Lipid rafts have different sizes depending on membrane composition: a time-resolved fluorescence resonance energy transfer study

The ternary lipid system palmitoylsphingomyelin (PSM)/palmitoyloleoylphosphatidylcholine (POPC)/cholesterol is a model for lipid rafts. Previously the phase diagram for that mixture was obtained, establishing the composition and boundaries for lipid rafts. In the present work, this system is further studied in order to characterize the size of the rafts. For this purpose, a time-resolved fluorescence resonance energy transfer (FRET) methodology, previously applied with success to a well-characterized phosphatidylcholine/cholesterol binary system, is used. It is concluded that: (1) the rafts on the low raft fraction of the raft region are small (below 20 nm), whereas on the other side the domains are larger; (2) on the large domain region, the domains reach larger sizes in the ternary system (> approximately 75-100 nm) than in binary systems phosphatidylcholine/cholesterol (between approximately 20 and approximately 75-100 nm); (3) the raft marker ganglioside G(M1) in small amounts (and excess cholera toxin subunit B) does not affect the general phase behaviour of the lipid system, but can increase the size of the rafts on the small to intermediate domain region. In summary, lipid-lipid interactions alone can originate lipid rafts on very different length scales. The conclusions presented here are consistent with the literature concerning both model systems and cell membrane studies.     

Proteomic analysis of plasma membrane lipid rafts of HL-60 cells

Neutrophils acquire phagocytic activity as they differentiate. Recently, plasma membrane lipid rafts have been shown to play important roles in the process of phagocytosis in neutrophils. To characterize the proteins involved in phagocytosis and to elucidate the process by which they acquire phagocytic activity, we investigated by nano-LC-MS/MS analysis the changes in protein composition of plasma membrane lipid rafts during DMSO-induced differentiation of the human leukemia cell line HL-60 cells into neutrophilic lineage. Based on the spectrum counts of 147 proteins identified, 25 proteins were upregulated and 49 were downregulated by DMSO treatment. CD11b/CD18 subunits of beta2-integrin Mac-1, CD35, and GPI-80, which are known to be upregulated during differentiation, were dominantly detected in the lipid rafts of DMSO-treated cells. Many known membrane proteins, G proteins, and cytoskeletal proteins were also detected and they showed characteristic distributions. Absolute quantification of nine proteins in the lipid rafts using internal standard peptides labeled with stable isotopes showed that the amount of protein almost corresponded to the results obtained by spectrum count. Identified proteins, expression of which was altered by DMSO treatment, are expected to be candidate proteins involved in differentiation and functions of neutrophils.     

Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts

Biological membranes are compartmentalized into microdomains that exhibit particular lipid and protein compositions. Membrane microdomains, such as tetraspanin-enriched microdomains and lipid rafts, have been suggested to play a role in a variety of physiological and pathological processes. Therefore, the characterization of the protein compositions of these microdomains, which is the focus of this review, appears to be a crucial step to better understanding their function. Proteomics has recently allowed the characterization of tetraspanin-enriched microdomains in colon cancer cells. This demonstrated the presence of different categories of membrane proteins and suggested a variation in the composition of tetraspanin-enriched microdomains during tumor progression. On the other hand, proteomics has permitted the identification of hundreds of proteins in lipid rafts of different origins. However, the diversity of methodologies in sample preparation and of strategies in protein identification led to a broad variability in the data obtained. These methodological issues are discussed. Moreover, proteomics has revealed that different sets of proteins were present in tetraspanin-enriched microdomains as compared with lipid rafts, strengthening the idea that these microdomains are distinct structures.     

Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane

A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol. Phospho- and glycoglycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane proteins were separated by one- and two-dimensional gel electrophoresis and identified by tandem mass spectrometry or use of specific antibody. The data clearly indicate that tobacco microdomains are able to recruit a specific set of the plasma membrane proteins and exclude others. We demonstrate the recruitment of the NADPH oxidase after elicitation by cryptogein and the presence of the small G protein NtRac5, a negative regulator of NADPH oxidase, in lipid rafts.     

