Dynamics and size of cross-linking-induced lipid nanodomains in model membranes

Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Förster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of ～8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation.     

Lo/Ld phase coexistence modulation induced by GM1

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting “raft-like” L_o domain size and the search for L_o nanodomains as precursors in L_o microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the L_o/L_d lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1 mol % abolishes the formation of the micrometer-scale L_o domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a L_o/L_d lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C₁₂NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale L_o phase domains over an L_d phase is determined, and is shifted to higher values when the GM1 content increases. A “morphological” phase diagram could be made, and it displays three regions corresponding respectively to L_o/L_d micrometric phase separation, L_o/L_d nanometric phase separation, and a homogeneous L_d phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, “arresting” domain growth and thereby stabilizing L_o nanodomains.     

Raft composition at physiological temperature and pH in the absence of detergents

Biological rafts were identified and isolated at 37°C and neutral pH. The strategy for isolating rafts utilized membrane tension to generate large domains. For lipid compositions that led only to microscropically unresolvable rafts in lipid bilayers, membrane tension led to the appearance of large, observable rafts. The large rafts converted back to small ones when tension was relieved. Thus, tension reversibly controls raft enlargement. For cells, application of membrane tension resulted in several types of large domains; one class of the domains was identified as rafts. Tension was generated in several ways, and all yielded raft fractions that had essentially the same composition, validating the principle of tension as a means to merge small rafts into large rafts. It was demonstrated that sphingomyelin-rich vesicles do not rise during centrifugation in sucrose gradients because they resist lysis, necessitating that, contrary to current experimental practice, membrane material be placed toward the top of a gradient for raft fractionation. Isolated raft fractions were enriched in a GPI-linked protein, alkaline phosphatase, and were poor in Na⁺-K⁺ ATPase. Sphingomyelin and gangliosides were concentrated in rafts, the expected lipid raft composition. Cholesterol, however, was distributed equally between raft and nonraft fractions, contrary to the conventional view.     

Effect of line tension on the lateral organization of lipid membranes

The principles of organization and functioning of cellular membranes are currently not well understood. The raft hypothesis suggests the existence of domains or rafts in cell membranes, which behave as protein and lipid platforms. They have a functional role in important cellular processes, like protein sorting or cell signaling, among others. Theoretical work suggests that the interfacial energy at the domain edge, also known as line tension, is a key parameter determining the distribution of domain sizes, but there is little evidence of how line tension affects membrane organization. We have investigated the effects of the line tension on the formation and stability of liquid ordered domains in model lipid bilayers with raft-like composition by means of time-lapse confocal microscopy coupled to atomic force microscopy. We varied the hydrophobic mismatch between the two phases, and consequently the line tension, by modifying the thickness of the disordered phase with phosphatidylcholines of different acyl chain length. The temperature of domain formation, the dynamics of domain growth, and the distribution of domain sizes depend strongly on the thickness difference between the domains and the surrounding membrane, which is related to line tension. When considering line tension calculated from a theoretical model, our results revealed a linear increase of the temperature of domain formation and domain growth rate with line tension. Domain budding was also shown to depend on height mismatch. Our experiments contribute significantly to our knowledge of the physical-chemical parameters that control membrane organization. Importantly, the general trends observed can be extended to cellular membranes.     

Ganglioside G(M1)-mediated amyloid-beta fibrillogenesis and membrane disruption

There is increasing evidence that a class of cell membrane glycolipids, gangliosides, can mediate the fibrillogenesis and toxicity of Alzheimer's disease amyloid-beta peptide (Abeta). Using lipid monolayers and vesicles as model membranes, we measured the insertion of Abeta into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-ganglioside GM1 monolayers to probe Abeta-GM1 interactions, imaged the effects of Abeta insertion on monolayer morphology, and measured the rate of Abeta fibril formation when incubated with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-GM1 vesicles. Furthermore, the location of Abeta association in the monolayer was assessed by dual-probe fluorescence experiments. Abeta exhibited direct and favorable interactions with GM1 as Abeta insertion monotonically increased with GM1 concentration, despite increases in monolayer rigidity at low GM1 levels. At low GM1 concentrations, Abeta preferentially inserted into the disordered, liquid expanded phase. At higher GM1 concentrations, Abeta inserted more uniformly into the monolayer, resulting in no detectable preferences for either the disordered or condensed phase. Abeta insertion led to the disruption of membrane morphology, specifically to the expansion of the disordered phase at low GM1 concentrations and significant disruption of the condensed domains at higher GM1 concentrations. During incubation with POPC vesicles containing physiological levels of GM1, the association of Abeta with vesicles seeded the formation of Abeta fibrils. In conclusion, favorable interactions between Abeta and GM1 in the cell membrane may provide a mechanism for Abeta fibrillogenesis in vivo, and Abeta-induced disruption of the cell membrane may provide a pathway by which Abeta exerts toxicity.     

