Plasma Membrane Lipid Domains Research Articles

Fungal Plasma Membrane Lipid Domains Alterations Upon Drug Exposure

The biophysical properties of the plasma membrane of the filamentous fungus Neurospora crassa was characterized using fluorescence spectroscopy. We have taken advantage of a strategy relying on the combined use of different membrane probes and photophysical properties to monitor, in vivo, thebiophysical changes upon a drug challenge with staurosporine, a programmed cell death-inducing drug, in N. crassa asexual spores (conidia). An increase in the rigidity of highly ordered membrane domains was observed, possibly associated with the increment on the levels of sphingolipids with smaller headgroups, allowing a tighter packing of the lipid acyl chains.

Differential impact of synthetic antitumor lipid drugs on the membrane organization of phosphatidic acid and diacylglycerol monolayers

Anti-tumour lipids are synthetic analogues of lysophosphatidylcholine. These drugs are both cytotoxic and cytostatic, and more interestingly, exert these effects preferentially in tumour cells. While the exact mechanism of action isn’t fully elucidated, these drugs appear to preferentially partition into rigid lipid domains in cell membranes. Upon insertion, the compounds alter membrane domain organization, disrupt normal signal transduction, and cause cell death. Recently, it has been reported that these drugs induce accumulation of diacylglycerol in yeast cells which in turn sensitizes cells to the drugs. Conversely, phosphatidic acid accumulation appears to protect cells against the drugs. In the current work, the aim was to compare the biophysical effects of the drugs edelfosine, miltefosine and perifosine on monolayers of dimyristoyl phosphatidic acid, dimyristoyl glycerol and an equimolar mixture, to understand how these lipids modulate the mode of action. Surface pressure – area isotherms, compression moduli and Brewster angle microscopy were used to compare drug effects on lipid packing, monolayer compressibility and lateral domain organization of these films. Results suggest that edelfosine and miltefosine have stabilizing effects on all of the monolayers, while perifosine destabilizes dimyristoyl glycerol and the equimolar mixture. Additionally, all three drugs change the morphology of the domains observed. Based on these results the stabilization of diacylgylcerol by edelfosine and miltefosine may contribute to the mode of action as diacylglycerol is a known disruptor of bilayers. Perifosine however does not stabilize diacylglycerol, and therefore cell death may occur through a more direct inhibition of specific signal transduction. These results suggest that perifosine may illicit cytotoxicity through a different mechanism compared to the other antitumor lipid drugs.

Phase separation in pore-spanning membranes induced by differences in surface adhesion.

Lipid domains in plasma membranes act as molecular sorting platforms for e.g., signalling processes. In model membranes, such as freestanding or supported bilayers, some lipid domains with defined chemical composition, lipid packing and physical behaviour can be reproduced. However, in vivo, the plasma membrane experiences a proteinaceous scaffold underneath, which can sort, compartmentalize and recruit components within the membrane. The influence of such scaffolds on the phase behaviour of lipid membranes has been barely studied. Here, we investigated the partial attachment of a membrane to a support and its influence on the phase behaviour using pore-spanning membranes (PSMs). PSMs were prepared on SiOx=1-2 functionalized silicon substrates with 1.2 μm-sized pores by spreading giant unilamellar vesicles (GUVs) composed of DOPC/sphingomyelin (1 : 1) with different cholesterol concentrations. Using two different fluorophores, PSMs were visualized by fluorescence microscopy allowing us to distinguish between different membrane phases, a gel (lβ), a liquid ordered (lo), and a liquid disordered (ld) phase. At low cholesterol concentrations, coexistence of lβ and ld was found, while at higher cholesterol concentrations, coexistence of lo and ld was predominant. Below the mixing temperature, determined by temperature scans, the more ordered phase was always found in the freestanding PSMs, whereas the ld-phase was present in the supported PSMs. We attribute this lipid sorting to a stronger adhesion of the ld-phase lipids to the underlying scaffold. The difference in adhesion alters the phase behaviour from a nominal DOPC/sphingomyelin (1 : 1) mixture to a DOPC/sphingomyelin (1 : 2-1 : 4) mixture compared to phase diagrams obtained from GUVs highlighting the importance of differential adhesive surfaces on lipid domain formation.

