Non-raft Phase Research Articles

Lipid Peroxidation Enhances LO/LD Domain Phase Separation in Giant Plasma Membrane Vesicles

Lipid peroxidation is an oxidative process in which free radicals lead to the degradation of membrane lipids. The prime action of oxidative reactions in biomembranes is to increase reactive oxygen species (ROS) and form fatty acid hydroperoxides. This leads to changes in membrane fluidity, disruption of plasma membrane integrity, and impairment of lipid-protein interactions that ultimately facilitate the modification of nucleic acids and proteins by the release of reactive aldehydes and bioactive products, thus disrupting gene transcription and signal transduction. Lipid peroxidation has been linked to a number of diseases such as Alzheimer's disease. However, the effects of lipid peroxidation on plasma membrane lipids and proteins are still incompletely understood. Therefore, we used giant plasma membrane vesicles (GPMVs) as a model system to examine lipid peroxidation-induced changes in plasma membrane phase behavior. In particular, we investigated how lipid peroxidation induced using a combination of Fe(II) and Cumene hydroperoxide alters membrane physical properties, especially lipid rafts (Lo) and non-raft (Ld) phases. We find that lipid peroxidation causes dramatic enhancement of phase separation at room temperature that is accompanied by a significant increase in the fraction of the non-raft phase. These effects were concentration dependent, and similar results were observed in GPMVs derived from different cell types. Interestingly, lipid peroxidation also decreased the affinity of the model raft protein YFP-GL-GPI for raft domains, indicating that protein composition of membrane domains is also impacted by lipid peroxidation. Taken together, these findings highlight the utility of this approach to study the impact of lipid peroxidation on cell membranes and suggest a potential mechanism by which lipid peroxidation can influence cellular functions.

Structural Determinants of Raft Partitioning for Single-Pass Transmembrane Proteins

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.

Dietary Fats Remodel Plasma Membrane Lipidome and Physical Properties to Regulate Phase Separation in Biological Membranes

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.

The Biophysics of Living Membranes: Protein Partitioning and Functional Differentiation in Ordered Plasma Membrane Domains

The lipid raft hypothesis posits that ordered, lateral membrane domains are important for the regulation of plasma membrane (PM) structure and function in eukaryotes. Giant Plasma Membrane Vesicles (GPMVs) are isolated plasma membranes that microscopically separate into coexisting ordered and disordered phases, facilitating experimental analysis of the composition and physical properties of ordered raft domains in biological membranes. In these studies, we analyze the biophysical and lipidomic differentiation of the plasma membrane in Mesenchymal Stem Cells (MSC) as the cells undergo differentiation into adipocytes and osteoblasts. During differentiation, dramatic remodeling the PM lipidome, including lipid acyl chain length and unsaturation, results inchanges to membrane order and phase separation. These observations elucidate the compositional determinants of biophysical properties in biological membranes, as well as identify lineage-specific PM features. These results facilitate rational remodeling of membrane phenotypes to direct differentiation, as supplementation with ꙍ-3 docosahexaneoic acid (DHA) promoted the osteoblastic PM phenotype and potentiated osteogenic differentiation. The differences in the physical properties of the coexisting domains lead to preferential protein partitioning between them. We evaluate the structural determinants of raft partitioning of a model transmembrane protein, and find that raft phase partitioning is related to features of the protein's transmembrane domain (TMD), namely the hydrophobic length and surface area of the TMD. Longer TMDs impart greater raft association while TMDs with larger, bulkier amino acid side chains prefer the non-raft phase. We present a simple physical model wherein raft partitioning is driven by phase-dependent differences in interfacial energy between the TMD and its surrounding lipid matrix, and find excellent quantitative agreement with observations that provide the first predictions of protein-lipid surface tension. These results point the way to a general rule for raft partitioning of transmembrane proteins.

Proteomic Profiling of Detergent Resistant Membranes (Lipid Rafts) of Prostasomes

Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.

Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy

Sphingomyelin (SM) and cholesterol (chol)-rich domains in cell membranes, called lipid rafts, are thought to have important biological functions related to membrane signaling and protein trafficking. To visualize the distribution of SM in lipid rafts by means of Raman microscopy, we designed and synthesized an SM analog tagged with a Raman-active diyne moiety (diyne-SM). Diyne-SM showed a strong peak in a Raman silent region that is free of interference from intrinsic vibrational modes of lipids and did not appear to alter the properties of SM-containing monolayers. Therefore, we used Raman microscopy to directly visualize the distribution of diyne-SM in raft-mimicking domains formed in SM/dioleoylphosphatidylcholine/chol ternary monolayers. Raman images visualized a heterogeneous distribution of diyne-SM, which showed marked variation, even within a single ordered domain. Specifically, diyne-SM was enriched in the central area of raft domains compared with the peripheral area. These results seem incompatible with the generally accepted raft model, in which the raft and nonraft phases show a clear biphasic separation. One of the possible reasons is that gradual changes of SM concentration occur between SM-rich and -poor regions to minimize hydrophobic mismatch. We believe that our technique of hyperspectral Raman imaging of a single lipid monolayer opens the door to quantitative analysis of lipid membranes by providing both chemical information and spatial distribution with high (diffraction-limited) spatial resolution.

Perturbation of Plasma Membrane Physical Properties by Endogenous and Exogenous Mediators Affects Cell Function

In mammalian plasma membranes (PM), ordered, lipid-driven lateral domains - known as lipid rafts - have been implicated as central regulators of a broad range of cell functions and pathophysiological processes. Recent observations suggest these domains comprise a major fraction of mammalian plasma membranes, so rather than isolated islands of distinct composition, they may be fundamental to PM structure and function. However, direct demonstrations of lipid raft involvement in specific cell functions remain elusive because of artifact-prone, indirect, and poorly characterized experimental paradigms for raft perturbation. Giant Plasma Membrane Vesicles (GPMVs) are isolated PMs that separate into coexisting raft and non-raft phases in a temperature dependent manner, with the miscibility transition temperature providing a quantitative estimate of the stability of raft domains in live cells. We have evaluated a number of endogenous and exogenous mediators of the plasma membrane phenotype - defined as the fluidity of PM and the stability of lipid raft domains - and measured the effects of these perturbations on cell function. Cell autonomously, we find that differentiation of Mesenchymal Stem Cells (MSC) causes rapid divergence of both aspects of the PM phenotype. Inversely, perturbation of PM physical properties by ω-3 poly-unsaturated fatty acids (PUFAs) affects MSC differentiation, promoting the osteogenic over the adipogenic phenotype. Extending these studies to other cell functions, we have discovered a variety of exogenous mediators of the PM phenotype (in addition to ω-3 PUFAs) and find that any deviation from optimal raft stability has a suppressive effect on antigen-mediated mast cell activation. These observations provide a quantitative framework to measure the PM phenotype, identify exogenous fatty acids as potential mediators of this phenotype, and demonstrate the effect of perturbations of PM physical properties on cell function.

Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.

Use of lipid rafting for the analysis of human basophil activation by flow cytometry

