental observations and physical model establish general rules for raft partitioning of TMDs and support the central role of rafts in membrane traffic., Lipid rafts are plasma membrane domains that specifically recruit particular proteins. Here, the authors show that the surface area, length and palmitoylation of single-pass transmembrane domains are crucial for raft partitioning and propose a general model to predict protein association with rafts.

SUMMARYA fundamental feature of cellular plasma membranes (PM) is asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets, nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being ∼2-fold more unsaturated than the exoplasmic. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in asymmetric structures of protein transmembrane domains (TMD). These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.

Phospholipid asymmetry between the two plasma membrane leaflets has been discovered 40 years ago and since then, various physiological processes have been associated with asymmetric lipid distributions and changes to lipid asymmetry. Nevertheless, it remains unclear how lipid asymmetry affects the biophysical properties of individual leaflets and whether this putative biophysical asymmetry affects transmembrane proteins. To address these questions, we conducted a detailed analysis of asymmetric plasma membrane leaflet lipidomes and leaflet-specific biophysical properties. We further investigated whether distinct leaflets are maintained in intracellular organelles and how such intracellular membrane asymmetry may affect transmembrane protein localization and structure. Lipidomics revealed a striking disparity in lipid acyl chains, with the inner plasma membrane leaflet containing two-fold more acyl chain unsaturations than the outer leaflet. Consistent with the difference in unsaturation, we observed that the outer leaflet was highly packed, resembling a liquid ordered phase, whereas the inner leaflet was much more disordered. A bioinformatic analysis revealed that transmembrane domains (TMDs) of single-pass transmembrane proteins in the plasma membrane are broadly asymmetric in shape, with smaller accessible surface areas in the outer leaflet than the inner (i.e. thinner outside half of the TMD, fatter inside). We inferred that this shape asymmetry should facilitate insertion into the asymmetrically packed plasma membrane. We verified this hypothesis by creating TMD constructs with several shapes and discovered that proteins with a small accessible surface area in the outer leaflet preferred to localize at the plasma membrane regardless of their inner leaflet counterpart. This study delivers new insights into the structural organization of cell membranes and reveals that outer leaflet packing can drive protein localization to the plasma membrane.

Eukaryotic plasma membranes are compartmentalized into functional lateral domains, including lipid-driven membrane rafts. Rafts are involved in most plasma membrane functions by selective recruitment and retention of specific proteins. However, the structural determinants of transmembrane protein partitioning to raft domains are not fully understood. Hypothesizing that protein transmembrane domains (TMDs) determine raft association, here we directly quantify raft affinity for dozens of TMDs. We identify three physical features that independently affect raft partitioning, namely TMD surface area, length, and palmitoylation. We rationalize these findings into a mechanistic, physical model that predicts raft affinity from the protein sequence. Application of these concepts to the human proteome reveals that plasma membrane proteins have higher raft affinity than those of intracellular membranes, consistent with raft-mediated plasma membrane sorting. Overall, our experimental observations and physical model establish general rules for raft partitioning of TMDs and support the central role of rafts in membrane traffic.

Increasing evidence supports the existence of lateral nanoscopic lipid domains in plasma membranes, known as lipid rafts. These domains preferentially recruit membrane proteins and lipids to facilitate their interactions and thereby regulate transmembrane signaling and cellular homeostasis. The functionality of raft domains is intrinsically dependent on their selectivity for specific membrane components; however, while the physicochemical determinants of raft association for lipids are known, very few systematic studies have focused on the structural aspects that guide raft partitioning of proteins. In this review, we describe biophysical and thermodynamic aspects of raft-mimetic liquid ordered phases, focusing on those most relevant for protein partitioning. Further, we detail the variety of experimental models used to study protein-raft interactions. Finally, we review the existing literature on mechanisms for raft targeting, including lipid post-translational modifications, lipid binding, and transmembrane domain features. We conclude that while protein palmitoylation is a clear raft-targeting signal, few other general structural determinants for raft partitioning have been revealed, suggesting that many discoveries lie ahead in this burgeoning field.

