Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C₁₂E₈) at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C₁₂E₈ DRMs prepared at 4°C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C₁₂E₈ DRMs. However, contrary to the 4°C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C₁₂E₈ treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C₁₂E₈ to the study of DRMs.     

Membrane raft actin deficiency and altered Ca2+-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca2+-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.     

Membrane raft actin deficiency and altered Ca-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to < 10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca²⁺-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.     

Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid

Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.     

Transport mechanisms in Plasmodium-infected erythrocytes: lipid rafts and a tubovesicular network

The mature human erythrocyte is a simple cell that is devoid of intracellular organelles and does not show endocytic or phagocytic activity at the plasma membrane. However, following infection by Plasmodium, the erythrocyte undergoes several morphological and functional changes. Parasite-derived proteins are exported into the erythrocyte cytoplasm and to the membrane, while several proteins are localised to the parasitophorous vacuolar membrane and to the tubovesicular membranous network structures surrounding the parasite. Recent evidence indicates that multiple host proteins, independent of the type of their membrane anchor, that exist in detergent-resistant membrane (DRM) rafts or microdomains enter this apicomplexan vacuole. The internalised host components along with the parasite-encoded transmembrane protein PfEXP1 can be detected as DRM rafts in the vacuole. It appears that in Plasmodium-infected erythrocytes lipid rafts may play a role in endovacuolation and macromolecular transport.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

Resistance of Human Erythrocyte Membranes to Triton X-100 and C<Subscript>12</Subscript>E<Subscript>8</Subscript>

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4°C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C₁₂E₈. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C₁₂E₈, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C₁₂E₈ in comparison to intact cells, while the difference in the S values between Triton X-100 and C₁₂E₈ DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C₁₂E₈ detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.     

Lipid rafts and malaria parasite infection of erythrocytes

Infection of human erythrocytes by the malarial parasite, Plasmodium falciparum, results in complex membrane sorting and signaling events in the mature erythrocyte. These events appear to rely heavily on proteins resident in erythrocyte lipid rafts. Over the past five years, we and others have undertaken a comprehensive characterization of major proteins present in erythrocyte detergent-resistant membrane lipid rafts and determined which of these proteins traffic to the host-derived membrane that bounds the intraerythrocytic parasite. The data suggest that raft association is necessary but not sufficient for vacuolar recruitment, and that there is likely a mechanism of active uptake of a subset of erythrocyte detergent-resistant membrane proteins. Of the ten internalized proteins, few have been evaluated for a role in malarial entry. The beta(2)-adrenergic receptor and heterotrimeric G protein G(s) signaling pathway proteins regulate invasion. The implications of these differences are discussed. In addition, the latter finding indicates that erythrocytes possess important signaling pathways. These signaling cascades may have important influences on in vivo malarial infection, as well as on erythrocyte membrane flexibility and adhesiveness in sickle cell anemia. With respect to malarial infection, host signaling components alone are not sufficient to induce formation of the malarial vacuole. Parasite proteins are likely to have a major role in making the intraerythrocytic environment conducive for vacuole formation. Such interactions should be the focus of future efforts to understand malarial infection of erythrocytes since host- and parasite-targeted interventions are urgently needed to combat this terrible disease.     

Identification of a stomatin orthologue in vacuoles induced in human erythrocytes by malaria parasites. A role for microbial raft proteins in apicomplexan vacuole biogenesis

When the human malaria parasite Plasmodium falciparum infects erythrocytes, proteins associated with host-derived detergent-resistant membrane (DRM) rafts are selectively recruited into the newly formed vacuole, but parasite proteins that contribute to raft-based vacuole development are unknown. In mammalian cells, DRM-associated integral membrane proteins such as caveolin-1 and flotillin-1 that form oligomers have been linked to the formation of DRM-based invaginations called caveolae. Here we show that the P. falciparum genome does not encode caveolins or flotillins but does contain an orthologue of human band 7 stomatin, a protein known to oligomerize, associate with non-caveolar DRMs and is distantly related to flotillins. Stomatins are members of a large protein family conserved in evolution and P. falciparum (Pf) stomatin appears to be a prokaryotic-like molecule. Evidence is presented that it associates with DRMs and may oligomerize, suggesting that these features are conserved in the stomatin family. Further, Pfstomatin is an integral membrane protein concentrated at the apical end of extracellular parasites, where it co-localizes with invasion-associated rhoptry organelles. A resident rhoptry protein, RhopH2 also resides in DRMs. This provides the first evidence that rhoptries of an apicomplexan parasite contain DRM rafts. Further, when the parasite invades erythrocytes, rhoptry Pfstomatin and RhopH2 are inserted into the newly formed vacuole. Thus, like caveolin-1 and flotillin-1, a stomatin may also associate with non-clathrin coated, DRM-enriched vacuoles. We propose a new model of invasion and vacuole formation involving DRM-based interactions of both host and parasite molecules.     

Evidence for a physiological role for membrane rafts in human platelets.

We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (diI-C(18)) that preferentially partitions into gel-like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30 degrees C in intact platelets, which we have assigned as the liquid-ordered to the liquid-disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin-enriched rafts could be observed as two phase transitions at around 15 and 30 degrees C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent-resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi-functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI-linked protein CD55 and the major platelet integrin alpha(IIb)beta(3a) did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation.     

Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage

The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC-MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids-containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression.     