Plasma Membrane Lipid Domains as Platforms for Vesicle Biogenesis and Shedding?

Extracellular vesicles (EVs) contribute to several pathophysiological processes and appear as emerging targets for disease diagnosis and therapy. However, successful translation from bench to bedside requires deeper understanding of EVs, in particular their diversity, composition, biogenesis and shedding mechanisms. In this review, we focus on plasma membrane-derived microvesicles (MVs), far less appreciated than exosomes. We integrate documented mechanisms involved in MV biogenesis and shedding, focusing on the red blood cell as a model. We then provide a perspective for the relevance of plasma membrane lipid composition and biophysical properties in microvesiculation on red blood cells but also platelets, immune and nervous cells as well as tumor cells. Although only a few data are available in this respect, most of them appear to converge to the idea that modulation of plasma membrane lipid content, transversal asymmetry and lateral heterogeneity in lipid domains may play a significant role in the vesiculation process. We suggest that lipid domains may represent platforms for inclusion/exclusion of membrane lipids and proteins into MVs and that MVs could originate from distinct domains during physiological processes and disease evolution.

Contribution of plasma membrane lipid domains to red blood cell (re)shaping

Although lipid domains have been evidenced in several living cell plasma membranes, their roles remain largely unclear. We here investigated whether they could contribute to function-associated cell (re)shaping. To address this question, we used erythrocytes as cellular model since they (i) exhibit a specific biconcave shape, allowing for reversible deformation in blood circulation, which is lost by membrane vesiculation upon aging; and (ii) display at their outer plasma membrane leaflet two types of submicrometric domains differently enriched in cholesterol and sphingomyelin. We here reveal the specific association of cholesterol- and sphingomyelin-enriched domains with distinct curvature areas of the erythrocyte biconcave membrane. Upon erythrocyte deformation, cholesterol-enriched domains gathered in high curvature areas. In contrast, sphingomyelin-enriched domains increased in abundance upon calcium efflux during shape restoration. Upon erythrocyte storage at 4 °C (to mimick aging), lipid domains appeared as specific vesiculation sites. Altogether, our data indicate that lipid domains could contribute to erythrocyte function-associated (re)shaping.

The Molecular Basis of Toxins' Interactions with Intracellular Signaling via Discrete Portals.

An understanding of the molecular mechanisms by which microbial, plant or animal-secreted toxins exert their action provides the most important element for assessment of human health risks and opens new insights into therapies addressing a plethora of pathologies, ranging from neurological disorders to cancer, using toxinomimetic agents. Recently, molecular and cellular biology dissecting tools have provided a wealth of information on the action of these diverse toxins, yet, an integrated framework to explain their selective toxicity is still lacking. In this review, specific examples of different toxins are emphasized to illustrate the fundamental mechanisms of toxicity at different biochemical, molecular and cellular- levels with particular consideration for the nervous system. The target of primary action has been highlighted and operationally classified into 13 sub-categories. Selected examples of toxins were assigned to each target category, denominated as portal, and the modulation of the different portal’s signaling was featured. The first portal encompasses the plasma membrane lipid domains, which give rise to pores when challenged for example with pardaxin, a fish toxin, or is subject to degradation when enzymes of lipid metabolism such as phospholipases A2 (PLA2) or phospholipase C (PLC) act upon it. Several major portals consist of ion channels, pumps, transporters and ligand gated ionotropic receptors which many toxins act on, disturbing the intracellular ion homeostasis. Another group of portals consists of G-protein-coupled and tyrosine kinase receptors that, upon interaction with discrete toxins, alter second messengers towards pathological levels. Lastly, subcellular organelles such as mitochondria, nucleus, protein- and RNA-synthesis machineries, cytoskeletal networks and exocytic vesicles are also portals targeted and deregulated by other diverse group of toxins. A fundamental concept can be drawn from these seemingly different toxins with respect to the site of action and the secondary messengers and signaling cascades they trigger in the host. While the interaction with the initial portal is largely determined by the chemical nature of the toxin, once inside the cell, several ubiquitous second messengers and protein kinases/ phosphatases pathways are impaired, to attain toxicity. Therefore, toxins represent one of the most promising natural molecules for developing novel therapeutics that selectively target the major cellular portals involved in human physiology and diseases.

Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterol nanoclusters.

The self-assembly of HIV-1 Gag polyprotein at the inner leaflet of the cell host plasma membrane is the key orchestrator of virus assembly. The binding between Gag and the plasma membrane is mediated by specific interaction of the Gag matrix domain and the PI(4,5)P2 lipid (PIP2). It is unknown whether this interaction could lead to local reorganization of the plasma membrane lipids. In this study, using model membranes, we examined the ability of Gag to segregate specific lipids upon self-assembly. We show for the first time that Gag self-assembly is responsible for the formation of PIP2 lipid nanoclusters, enriched in cholesterol but not in sphingomyelin. We also show that Gag mainly partition into liquid-disordered domains of these lipid membranes. Our work strongly suggests that, instead of targeting pre-existing plasma membrane lipid domains, Gag is more prone to generate PIP2/Cholesterol lipid nanodomains at the inner leaflet of the plasma membrane during early events of virus assembly.

Reorganization of plasma membrane lipid domains during conidial germination

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted.Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth.

Structural determinants of protein partitioning into ordered membrane domains and lipid rafts

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.

Imaging lipid domains in cell membranes: the advent of super-resolution fluorescence microscopy

The lipid bilayer of model membranes, liposomes reconstituted from cell lipids, and plasma membrane vesicles and spheres can separate into two distinct liquid phases to yield lipid domains with liquid-ordered and liquid-disordered properties. These observations are the basis of the lipid raft hypothesis that postulates the existence of cholesterol-enriched ordered-phase lipid domains in cell membranes that could regulate protein mobility, localization and interaction. Here we review the evidence that nano-scaled lipid complexes and meso-scaled lipid domains exist in cell membranes and how new fluorescence microscopy techniques that overcome the diffraction limit provide new insights into lipid organization in cell membranes.

Recent Advances in the Study of Glycosphingolipids

Glycosphingolipids (GSLs) are present in all mammalian cell plasma membranes and intracellular membrane structures. They are especially concentrated in plasma membrane lipid domains that are specialized for cell signaling. Plasma membranes show typical structures called rafts and caveola domain structures, with large amounts of sphingolipids, cholesterol, and sphingomyelin in the cell membranes. Plasma membranes have two faces, many kinds of receptors for intercellular signal transducers such as GPI-anchored proteins on the exoplasmic faces of the rafts/caveolae and src family kinases on the cytosolic face. Thus they play a role in transmembrane signal transduction, following the phosphorylation of some substrates and gene expression. On the other hand, their functions have become clear through the study of gene-manipulated mice. For further advances, a visual method to display diversity of biological functions is necessary. For this purpose, the use of high-performance microscopes and live cell imaging technologies are useful for more detailed understanding.

Glycosphingolipid Modification: Structural Diversity, Functional and Mechanistic Integration of Diabetes

Glycosphingolipids (GSLs) are present in all mammalian cell plasma membranes and intracellular membrane structures. They are especially concentrated in plasma membrane lipid domains that are specialized for cell signaling. Plasma membranes have typical structures called rafts and caveola domain structures, with large amounts of sphingolipids, cholesterol, and sphingomyelin. GSLs are usually observed in many organs ubiquitously. However, GSLs, including over 400 derivatives, participate in diverse cellular functions. Several studies indicate that GSLs might have an effect on signal transduction related to insulin receptors and epidermal growth factor receptors. GSLs may modulate immune responses by transmitting signals from the exterior to the interior of the cell. Guillain-Barré syndrome is one of the autoimmune disorders characterized by symmetrical weakness in the muscles of the legs. The targets of the immune response are thought to be gangliosides, which are one group of GSLs. Other GSLs may serve as second messengers in several signaling pathways that are important to cell survival or programmed cell death. In the search for clear evidence that GSLs may play critical roles in various biological functions, many researchers have made genetically engineered mice. Before the era of gene manipulation, spontaneous animal models or chemical-induced disease models were used.