Dietary lipids change membrane phenotypes, which can be used to affect lineage specification in stem cells., Mammalian cells produce hundreds of dynamically regulated lipid species that are actively turned over and trafficked to produce functional membranes. These lipid repertoires are susceptible to perturbations from dietary sources, with potentially profound physiological consequences. However, neither the lipid repertoires of various cellular membranes, their modulation by dietary fats, nor their effects on cellular phenotypes have been widely explored. We report that differentiation of human mesenchymal stem cells (MSCs) into osteoblasts or adipocytes results in extensive remodeling of the plasma membrane (PM), producing cell-specific membrane compositions and biophysical properties. The distinct features of osteoblast PMs enabled rational engineering of membrane phenotypes to modulate differentiation in MSCs. Specifically, supplementation with docosahexaenoic acid (DHA), a lipid component characteristic of osteoblast membranes, induced broad lipidomic remodeling in MSCs that reproduced compositional and structural aspects of the osteoblastic PM phenotype. The PM changes induced by DHA supplementation potentiated osteogenic differentiation of MSCs concurrent with enhanced Akt activation at the PM. These observations prompt a model wherein the DHA-induced lipidome leads to more stable membrane microdomains, which serve to increase Akt activity and thereby enhance osteogenic differentiation. More broadly, our investigations suggest a general mechanism by which dietary fats affect cellular physiology through remodeling of membrane lipidomes, biophysical properties, and signaling.

Saponins, amphiphiles of natural origin with numerous biological activities, are widely used in the cosmetic and pharmaceutical industry. Some saponins exhibit relatively selective cytotoxic effects on cancer cells but the tendency of saponins to induce hemolysis limits their anticancer potential. This review focused on the effects of saponin activity on membranes and consequent implications for red blood and cancer cells. This activity seems to be strongly related to the amphiphilic character of saponins that gives them the ability to self-aggregate and interact with membrane components such as cholesterol and phospholipids. Membrane interactions of saponins with artificial membrane models, red blood and cancer cells are reviewed with respect to their molecular structures. The review considered the mechanisms of these membrane interactions and their consequences including the modulation of membrane dynamics, interaction with membrane rafts, and membrane lysis. We summarized current knowledge concerning the mechanisms involved in the interactions of saponins with membrane lipids and examined the structure activity relationship of saponins regarding hemolysis and cancer cell death. A critical analysis of these findings speculates on their potential to further develop new anticancer compounds.

The interactions of triterpenoid monodesmosidic saponins, α-hederin and δ-hederin, with lipid membranes are involved in their permeabilizing effect. Unfortunately, the interactions of these saponins with lipid membranes are largely unknown, as are the roles of cholesterol or the branched sugar moieties (two for α-hederin and one for δ-hederin) on the aglycone backbone, hederagenin. The differences in sugar moieties are responsible for differences in the molecular shape of the saponins and the effects on membrane curvature that should be the most positive for α-hederin in a transbilayer direction. In large unilamellar vesicles and monocyte cells, we showed that membrane permeabilization was dependent on the presence of membrane cholesterol and saponin sugar chains, being largest for α-hederin and smallest for hederagenin. In the presence of cholesterol, α-hederin induced the formation of nonbilayer phases with a higher rate of Brownian tumbling or lateral diffusion. A reduction of Laurdan's generalized polarization in relation to change in order of the polar heads of phospholipids was observed. Using giant unilamellar vesicles, we visualized the formation of wrinkled borders, the decrease in liposome size, budding, and the formation of macroscopic pores. All these processes are highly dependent on the sugars linked to the aglycone, with α-hederin showing a greater ability to induce pore formation and δ-hederin being more efficient in inducing budding. Hederagenin induced intravesicular budding but no pore formation. Based on these results, a curvature-driven permeabilization mechanism dependent on the interaction between saponin and sterols and on the molecular shape of the saponin and its ability to induce local spontaneous curvature is proposed.

Biological membranes contain a broad variety of lipid species whose individual physicochemical properties and collective interactions ultimately determine membrane organization. A key aspect of the organization of cellular membranes is their lateral subdivision into domains of distinct structure and composition. The most widely studied membrane domains are lipid rafts, which are the biological manifestations of liquid-ordered phases that form in sterol-containing membranes. Detailed studies of biomimetic membrane mixtures have yielded wide-ranging insights into the physical principles behind lipid rafts; however, these simplified models do not fully capture the diversity and complexity of the mammalian lipidome, most notably in their exclusion of polyunsaturated lipids. Here, we assess the role of lipid acyl chain unsaturation as a driving force for phase separation using coarse-grained molecular dynamics (CGMD) simulations validated by model membrane experiments. The clear trends in our observations and good qualitative agreements between simulations and experiments support the conclusions that highly unsaturated lipids promote liquid-liquid domain stability by enhancing the differences in cholesterol content and resulting lipid chain order between the coexisting domains. These observations reveal the important role of non-canonical biological lipids in the physical properties of membranes, showing that lipid polyunsaturation is a driving force for liquid-liquid phase separation.