Distribution of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine/cholesterol monolayers: a model for lipid rafts

The distribution of low concentrations of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol monolayers supported on mica has been studied using atomic force microscopy (AFM). The monolayers studied correspond to a pure gel phase and a mixture of liquid-expanded (LE) and liquid-condensed (LC) phases for DPPC and to a single homogeneous liquid-ordered phase for 2:1 DPPC/cholesterol. The addition of 2.5-5% GM1 to phase-separated DPPC monolayers resulted in small round ganglioside-rich microdomains in the center and at the edges of the LC domains. Higher amounts of GM1 (10%) give numerous filaments in the center of the LC domains and larger patches at the edges. A gel phase DPPC monolayer containing GM1 showed large domains containing a network of GM1-rich filaments. The addition of GM1 to a liquid-ordered 2:1 DPPC/cholesterol monolayer gives small, round domains that vary in size from 50 to 150 nm for a range of surface pressures. Larger amounts of GM1 lead to coalescence of the small, round domains to give longer filaments that cover 30-40% of the monolayer surface for 10 mol % GM1. The results indicate that biologically relevant GM1 concentrations lead to submicron-sized domains in a cholesterol-rich liquid-ordered phase that is analogous to that found in detergent-insoluble membrane fractions, and are thought to be important in membrane microdomains or rafts. This demonstrates that AFM studies of model monolayers and bilayers provide a powerful method for the direct detection of microdomains that are too small for study with most other techniques.     

More ordered, convex ganglioside-enriched membrane domains: the effects of GM1 on sphingomyelin bilayers containing a low level of cholesterol

The special physical state of the sphingolipid-enriched membranes with characteristic lipid composition, presently one of the most controversial foci in cell biology, provides the essential environment for the proteins inside to be involved in the related physiological processes. The role of gangliosides, an important component of the membranes, deserves attention. The present investigation using several biophysical techniques indicates that ganglioside GM(1) induces the phase separation in the sphingomyelin membrane with 5 mol% cholesterol and regulates the membrane structure. The results of differential scanning calorimetry show that a higher T(m), GM(1)-rich phase emerges behind the lower T(m), sphingomyelin-rich phase with the incorporation of GM(1) into the sphingomyelin/cholesterol bilayers; and the GM(1)-rich phase dominates the membrane when the proportion of GM(1) reaches about 20 mol%. Fluorescence quenching further shows that the separation of the two domains is independent of temperature, occurring both in the gel phase and in the liquid phase. Laser Raman spectroscopy and fluorescence polarization suggest that the order of hydrocarbon chains increases and membrane fluidity decreases with increase in GM(1) content. Use of the fluorescence probe merocyanine-540 and electron microscopy reveals that the insertion of GM(1) leads to an increase in the spatial density of the lipid headgroups and a decrease in the curvature of the sphingomyelin/cholesterol bilayers. In sums, both the hydrophilic sugar heads and the hydrophobic hydrocarbon chains of GM(1) contribute to the regulation of membrane architecture. We suggest that the convex curvature of ganglioside-enriched membrane could be involved in forming and maintaining the characteristic flask-shaped invagination of caveolae.     