Endothelial Cell Membrane Sensitivity to Shear Stress is Lipid Domain Dependent

Blood flow-associated shear stress causes physiological and pathophysiological biochemical processes in endothelial cells that may be initiated by alterations in plasma membrane lipid domains characterized as liquid-ordered (l(o)), such as rafts or caveolae, or liquid-disordered (l(d)). To test for domain-dependent shear sensitivity, we used time-correlated single photon counting instrumentation to assess the photophysics and dynamics of the domain-selective lipid analogues DiI-C(12) and DiI-C(18) in endothelial cells subjected to physiological fluid shear stress. Under static conditions, DiI-C(12) fluorescence lifetime was less than DiI-C(18) lifetime and the diffusion coefficient of DiI-C(12) was greater than the DiI-C(18) diffusion coefficient, confirming that DiI-C(12) probes l(d), a more fluid membrane environment, and DiI-C(18) probes the l(o) phase. Domains probed by DiI-C(12) exhibited an early (10 s) and transient decrease of fluorescence lifetime after the onset of shear while domains probed by DiI-C(18) exhibited a delayed decrease of fluorescence lifetime that was sustained for the 2 min the cells were subjected to flow. The diffusion coefficient of DiI-C(18) increased after shear imposition, while that of DiI-C(12) remained constant. Determination of the number of molecules (N) in the control volume suggested that DiI-C(12)-labeled domains increased in N immediately after step-shear, while N for DiI-C(18)-stained membrane transiently decreased. These results demonstrate that membrane microdomains are differentially sensitive to fluid shear stress.

The Human Concentrative Nucleoside Transporter-3 C602R Variant Shows Impaired Sorting to Lipid Rafts and Altered Specificity for Nucleoside-Derived Drugs

The human concentrative nucleoside transporter-3 C602R (hCNT3C602R), a recently identified human concentrative nucleoside transporter-3 (hCNT3) variant, has been shown to interact with natural nucleosides with apparent K(m) values similar to those of the wild-type transporter, although binding of one of the two sodium ions required for nucleoside translocation is impaired, resulting in decreased V(max) values (Mol Pharmacol 73:379-386, 2008). We have further analyzed the properties of this hCNT3 variant by determining its localization in plasma membrane lipid domains and its interaction with nucleoside-derived drugs used in anticancer and antiviral therapies. When expressed heterologously in HeLa cells, wild-type hCNT3 localized to both lipid raft and nonlipid raft domains. Treatment of cells with the cholesterol-depleting agent methyl-beta-cyclodextrin resulted in a marked decrease in hCNT3-related transport activity that was associated with the loss of wild-type hCNT3 from lipid rafts. It is noteworthy that although exogenously expressed hCNT3C602R was present in nonlipid raft domains at a level similar to that of the wild-type transporter, the mutant transporter was present at much lower amounts in lipid rafts. A substrate profile analysis showed that interactions with a variety of nucleoside-derived drugs were altered in the hCNT3C602R variant and revealed that sugar hydroxyl residues are key structural determinants for substrate recognition by the hCNT3C602R variant.

Detection of Lipid Domains in Model and Plasma Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate that lipid domains in plasma membranes of mammalian cells are short lived and in the submicron range. To unravel the lateral heterogeneity of cellular membranes, in particular of mammalian plasma membranes, at this scale various techniques of fluorescence spectroscopy and microscopy have been applied. Measuring fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in Giant Unilamellar Vesicles (GUVs) and the plasma membrane of mammalian cells by Fluorescence Lifetime Imaging (FLIM). For GUVs forming microscopically visible lipid domains the fluorescence lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct being about 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM. Application of this approach to mammalian cells revealed that while the fluorescence lifetime is sensitive to the composition of the plasma membrane, distinct lipid domains as found for GUVs were not detected. A broad distribution of the long lifetime was found for C6-NBD-PC inserted in the plasma membrane of these cells centred around 11 ns. However, FLIM studies on lipid domain forming giant vesicles derived from the plasma membrane of HeLa-cells rather support a recent hypothesis that an ensemble of lipid domains being of submicroscopic size exist in the plasma membrane.