In perspective of reducing the mortality of cancer, there is a high interest in compounds which act on multiple cellular targets and therefore prevent the appearance of cancer resistances. Saponins and α-hederin, an oleanane-type saponin, induce cancer cell death through different pathways, including apoptosis and membrane permeabilization. Unfortunately, the mechanism by which cell death is induced is unknown. We hypothesized that the activity of α-hederin mainly depends on its interaction with membrane cholesterol and therefore investigated the cholesterol and saponin-structure dependency of apoptosis and membrane permeabilization in two malignant monocytic cell lines. Apoptotic cell death and membrane permeabilization were significantly reduced in cholesterol-depleted cells. Permeabilization further depended upon the osidic side chain of α-hederin and led to extracellular calcium influx and nuclear fragmentation, with only the latter being susceptible to caspase inhibitors. Membrane order, measured by laurdan generalized polarization imaging, was neither reduced by α-hederin nor its aglycone hederagenin suggesting that their activity was not related to membrane cholesterol extraction. However, a radical change in morphology, including the disappearance of pseudopodes was observed upon incubation with α-hederin. Our results suggest that the different activities of α-hederin mainly depend on its interaction with membrane cholesterol and consequent pore formation.

The plasma membrane (PM) serves as the functional interface between a cell and its environment, hosting extracellular signal transduction and nutrient transport among a variety of other processes. To support this extensive functionality, PMs are organized into lateral domains, including ordered, lipid-driven assemblies termed lipid rafts. Although the general requirements for ordered domain formation are well established, how these domains are regulated by cell-endogenous mechanisms or exogenous perturbations has not been widely addressed. In this context, an intriguing possibility is that dietary fats can incorporate into membrane lipids to regulate the properties and physiology of raft domains. Here, we investigate the effects of polyunsaturated fats on the organization of membrane domains across a spectrum of membrane models, including computer simulations, synthetic lipid membranes, and intact PMs isolated from mammalian cells. We observe that the ω-3 polyunsaturated fatty acid docosahexaenoic acid is robustly incorporated into membrane lipids, and this incorporation leads to significant remodeling of the PM lipidome. Across model systems, docosahexaenoic acid-containing lipids enhance the stability of ordered raft domains by increasing the order difference between them and coexisting nonraft domains. The relationship between interdomain order disparity and the stability of phase separation holds for a spectrum of different perturbations, including manipulation of cholesterol levels and high concentrations of exogenous amphiphiles, suggesting it as a general feature of the organization of biological membranes. These results demonstrate that polyunsaturated fats affect the composition and organization of biological membranes, suggesting a potential mechanism for the extensive effects of dietary fat on health and disease.

The spatial organization of biological membranes is crucial for their extensive functionality. One example of such organization is the partitioning of mammalian plasma membranes (PMs) into lipid-driven ordered domains termed membrane rafts. While the physicochemical interactions responsible for raft formation are clearly established, the specific properties and biological consequences of these domains remain unclear. In particular, little is known about how the properties of raft domains are regulated, either by endogenous mechanisms or exogenous perturbations. In this context, an intriguing question concerns the effect of dietary fats on the lipid composition, biophysical properties, and ultimate function of membrane domains. Here, we investigate the consequences of the ω-3 polyunsaturated fatty acid docosohexaenoic acid (DHA) on the biochemical and biophysical properties of the mammalian PM. By detailed lipidomic analysis, we observe that DHA is robustly incorporated into specific PM lipids leading to wholesale remodeling of the lipidome, with saturated lipids and cholesterol up-regulated as a homeoviscous compensatory response to the disordering effect of polyunsaturated lipids. The combined effect of DHA incorporation and compensatory response result in biophysical remodeling of the PM, which we address in membrane model systems ranging from coarse-grained molecular dynamics simulations to intact PMs isolated from live cells. In all systems, DHA-containing lipids promote liquid-liquid phase separation by disordering non-raft domains and thereby increasing the differences between coexisting raft and non-raft phases. This relationship between interdomain disparity and stability of phase separation holds for a variety of perturbations, including manipulation of cholesterol levels and treatment by exogenous amphiphiles, suggesting it as a general design feature of the organization of biological membranes.