P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.     

Contamination of nuclear fractions with plasma membrane lipid rafts

Subcellular fractionation is central to a range of cell biological, biochemical and proteomic studies. Purification of nuclear-enriched fractions is critical for studies on nuclear structure and function. Here we show that detergent-based nuclear isolation methods cause the redistribution of proteins associated with plasma membrane lipid rafts into nuclear fractions. The glycosyl-phosphatidylinositol (GPI)-anchored prion protein (PrP(C)) and a GPI-anchored construct of angiotensin converting enzyme (GPI-ACE), as well as the lipid raft markers flotillin-1 and -2, were present in the nuclear fractions derived using three different subcellular fractionation protocols. Incubation of intact cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves GPI-anchored proteins from the cell surface, significantly reduced the amount of PrP(C) and GPI-ACE in the nuclear fraction. Buoyant sucrose density gradient centrifugation in the presence of Triton X-100 of the nuclear fraction resulted in a significant proportion of the GPI-anchored proteins being recovered in the low density lipid raft fractions. These data indicate that the nuclear fraction isolated using such subcellular fractionation protocols is contaminated with components of plasma membrane lipid rafts and raises questions as to the integrity of the nuclear fraction isolated by such protocols for use in detailed cell biological studies and proteomics analysis.     

Gag induces the coalescence of clustered lipid rafts and tetraspanin-enriched microdomains at HIV-1 assembly sites on the plasma membrane

The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Förster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominately at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly.     

The tricyclic antidepressant imipramine induces autophagic cell death in U-87MG glioma cells

In this study, we investigated the antitumor effects of the tricyclic antidepressant 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1-amine (imipramine) on glioma cells. We found that exposure of U-87MG cells to imipramine resulted in the inhibition of PI3K/Akt/mTOR signaling, reduction of clonogenicity, and induction of cell death. Imipramine stimulated the formation of acidic vesicular organelles, the conversion of LC3-I to LC3-II, and the redistribution of LC3 to autophagosomes, suggesting that it stimulates the progression of autophagy. It did not, however, induce apoptosis. We further showed that knockdown of Beclin-1 using siRNA abrogated imipramine-induced cell death. These results suggest that imipramine exerts antitumor effects on PTEN-null U-87MG human glioma cells by inhibiting PI3K/Akt/mTOR signaling and by inducing autophagic cell death.     

Galactocerebroside mediated transmembrane Ca2+ signalling in U-87 MG cells: Possible involvement of phosphoinositides

Antibody to galactocerebroside (anti- GalC) has been shown to evoke a Ca²⁺ response in cultured glioma U- 87 MG cells. The rise in [Ca²⁺]_i was due to release of Ca²⁺ from the intracellular stores and influx through the plasma membrane. The rise in [Ca²⁺]_i was markedly inhibited by neomycin sulphate and phorbol dibutyrate suggesting the involvement of phosphoinositides in Ca²⁺ mobilization. The Ca²⁺ response induced by anti- GalC was rapidly desensitized and repeated addition of anti- GalC did not elevate the [Ca²⁺]_i. Heterologous desensitization was observed with bradykinin and adenosine triphosphate. The intracellular Ca²⁺ store mobilized by anti- GalC appears to be the IPin3 sensitive pool of endoplasmic reticulum. The influx of Ca²⁺ is mediated by a channel. The Ca²⁺ influx was also prevented by pretreatment of cells with neomycin sulphate or phorbol dibutyrate. We propose that galactocerebroside may be associated with phospholipase C or other proteins linked to the phosphoinositide pathway of transmembrane signalling and anti- GalC activates the breakdown of phosphoinositides and thus mobilizes Ca²⁺ in U-87 MG cells.     