Detection of Lipid Domains in Model and Cell Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being approximately 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain-forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-d-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.

Interactions between apolipoprotein A-I and plasma membrane lipid domains, leading to phospholipid and cholesterol export

Interactions between apolipoprotein A-I and plasma membrane lipid domains, leading to phospholipid and cholesterol export

Characterization and Application of a New Optical Probe for Membrane Lipid Domains

In this article, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUVs), its emission spectrum shifts up to 30 nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60-nm difference in emission spectra for cholesterol containing LUVs in the liquid-ordered state versus cholesterol-free LUVs in the liquid-disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and nonlinear optical microscopy. The dye shows green and red fluorescence in liquid-ordered and -disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a sevenfold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye for reporting lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.

Lipid raft-enriched stem cell-like keratinocytes in the epidermis, hair follicles and sinus tracts in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a disease, that causes considerable morbidity in patients. A histological hallmark of the disorder is the formation of sinus tracts in the dermis and the subcutis. Biologically, they represent a poorly understood phenomenon involving the infiltrative growth of proliferating non-malignant keratinocytes. Lipid domains in plasma membranes (lipid rafts) play a role in the function of growth factors and are suspected of having a pathogenic role in cell migration and invasive growth. Using HS as a model, the presence of lipid rafts was studied using cholera toxin conjugated with FITC (CTx-FITC) and antibeta1 integrin (CD29)-CyChrome conjugate fluorescence staining of unfixed and acetone-fixed cryostat sections of lesional and paralesional skin samples. The double-labeled skin samples were observed in the confocal laser-scanning fluorescence microscope. Samples were obtained from five patients with HS. The lesional epidermis of HS contained three populations of keratinocytes: CD29(bright)CTx(dim), CD29(dim)CTx(bright) and a third hitherto unseen population containing double-positive CD29(bright)CTx(bright) cells. The CD29(bright)CTx(dim) population resembles the earlier described epidermal stem-like cells, while the CD29(dim)CTx(bright) basal keratinocytes overlap with the transit-amplifying cell pool. The new population of double-positive CD29(bright)CTx(bright) cells was localized on the slopes of the papillas, focally in the suprabasal epidermal layers, in some hair follicles and in the majority of sinus tracts. Such double-positive cells have not previously been encountered by us in normal epidermis and hair follicles. Using HS as a model, it is suggested that the keratinocytes involved in sinus tract formation are CD29(bright)CTx(bright) cells. Owing to the physical proximity of the cells, it is hypothesised that the described CD29(bright)CTx(bright) cells result from an increased expression of CD29 on the CTx(bright) cells. It is likely that the double-positive CD29(bright)CTx(bright) cells emerge due to the influence of local inflammatory cytokines. Sinus tract formation may represent an aberrant epidermal repair response executed by the activated CD29(bright)CTx(bright) keratinocytes capable of non-malignant infiltrative growth in the dermis and subcutis.

Interaction of the bisbiguanides chlorhexidine and alexidine with phospholipid vesicles: evidence for separate modes of action.

Strains of Providencia stuartii with demonstrated resistance towards chlorhexidine did not show such resistance towards either of the related biguanide antiseptics, alexidine or vantocil. Alexidine promoted a significantly faster alteration in the permeability of Escherichia coli cell membranes towards various metal cations than chlorhexidine. Differential thermal analysis of various mixed lipid vesicles and pure phospholipid vesicles showed alexidine to share with vantocil the property of producing lipid phase separation and domain formation. It is suggested that the nature of the end-group on the biguanides affects the ability to produce lipid domains in cell membranes and this this might, in turn, affect activity and resistance patterns observed.