Human leukocyte activation induced by specific and non-specific stimuli is characterized by the formation of lipid rafts defined as lipid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. These lipid rafts are formed in parallel with profound membrane reorganization. Analyse the rafting and non-rafting proteins present on the activated and resting basophil membrane and study their interest for the flow cytometric analysis of basophil activation. Human basophils obtained from samples used for diagnostic cellular tests such as basophil or lymphocyte activation tests were stimulated either by the formyl-methionyl-leucyl-phenylalanine peptide (fMLP), by an anti-IgE or by an allergen. After 40 min at 37 degrees C, they were labelled by different antibodies conjugated to fluorescent dyes as an anti-IgE FITC, an anti-CCR3 PE, an anti-CD63, an anti-CD203c PE, an anti-11b, annexin V FITC or by cholera toxin FITC. Moreover, several experiments were analysed using an Amnis cytometer, allowing one to obtain the picture of the analysed cells. Anti-IgE or specific allergen elicits a membrane neo expression of CD63 at a high density and is poorly represented on resting basophil membrane. Upon an IgE-dependant activation some of the markers already present on resting basophil membrane, as CD203c, are up regulated and others, such as the IgE/IgE FcepsilonRI receptor and CCR3 are down regulated and submitted to the formation of clusters demonstrated by the pictures taken with the Amnis cytometer. For non-IgE dependant activators, such as fMLP, the picture was different since IgE was not down regulated, whereas CCR3 was down regulated. As demonstrated using annexin V or the cholera toxin used for analysing apoptosis, these phenomenon were paralleled by the formation of lipid rafts, gangliosides domains, such as GM1, which is accessible from the extra cellular medium. Basophil activation leads to membrane events close to the apoptosis phenomenon. The flow cytometric analysis of these membrane events may lead to protocols for allergen-induced activation and, may significantly increase cellular test sensitivity, particularly for drugs allergy diagnosis for which the usual protocols, such as those using CD63 alone, are insufficiently sensitive.

Ganglioside GM1 increases line tension at raft boundary in model membranes

Gangliosides are significant participants in suppression of immune system during tumor processes. It was shown that they can induce apoptosis of T-lymphocytes in a raft-dependent manner. Fluorescence confocal microscopy was used to study distribution and influence of ganglioside GM1 on raft properties in giant unilamellar vesicles. Both raft and non-raft phase markers were utilized. No visible phase separation was observed without GM1 unless lateral tension was applied to the membrane. At 2 mol % of GM1 large domains appeared indicating macroscopic phase separation. Increase of GM1 content to 5 mol % resulted in shape transformation of the domains consistent with growth of line tension at the domain boundary. At 10 mol % of GM1 almost all domains were pinched out from vesicles, forming their own homogeneous liposomes. Estimations showed that the change of the GM1 content from 2 to 5–10 mol % resulted in a several-fold increase of line tension. This finding provides a possible mechanism of apoptosis induction by GM1. Incorporation of GM1 into a membrane leads to an increase of the line tension. This results in a growth of the average size of rafts due to coalescence or merger of small domains. Thus, necessary proteins can find themselves in one common raft and start the corresponding cascade of reactions.

Influence Of Ganglioside GM1 On Formation And Properties Of Rafts In Lipid Membranes

Gangliosides are the universal components of membranes of eukaryotic cells playing a number of important regulatory functions. It has been previously demonstrated that release of certain gangliosides from malignant cells can be responsible for inducing apoptosis of immunocompetent cells. One of the possible mechanisms includes raft formation, oligomerization of pro-apoptotic receptors with consequential apoptosis signal transduction. In particular, GM1 and GM3 can both form their own microdomains and partition to rafts and caveolae in membranes. Since investigation of raft properties in living cells is greatly complicated by small size of rafts, and other factors, giant unilamellar vesicles (GUV) made of biological membrane mimicking mixture of lipids were used as a model system.Fluorescence confocal microscopy was employed to study distribution of ganglioside GM1 in GUV and its influence on formation and properties of rafts in bilayer lipid membranes. Both raft and non-raft phase markers were added to membrane-forming lipid mixture. No visible phase separation was observed without GM1 unless lateral tension was applied to the membrane by osmotic stress. At 2 mol% concentration of GM1, large domains appeared indicating macroscopic phase separation. Increase of GM1 content to 5 mol% resulted in transformations of the domain shape consistent with significant growth of line tension at the domain boundary. On further increase of GM1 concentration to 10 mol%, almost all domains were pinched out from the vesicles, forming their own homogeneous liposomes. Estimates indicated that changing GM1 content from 2 mol% to 5-10 mol% resulted in the increase of the line tension at domain boundary by several hundred percent.