Isolation of nano-meso scale detergent resistant membrane that has properties expected of lipid 'rafts'

This review assesses problems that confound attempts to isolate 'raft' domains from cell membranes, focusing in particular upon the isolation of detergent resistant membrane (DRM). Despite its widespread use, this technique is rightly viewed with skepticism by many membrane biochemists and biophysics for reasons that include the inability to isolate DRMs at 37°C, the temperature at which their lipids are supposed to be ordered and so exclude detergents. If solubilization is done in an ionic buffer that preserves the lamellar phase of the metastable inner leaflet lipids, DRMs can readily be isolated at 37°C, and these have many properties expected of lipid rafts. However, to date these DRMs have remained somewhat larger than current concepts of rafts. We describe an adaptation of this method that purifies nano-meso scale DRMs, and could be a significant step towards purifying the membrane of individual 'rafts'.     

Delivery of the malaria virulence protein PfEMP1 to the erythrocyte surface requires cholesterol-rich domains

The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or "rafts" have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-beta-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d=6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 degrees C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 degrees C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

NGF causes TrkA to specifically attract microtubules to lipid rafts

Membrane protein sorting is mediated by interactions between proteins and lipids. One mechanism that contributes to sorting involves patches of lipids, termed lipid rafts, which are different from their surroundings in lipid and protein composition. Although the nerve growth factor (NGF) receptors, TrkA and p75(NTR) collaborate with each other at the plasma membrane to bind NGF, these two receptors are endocytosed separately and activate different cellular responses. We hypothesized that receptor localization in membrane rafts may play a role in endocytic sorting. TrkA and p75(NTR) both reside in detergent-resistant membranes (DRMs), yet they responded differently to a variety of conditions. The ganglioside, GM1, caused increased association of NGF, TrkA, and microtubules with DRMs, but a decrease in p75(NTR). When microtubules were induced to polymerize and attach to DRMs by in vitro reactions, TrkA, but not p75(NTR), was bound to microtubules in DRMs and in a detergent-resistant endosomal fraction. NGF enhanced the interaction between TrkA and microtubules in DRMs, yet tyrosine phosphorylated TrkA was entirely absent in DRMs under conditions where activated TrkA was detected in detergent-sensitive membranes and endosomes. These data indicate that TrkA and p75(NTR) partition into membrane rafts by different mechanisms, and that the fraction of TrkA that associates with DRMs is internalized but does not directly form signaling endosomes. Rather, by attracting microtubules to lipid rafts, TrkA may mediate other processes such as axon guidance.     

Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells. methyl-beta-cyclodextrin does not affect cell surface transport of a GPI-anchored protein

Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergent-resistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-beta-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-beta-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties.     

FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires plasma membrane rafts

We previously reported that lipid rafts are involved in long-chain fatty acid (LCFA) uptake in 3T3-L1 adipocytes. The present data show that LCFA uptake does not depend on caveolae endocytosis because expression of a dominant negative mutant of dynamin had no effect on uptake of [3H]oleic acid, whereas it effectively prevented endocytosis of cholera toxin. Isolation of detergent-resistant membranes (DRMs) from 3T3-L1 cell homogenates revealed that FAT/CD36 was expressed in both DRMs and detergent-soluble membranes (DSMs), whereas FATP1 and FATP4 were present only in DSMs but not DRMs. Disruption of lipid rafts by cyclodextrin and specific inhibition of FAT/CD36 by sulfo-N-succinimidyl oleate (SSO) significantly decreased uptake of [3H]oleic acid, but simultaneous treatment had no additional or synergistic effects, suggesting that both treatments target the same mechanism. Indeed, subcellular fractionation demonstrated that plasma membrane fatty acid translocase (FAT/CD36) is exclusively located in lipid rafts, whereas intracellular FAT/CD36 cofractionated with DSMs. Binding assays confirmed that [3H]SSO predominantly binds to FAT/CD36 within plasma membrane DRMs. In conclusion, our data strongly suggest that FAT/CD36 mediates raft-dependent LCFA uptake. Plasma membrane lipid rafts might control LCFA uptake by regulating surface availability of FAT/CD36.     

Active gamma-secretase is localized to detergent-resistant membranes in human brain

Several lines of evidence suggest that polymerization of the amyloid beta-peptide (Abeta) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Abeta is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by beta-secretase and gamma-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. gamma-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that gamma-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied gamma-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in gamma-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the gamma-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the gamma-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in gamma-secretase activity, suggesting that DRMs from fresh rat brain may be useful for gamma-secretase activity studies. Finally, confocal microscopy showed co-localization of gamma-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active gamma-secretase complex is localized to lipid rafts in human brain.     

Characteristics of morphological differences of detergent-resistant membrane domains isolated from different cells and investigated by atomic force microscopy

Planar rafts and caveolae are specific membrane clusters that contain high concentrations of cholesterol and lipids consisting of saturated fatty acids. These clusters are resistant to detergents and are known as “detergent-resistant membrane domains” (DRMs). Their morphology and size were studied by atomic force microscopy (AFM). Planar rafts extracted by Lubrol WX from monocytes of healthy donors are 150.6 ± 68.6 nm in diameter and 5.7 ± 2.9 nm in height, while caveolae are 87.3 ± 46.1 nm in diameter and 9.4 ± 5.4 nm in height. Significant differences in size and morphology were found between DRMs isolated from monocytes of healthy donors and patients with myocardial infarction, as well as between DRMs of monocytes and endothelial cells. The morphology dynamics of the isolated planar rafts and caveolae indicates that they quickly aggregate during storage; therefore, in order to assess the actual DRM size and morphology it is necessary to investigate them immediately after isolation.     

Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions

Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.