Alkylphospholipids deregulate cholesterol metabolism and induce cell-cycle arrest and autophagy in U-87 MG glioblastoma cells

Glioblastoma is the most common malignant primary brain tumour in adults and one of the most lethal of all cancers. Growing evidence suggests that human tumours undergo abnormal lipid metabolism, characterised by an alteration in the mechanisms that regulate cholesterol homeostasis. We have investigated the effect that different antitumoural alkylphospholipids (APLs) exert upon cholesterol metabolism in the U-87 MG glioblastoma cell line. APLs altered cholesterol homeostasis by interfering with its transport from the plasma membrane to the endoplasmic reticulum (ER), thus hindering its esterification. At the same time they stimulated the synthesis of cholesterol from radiolabelled acetate and its internalisation from low-density lipoproteins (LDLs), inducing both 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and LDL receptor (LDLR) genes. Fluorescent microscopy revealed that these effects promoted the accumulation of intracellular cholesterol. Filipin staining demonstrated that this accumulation was not confined to the late endosome/lysosome (LE/LY) compartment since it did not colocalise with LAMP2 lysosomal marker. Furthermore, APLs inhibited cell growth, producing arrest at the G2/M phase. We also used transmission electron microscopy (TEM) to investigate ultrastructural alterations induced by APLs and found an abundant presence of autophagic vesicles and autolysosomes in treated cells, indicating the induction of autophagy. Thus our findings clearly demonstrate that antitumoural APLs interfere with the proliferation of the glioblastoma cell line via a complex mechanism involving cholesterol metabolism, cell-cycle arrest or autophagy. Knowledge of the interrelationship between these processes is fundamental to our understanding of tumoural response and may facilitate the development of novel therapeutics to improve treatment of glioblastoma and other types of cancer.     

Optical techniques for imaging membrane lipid microdomains in living cells

Lateral organisation of cellular membranes, particularly the plasma membrane, is of benefit to the cell as it allows complicated cellular processes to be regulated and efficient. For example, trafficking and secretion of molecules can be targeted and directed, cells polarised and signalling events modulated and propagated. The fluid mosaic model allows for significant heterogeneity on the part of the lipids themselves and of membrane associated proteins. By exploiting the tendency of complex lipid bilayers to undergo spontaneous or induced phase-separation into non-miscible domains, the cell could achieve this desired spatial organisation. While phase-separation is readily observed in simple, artificial bilayers, its occurrence in physiological membranes remains controversial. This stems mainly from our inability to image lipid microdomains directly - possibly due to their small size, short lifespan and/or morphological similarity to the bulk membrane. In this review, we seek to examine the techniques used to try to image membrane lipid microdomains, concentrating mainly on optical microscopy techniques that are applicable to live cells. We also look at novel emerging instruments and methods that promise to overcome our current technological limitations and shed new light on these important structures.     

Tea polyphenol epigallocatechin-3-gallate associates with plasma membrane lipid rafts: lipid rafts mediate anti-allergic action of the catechin

High-affinity IgE receptor FcepsilonRI is key molecule in the IgE-mediated allergic reactions. Epigallocatechin-3-gallate (EGCG) has a suppressive effect of the expression of the FcepsilonRI. We show here that EGCG highly associates with plasma membrane microdomains, lipid rafts. The disruption of these lipid rafts caused a reduction of the amount of raft-associated EGCG and the FcepsilonRI -suppressive effect of EGCG. These results suggest that the interaction between EGCG and the lipid rafts is important for EGCG's ability to downregulate FcepsilonRI expression.     

Role of plasma membrane lipid microdomains in respiratory syncytial virus filament formation