Focal junctions retard lateral movement and disrupt fluid phase connectivity in the plasma membrane

In cultured keratinocytes, focal junctions are enriched in major constituents of lipid rafts, such as GM 1 ganglioside, phosphoinositides, caveolins and flotillins. We have therefore speculated that focal junctions represent superrafts formed by coalescence of microdomains into large areas containing liquid-ordered ( L o) lipids. Indeed, values of maximal fluorescence recovery after photobleaching revealed that the long-range mobility of cholera toxin B subunit (CTB, marker of L o) was ∼1.5-fold retarded within the focal junctions compared to the surrounding membrane. However, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI-C 18:0), which specifically partitions to the liquid-disordered ( L d), non-raft phase, was also enriched in focal junctions and its mobility was slightly retarded. Cross-linking of GM 1 by CTB or raft aggregation by methyl-β-cyclodextrin further decreased the recovery of DiI-C 18:0. We propose a model in which focal junctions impose lateral heterogeneity in the plasma membrane by entrapment of lipid microdomains between dense arrays of immobilized transmembrane molecules which can enmesh otherwise freely percolating L d phase lipids.

Lipid Rafts and Nonrafts Mediate Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptotic and Nonapoptotic Signals in Non–Small Cell Lung Carcinoma Cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is capable of inducing apoptosis in non-small cell lung carcinoma (NSCLC). However, many of the human NSCLC cell lines are resistant to TRAIL, and TRAIL treatment of the resistant cells leads to the activation of nuclear factor-kappaB (NF-kappaB) and extracellular signal-regulated kinase 1/2 (ERK1/2). TRAIL can induce apoptosis in TRAIL-sensitive NSCLC cells through the induction of death-inducing signaling complex (DISC) assembly in lipid rafts of plasma membrane. In the DISC, caspase-8 is cleaved and initiates TRAIL-induced apoptosis. In contrast, TRAIL-DISC assembly in the nonraft phase of the plasma membrane leads to the inhibition of caspase-8 cleavage and NF-kappaB and ERK1/2 activation in TRAIL-resistant NSCLC cells. Receptor-interacting protein (RIP) and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP) mediates the DISC assembly in nonrafts and selective knockdown of either RIP or c-FLIP with interfering RNA redistributes the DISC from nonrafts to lipid rafts, thereby switching the DISC signals from NF-kappaB and ERK1/2 activation to caspase-8-initiated apoptosis. Chemotherapeutic agents inhibit c-FLIP expression, thereby enhancing the DISC assembly in lipid rafts for caspase-8-initiated apoptosis. These studies indicate that RIP and c-FLIP-mediated assembly of the DISC in nonrafts is a critical upstream event in TRAIL resistance and thus targeting of either RIP or c-FLIP may lead to the development of novel therapeutic strategies that can overcome TRAIL resistance in human NSCLC.

Phase coexistence and connectivity in the apical membrane of polarized epithelial cells

Although it is well described in model membranes, little is known about phase separation in biological membranes. Here, we provide evidence for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells. Phase connectivity was assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photobleaching in two polarized epithelial cell lines and one fibroblast cell line. In contrast to the fibroblast plasma membrane, in which all of the proteins diffused with similar characteristics, in the apical membrane of epithelial cells the proteins could be divided into two groups according to their diffusion characteristics. At room temperature ( approximately 25 degrees C), one group showed fast diffusion and complete recovery. The other diffused three to four times slower and, more importantly, displayed only partial recovery. Only the first group comprises proteins that are believed to be associated with lipid rafts. The partial recovery is not caused by topological constraints (microvilli, etc.), cytoskeletal constraints, or protein-protein interactions, because all proteins show 100% recovery in fluorescence recovery after photobleaching experiments at 37 degrees C. In addition, the raft-associated proteins cannot be coclustered by antibodies on the apical membrane at 12 degrees C. The interpretation that best fits these data is that the apical membrane of epithelial cells is a phase-separated system with a continuous (percolating) raft phase <25 degrees C in which isolated domains of the nonraft phase are dispersed, whereas at 37 degrees C the nonraft phase becomes the continuous phase with isolated domains of the raft phase dispersed in it.