Biological membranes contain a broad variety of lipid species whose individual physicochemical properties and collective interactions ultimately determine membrane organization. A key aspect of the organization of cellular membranes is their lateral subdivision into domains of distinct structure and composition. The most widely studied membrane domains are lipid rafts, which are the biological manifestation of the capacity of sterol-containing membranes to form liquid-ordered phases. Detailed studies of biomimetic membrane mixtures have yielded wide-ranging insights into the physical principles behind lipid rafts; however, these simplified models do not fully capture the diversity and complexity of the mammalian lipidome, most notably in their exclusion of polyunsaturated lipids. Here, we assess the role of lipid tail unsaturation as a driving force for phase separation using coarse-grained molecular dynamics simulations validated by model membrane experiments. The clear trends in our observations and good qualitative agreements between simulations and experiments support the conclusions that highly unsaturated lipids promote liquid-liquid phase separation by enhancing the differences in lipid chain order, domain thickness, cholesterol preference, and diffusivity between the coexisting domains. To determine which of these parameters is the key driver of phase separation, we tuned lipid compositions to independently vary membrane order and thickness. We find that interdomain order differences driven by acyl chain unsaturation are the key determinants of domain formation in model membranes. These observations begin to define the important role of non-canonical biological lipids on the physical properties of membranes, showing that lipid polyunsaturation is a driving force for liquid-liquid phase separation.

Eukaryotic plasma membranes (PMs) are believed to possess lateral lipid domains termed lipid rafts, which form functional platforms for membrane sorting and cell signalling. The function of these domains depends on their selective recruitment of specific membrane proteins. However the underlying structural features governing protein partition into lipid rafts remain unknown. In live cells, the nanoscopic size and subsecond lifetime of rafts presents great difficulty for measuring their properties and composition. Intact PMs isolated as Giant Plasma Membrane Vesicles (GPMVs) phase separate into two microscopic, stable lipid domains, with one of these domains possessing greater lipid order, lower diffusivity and enriching for canonical raft components. This microscopic raft phase separation thus presents an optimal system for measuring protein partitioning between coexisting membrane domains. Using GPMVs, we demonstrate that a protein's transmembrane domain (TMD) is a central determinant of raft affinity. A major factor governing raft partition was the length of the TMD, with longer TMDs preferring the thicker raft domains. Further, by systematic mutation of the TMD of LAT (Linker for activation of T-cells), we were able to determine amino acid sequences which are necessary and sufficient for raft partitioning and which highly resemble the α-helix oligomerization motif GxxxG. This resemblance suggested that TMD oligomerization is the structural basis for the discovered raft-partitioning motif. We confirmed this hypothesis by measure sequence-dependent TMD oligomerization by fluorescence lifetime microscopy (FLIM) combined with Forster resonance energy transfer (FRET). Finally, we demonstrate the biological significance of our observations by showing that raft partitioning determines sorting between organellar membranes. For the entire panel of TMD-variants, we observed a strong quantitative relationship between raft association and sorting to the plasma membrane, with non-raft mutants being sorted into the lysosomes for degradation.

Saponins, amphiphiles of natural origin with numerous biological activities, are widely used in the cosmetic and pharmaceutical industry. Some saponins exhibit relatively selective cytotoxic effects on cancer cells but the tendency of saponins to induce hemolysis limits their anticancer potential. This review focused on the effects of saponin activity on membranes and consequent implications for red blood and cancer cells. This activity seems to be strongly related to the amphiphilic character of saponins that gives them the ability to self-aggregate and interact with membrane components such as cholesterol and phospholipids. Membrane interactions of saponins with artificial membrane models, red blood and cancer cells are reviewed with respect to their molecular structures. The review considered the mechanisms of these membrane interactions and their consequences including the modulation of membrane dynamics, interaction with membrane rafts, and membrane lysis. We summarized current knowledge concerning the mechanisms involved in the interactions of saponins with membrane lipids and examined the structure activity relationship of saponins regarding hemolysis and cancer cell death. A critical analysis of these findings speculates on their potential to further develop new anticancer compounds.