The fusion protein (F) of respiratory syncytial virus (RSV) is the envelope glycoprotein responsible for the characteristic cytopathology of syncytium formation. RSV has been shown to bud from selective areas of the plasma membrane as pleomorphic virions, including both filamentous and round particles. With immunofluorescent microscopy, we demonstrated evidence of RSV filaments incorporating the fusion protein F and colocalizing with a lipid microdomain-specific fluorescent dye, 1,1-dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate. Western blot analysis of Triton X-100 cold-extracted membrane fractions confirmed the presence of RSV proteins within the lipid microdomains. RSV proteins also colocalized with cellular proteins associated with lipid microdomains, caveolin-1, and CD44, as well as with RhoA, a small GTPase. ADP-ribosylation of RhoA by Clostridium botulinum exotoxin inactivated RhoA signaling and resulted in the absence of RSV-induced syncytia despite no significant change in viral titer. We demonstrated an overall decrease in both the number and length of the viral filaments and a shift in the localization of F to nonlipid microdomain regions of the membrane in the presence of C3 toxin. This suggests that the selective incorporation of RSV proteins into lipid microdomains during virus assembly may lead to critical interactions of F with cellular proteins, resulting in microvillus projections necessary for the formation of filamentous virus particles and syncytium formation. Thus, manipulation of membrane lipid microdomains may lead to alterations in the production of viral filaments and RSV pathogenesis and provide a new pharmacologic target for RSV therapy.     

Assembly of staphylococcal leukocidin into a pore-forming oligomer on detergent-resistant membrane microdomains, lipid rafts, in human polymorphonuclear leukocytes

Staphylococcal leukocidin (Luk) consists of LukS and LukF, which cooperatively lyse human polymorphonuclear leukocytes (HPMNLs), monocytes, and macrophages. Here we found that LukS and LukF assembles into hetero-oligomeric pore complexes on the detergent-resistant membrane microdomains, lipid rafts of HPMNLs. When HPMNLs were treated with LukS alone, 24% of the added LukS was localized in lipid rafts. Furthermore, in HPMNLs treated with both LukS and LukF simultaneously, about 90% of high molecular-mass complexes of 100 kDa, which consists of LukS and LukF, were detected in the lipid raft fractions. In contrast, in HPMNLs treated with LukF alone, LukF was not localized in lipid rafts despite binding to the target cell membranes. Ten mM methyl-beta-cyclodextrin, a dysfunctioning agent of lipid rafts, completely inhibited assembly of Luk on lipid rafts, and resulted in null leukocytolytic activity of Luk. Hence, we concluded that assembly of LukS and LukF into the pore-complex occurs in lipid rafts in HPMNLs and that LukF can bind to LukS, which had already bound to lipid rafts, to assemble into hetero-oligomers.     

The plasma membrane ganglioside sialidase cofractionates with markers of lipid rafts

Gangliosides of the plasma membrane are important modulators of cellular functions. Recent reports have shown their enrichment in glycosphingolipid-containing membrane microdomains, called glycosphingolipid-signaling domain or rafts, which can be isolated due to their insolubility in Triton X-100 and flotation through a sucrose gradient. In previous work on neuroblastoma cells we had found that a ganglioside-specific sialidase activity of the plasma membrane controlled proliferation and differentiation through selective ganglioside desialylation. Assuming the ganglioside sialidase to be close to its substrates in the membrane, we investigated its association with detergent-insoluble microdomains in the neuroblastoma cell line SK-N-MC. The results show that the ganglioside sialidase codistributes with the raft markers ganglioside GM1, flotillin, src family kinases, and glycosylphosphatidylinositol-anchored proteins in a fraction containing about 2% of cellular protein. The association of the ganglioside sialidase with glycosphingolipid-enriched membrane fractions therefore is in support of a role of this glycosidase in ganglioside-dependent signaling processes.     

Proteome profile changes that are differentially regulated by lipid and protein phosphatase activities of tumor suppressor PTEN in PTEN-expressing U-87 MG human glioblastoma cells