Chondrocytes utilize a cholesterol-dependent lipid translocator to externalize phosphatidylserine

During endochondral ossification, growth plate chondrocytes release plasma membrane (PM) derived matrix vesicles (MV), which are the site of initial hydroxyapatite crystal formation. MV constituents which facilitate the mineralization process include the integral membrane ectoenzymes alkaline phosphatase (ALPase) and nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), along with a phosphatidylserine- (PS-) rich membrane surface that binds annexins and calcium, resulting in enhanced calcium entry into MV. In this study, we determined that chick growth plate MV were highly enriched in membrane raft microdomains containing high levels of cholesterol, glycophosphatidylinositol- (GPI-) anchored ALPase, and phosphatidylserine (PS) localized to the external leaflet of the bilayer. To determine how such membrane microdomains arise during chondrocyte maturation, we explored the role of PM cholesterol-dependent lipid assemblies in regulating the activities of lipid translocators involved in the externalization of PS. We first isolated and determined the composition of detergent-resistant membranes (DRMs) from chondrocyte PM. DRMs isolated from chondrocyte PM were enhanced in ganglioside 1 (GM1) and cholesterol as well as GPI-anchored ALPase. Furthermore, these membrane domains were enriched in PS (localized to the external leaflet of the bilayer) and had significantly higher ALPase activity than non-cholesterol-enriched domains. To understand the role of cholesterol-dependent lipid assemblies in the externalization of PS, we measured the activities of two lipid transporters involved in PS externalization, aminophospholipid translocase (APLT) and phospholipid scramblase (PLSCR1), during maturation of a murine chondrocytic cell line, N1511. In this report, we provide the first evidence that maturing chondrocytes express PLSCR1 and have scramblase activity. We propose that redistribution of PS is dependent on an increase in phospholipid scramblase activity and a decrease in APLT activity. Lastly, we show that translocator activity is most likely to be modulated by membrane cholesterol levels through a membrane raft microdomain.     

Stabilization of bilayer structure of raft due to elastic deformations of membrane

Line tension of the boundary of specific domains (rafts) rich in sphingomyelin was calculated. The line tension was calculated based on macroscopic theory of elasticity under assumption that the bilayer in raft is thicker than in the surrounding membrane. The calculations took into account the possibility of lateral shift of the domain boundaries located in different monolayers of the membrane. The line tension was associated with the energy of elastic deformations appearing in the vicinity of the boundary in order to compensate for the difference in the thickness of the monolayers. Spatial distribution of deformations and the line tension was calculated by minimization of elastic free energy of the system. Dependence of the line tension on the distance between the domains boundaries located in different monolayers was obtained. It was shown that the line tension is minimal if the distance is about 4 nm. Thus, membrane deformations stabilize the bilayer structure of rafts observed experimentally. The calculated value of line tension is about 0.6 pN for the difference between the monolayer thickness of raft and surrounding membrane of about 0.5 nm, which is in agreement with the experimental data available.     

HDLs induce raft domain vanishing in heterogeneous giant vesicles

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.     

Line tension and structure of raft boundary calculated from bending,tilt, and lateral compression/stretching

Bilayer thickness in membrane domains enriched with sphingomielin and cholesterol (known as “rafts”) is bigger than thickness of neighboring membrane. Monolayers need to deform to compensate the thicknesses difference in the vicinity of the raft boundary. Line tension of the boundary of rafts associated with elastic deformations originating from the compensation of the thickness mismatch is calculated in the frame-work of the elasticity theory. In the calculations deformations of splay, tilt and lateral stretching/compression are considered. It is assumed that raft consists of two monolayer domains situated in the different membrane monolayers; it is also assumed that the boundaries of domains can shift in the lateral direction with respect to relative to each other. Dependence of the boundary energy of raft on the value of the relative shift of the boundaries is calculated. It is shown that the boundary energy is minimal when shift is equal to 4.5 nm. Dependence of the optimal shift on the mismatch of the monolayer thicknesses of raft and surrounding membrane as well as membrane shape in the vicinity of boundary are calculated. The calculated values of line tension are in a good agreement with available experimental data. Taking into account deformation of stretching/compression increases the accuracy of calculations by 30%; this exceeds the uncertainty of the line tension measurements by modern techniques.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.     