Saponins and triterpenic acids have been shown to be able to interact with lipid membranes and domains enriched with cholesterol (rafts). How saponins are able to modulate lipid phase separation in membranes and the role of the sugar chains for this activity is unknown. We demonstrate in a binary membrane model composed of DMPC/Chol (3:1 mol/mol) that the saponin α-hederin and its aglycone presenting no sugar chain, the triterpenic acid hederagenin, are able to induce the formation of lipid domains. We show on multilamellar vesicles (MLV), giant unilamellar vesicles (GUV), and supported planar bilayers (SPB) that the presence of sugar units on the sapogenin accelerates domain formation and increases the proportion of sterols within these domains. The domain shape is also influenced by the presence of sugars because α-hederin and hederagenin induce the formation of tubular and spherical domains, respectively. These highly curved structures should result from the induction of membrane curvature by both compounds. In addition to the formation of domains, α-hederin and hederagenin permeabilize GUV. The formation of membrane holes by α-hederin comes along with the accumulation of lipids into nonbilayer structures in SPB. This process might be responsible for the permeabilizing activity of both compounds. In LUV, permeabilization by α-hederin was sterol-dependent. The biological implications of our results and the mechanisms involved are discussed in relation to the activity of saponins and triterpenic acids on membrane rafts, cancer cells, and hemolysis.

Transmembrane proteins (TMPs) comprise ∼30% of the mammalian proteome and mediate nearly all functions of the cell. The subcellular localization, trafficking, signalling, and enzymatic activity of many TMPs is regulated by their association with lipid-driven lateral membrane domains known as lipid rafts. However, the structural determinants of TMP partitioning to raft domains remain almost unknown. We hypothesized that structural features of the transmembrane domain (TMD) of single-pass TMPs would guide raft partitioning and raft-dependent sub-cellular trafficking. To explore TMD-dependent raft partitioning, we isolated plasma membranes as Giant Plasma Membrane Vesicles (GPMVs), a model system that allows direct observation of protein partitioning between raft/non-raft phases by fluorescence microscopy. We generated 96 different fluorescent mutants of a model TMP and quantified their partitioning as a function of TMD features, namely hydrophobic length and accessible surface area. Raft partitioning was related to TMD length, with longer TMDs imparting greater raft association, confirming previously untested predictions. Investigation of 74 mutants with constant TMD length revealed that raft partitioning was also inversely related to the TMD accessible surface area, with larger surface areas preferring the non-raft phase. To explain these results, we present a simple physical model wherein raft partitioning is driven by differences in interfacial energy between the TMD and its surrounding lipid matrix in raft and non-raft phases. This model is in quantitative agreement with experimental observations and provides first predictions of protein-lipid surface tensions differences between coexisting membrane phases. The model could successfully connect predicted raft partitioning with subcellular localization of single-pass TMPs in Eukaryota. These results point the way to a general rule for raft partitioning of TMPs and underline the central role of membrane domains in cellular traffic.

α-hederin is a triterpenoid saponin which has shown hemolytic and apoptotic properties. (Chwalek et al. 2006, Gauthier et al. 2009). Many saponins are known to interact with biological membranes on a cholesterol dependent or independent manner (Francis et al. 2002) but the underlying mechanisms need to be further explored. We have studied the effects of α-hederin on size, membrane permeability and fluidity in a LUV model composed of DMPC including 25% (mol/mol) of cholesterol or not. Size was determined by quasi-elastic light scattering spectroscopy. Permeability was evaluated by the release of calcein, entrapped at self-quenching concentrations in liposomes. Membrane dynamics at the hydrophobic core was evaluated by fluorescence anisotropy of diphenylhexatriene, upon increasing temperatures.α-hederin increased the size of cholesterol-rich liposomes and induced release of calcein entrapped within liposomes, on a time- and concentration-dependent manner. It was without effect when cholesterol was absent. α-hederin reduced the transition temperature on a concentration-dependent manner only in liposomes containing cholesterol but increased anisotropy beyond the transition temperature in both types of liposomes.Our data suggest that cholesterol is required for α-hederin's effects on liposome's size, permeability and transition temperature but probably not for the effect on membrane fluidity, when lipids are in fluid phase. The role of these effects for apoptosis induced by α-hederin is under investigation.

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.