The phosphatase and tensin homolog tumor suppressor (PTEN) belongs to a class of "gatekeeper" tumor suppressors together with p53, retinoblastoma and adenomatous polyposis. It is considered one of the most important tumor suppressors in the post p53 era. Previously to identify the molecules involved in the signaling network regulated by PTEN using proteomic tools, we reported global proteome profiles at different time points using the PTEN inducible NIH3T3 cells (Kim, S.-y., Kim, Y. S., Bahk, Y. Y., Mol. Cells 2003, 15, 396-405). However, the system had a critical limitation that NIH3T3 cell has endogenous wild-type PTEN and, thus to be exact, the induced PTEN could not give the answer about the real physiological roles of this tumor suppressor. Here, to find out PTEN-related protein network we have established various PTEN (wild-type, an activity inert C124G, and a lipid phosphatase deficient G129E)-expressing cell clones in U-87 MG human glioblastoma cells lacking detectable PTEN as a result of genetic lesions. In this biological context, we compared their morphological and expression patterns, and proteome images of each PTEN-expressing cell clone by 2-DE followed by identification with MALDI-TOF MS. We obtained some pieces of evidence that morphological change by PTEN expression is mediated by its protein phosphatase activity and their growth rate by the lipid phosphatase activity. The proteomic approaches showed that 30 proteins possibly correlated with PTEN's protein phosphatase activity (13 down-regulated and 17 up-regulated) and 20 with the lipid phosphatase activity (14 down-regulated and 6 up-regulated) were identified. Taken together, we conclude that the comparative analysis of proteome from various PTEN-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly caused by individual phosphatase activities of PTEN in vivo.     

L-type calcium channels and cytochrome b5 reductase are components of protein complexes tightly associated with lipid rafts microdomains of the neuronal plasma membrane

The presence of cytosolic calcium microcompartments in neurons is well established. L-type voltage calcium channels play a leading role in the rise of cytosolic calcium in the neuronal soma and are sensitive to redox modulation. In a recent work [Samhan-Arias, A.K., García-Bereguiaín, M.A., Martín-Romero, F.J. and Gutiérrez-Merino, C. (2009) Mol. and Cell. Neurosci. 40, 14–26], we have shown that cytochrome b₅ reductase, whose deregulation leads to an overshot of superoxide anion production at the neuronal plasma membrane that triggers apoptosis in primary cultures of cerebellar granule neurons in culture, forms a large mesh of redox centres associated with lipid rafts in these neurons. In this work, we have implemented the use of fluorescent antibodies as reagents for quantitative Förster resonance energy transfer measurements and analysis using fluorescence microscopy images of cerebellar granule neurons in culture. The results of this study show that L-type voltage-operated calcium channels are also enriched in lipid rafts associated protein microdomains at a distance between 10 and 100 nm from cytochrome b₅ reductase. The methodological improvements done in this work can be also valuable for the study of proteins compartmentalization within other subcellular microdomains in any cell type in culture.     

Lipid rafts in epithelial brush borders: atypical membrane microdomains with specialized functions

Epithelial cells that fulfil high-throughput digestive/absorptive functions, such as small intestinal enterocytes and kidney proximal tubule cells, are endowed with a dense apical brush border. It has long been recognized that the microvillar surface of the brush border is organized in cholesterol/sphingolipid-enriched membrane microdomains commonly known as lipid rafts. More recent studies indicate that microvillar rafts, in particular those of enterocytes, have some unusual properties in comparison with rafts present on the surface of other cell types. Thus, microvillar rafts are stable rather than transient/dynamic, and their core components include glycolipids and the divalent lectin galectin-4, which together can be isolated as "superrafts", i.e., membrane microdomains resisting solubilization with Triton X-100 at physiological temperature. These glycolipid/lectin-based rafts serve as platforms for recruitment of GPI-linked and transmembrane digestive enzymes, most likely as an economizing effort to secure and prolong their digestive capability at the microvillar surface. However, in addition to microvilli, the brush border surface also consists of membrane invaginations between adjacent microvilli, which are the only part of the apical surface sterically accessible for membrane fusion/budding events. Many of these invaginations appear as pleiomorphic, deep apical tubules that extend up to 0.5-1 microm into the underlying terminal web region. Their sensitivity to methyl-beta-cyclodextrin suggests them to contain cholesterol-dependent lipid rafts of a different type from the glycolipid-based rafts at the microvillar surface. The brush border is thus an example of a complex membrane system that harbours at least two different types of lipid raft microdomains, each suited to fulfil specialized functions. This conclusion is in line with an emerging, more varied view of lipid rafts being pluripotent microdomains capable of adapting in size, shape, and content to specific cellular functions.