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld)?+?liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). On giant unilamellar vesicles (GUVs) display a nanoscopic-to-macroscopic transition of Ld?+?Lo phase domains as POPC is replaced by DOPC, and this transition also depends on the cholesterol fraction. Line tension decreases with increasing cholesterol mole fractions in both lipid mixtures. For the ternary BSM/DOPC/Chol mixture, the published phase diagram [19] requires a modification to show that when cholesterol mole fraction is >~0.33, coexisting phase domains become nanoscopic.     

Thermally induced proliferation of pores in a model fluid membrane.

The growth of thermally induced pores in a two-dimensional model fluid membrane is investigated by Monte Carlo simulation. Holes appear in the membrane via an activated process, and their subsequent growth is controlled by an edge energy per unit length or line tension. The barrier height and line tension, together with a lateral tension, are the independent parameters of the model. In the resulting phase diagram, a rupture transition separates an intact membrane from a disintegrated state. The approach to the ruptured state shows distinct regimes. Reducing the barrier height at large line tension produces multiple, quasi-independent, small holes whose behavior is dominated by their edge energy, whereas at lower line tensions shape fluctuations of the holes facilitate their coalescence into a single large hole. At a small value of line tension and large barrier height, a single hole spontaneously permeabilizes the membrane in an entropically driven phase transition. Entropy dominates pore growth for line tensions not far below those measured for artificial vesicles. Permeabilization of lipid bilayers by certain peptides involves perturbing lipid-lipid cohesive energies, and our simulations show that at small line tensions the entropy of hole shape fluctuations destroys the model membrane's stability.     

Effects of integrin-mediated cell adhesion on plasma membrane lipid raft components and signaling

Anchorage dependence of cell growth, which is mediated by multiple integrin-regulated signaling pathways, is a key defense against cancer metastasis. Detachment of cells from the extracellular matrix triggers caveolin-1-dependent internalization of lipid raft components, which mediates suppression of Rho GTPases, Erk, and phosphatidylinositol 3-kinase in suspended cells. Elevation of cyclic adenosine monophosphate (cAMP) following cell detachment is also implicated in termination of growth signaling in suspended cells. Studies of integrins and lipid rafts, however, examined mainly ganglioside GM1 and glycosylphosphatidylinositol-linked proteins as lipid raft markers. In this study, we examine a wider range of lipid raft components. Whereas many raft components internalized with GM1 following cell detachment, flotillin2, connexin43, and Gα(s) remained in the plasma membrane. Loss of cell adhesion caused movement of many components from the lipid raft to the nonraft fractions on sucrose gradients, although flotillin2, connexin43, and H-Ras were resistant. Gα(s) lost its raft association, concomitant with cAMP production. Modification of the lipid tail of Gα(s) to increase its association with ordered domains blocked the detachment-induced increase in cAMP. These data define the effects of that integrin-mediated adhesion on the localization and behavior of a variety of lipid raft components and reveal the mechanism of the previously described elevation of cAMP after cell detachment.     

Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.     

Lipid rafts reconstituted in model membranes

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.     

Limited cholesterol depletion causes aggregation of plasma membrane lipid rafts inducing T cell activation

Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10–20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca²⁺. Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD–cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.     

Acyl chain unsaturation in PEs modulates phase separation from lipid raft molecules

By one hypothesis, phospholipids containing unsaturated fatty acids may be involved in phase separation from the lipid raft molecules sphingomyelin (SM) and cholesterol (CHOL). We tested the effect of increasing the number of double bonds in the acyl chains of phosphatidylethanolamines (PEs) on phase separation from SM/CHOL. The detergent extraction method was employed on various homoacid and heteroacid PEs in mixed vesicles composed of PE/SM/CHOL (1:1:1mol). The disaturated homoacid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0-16:0PE) showed the least solubility upon detergent extraction whereas maximal solubility was observed for the polyunsaturated homoacid 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6-22:6PE). Increasing the number of double bonds in the sn-2 position of heteroacid saturated-unsaturated PEs resulted in an increase in detergent solubility, which correlated with a general decrease in the gel-to-liquid crystalline phase transition temperature of the PEs. Our findings demonstrate that increasing unsaturation in PEs results in increased phase separation from SM/CHOL membranes, which may have implications for cellular signaling.