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Partial Retraction of: The EMBO Journal (2010) 29: 3607-3620. DOI: 10.1038/emboj.2010.237 | Published online 24 September 2010 Journal statement The journal contacted the authors in February 2022 about potential image insertions and duplications in Fig 4A and 4E. In the absence of source data, the authors are retracting Fig 4A, the lower panel of Fig 4E (LAMP1 immunoblot), and the following statements in the text that rely on these data: "Quantitative analysis showed that the percentage of Flotillin-1 associated with DRMs was increased in LSD endolysosomal membranes (Figure 4A), indicating an increased amount of cholesterol-enriched regions in these membrane samples." "LAMP1 also displayed a similar distribution profile in WT and LSD cells (Figure 4E)". Author statement The authors could not verify the aberrations in panel A of Fig 4 and the lower immunoblot (LAMP1) of 4E because the original source data are no longer available (12 years after publication, which is beyond the institute's 10-year data retention policy). The authors wish to clarify that the main conclusions of the paper are not affected by the retraction of Figure panels 4A and 4E for the following reasons: Figure panel 4A supports the observation that there are increased cholesterol-enhanced regions in LSD samples. This finding is also supported by data provided in figs 4B, 4C and 4D. Figure panel 4E: The LAMP1 blot in Fig 4E shows that the distribution of protein normally excluded from DRMs is not altered between Wt and LSD samples. This result is also supported by the upper blot in this panel (Transferrin receptor). The authors apologize for these errors and agree with this corrigendum; no response could be obtained from AL.
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The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.
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Bioquímica/métodos , Membrana Celular/química , Microdomínios da Membrana/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Corantes Fluorescentes/química , Lauratos/química , Organelas/química , Transição de Fase , Espectrometria de Fluorescência , TemperaturaRESUMO
The conservation of fluidity is a theme common to all cell membranes. In this study, an analysis of lipid packing was conducted via C-laurdan spectroscopy of cell surface membranes prepared from representative species of Bacteria and Eukarya. We found that despite their radical differences in composition (namely the presence and absence of membrane-rigidifying sterol) the membrane order of all taxa converges on a remarkably similar level. To understand how this similarity is constructed, we reconstituted membranes with either bacterial or eukaryotic components. We found that transmembrane segments of proteins have an important role in buffering lipid-mediated packing. This buffering ensures that sterol-free and sterol-containing membranes exhibit similar barrier properties.
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Bactérias/citologia , Membrana Celular/química , Eucariotos/citologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , RatosRESUMO
Theoretical studies predict hydrophobic matching between transmembrane domains of proteins and bilayer lipids to be a physical mechanism by which membranes laterally self-organize. We now experimentally study the direct consequences of mismatching of transmembrane peptides of different length with bilayers of different thicknesses at the molecular level. In both model membranes and simulations we show that cholesterol critically constrains structural adaptations at the peptide-lipid interface under mismatch. These constraints translate into a sorting potential and lead to selective lateral segregation of peptides and lipids according to their hydrophobic length.
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Colesterol/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Simulação de Dinâmica MolecularRESUMO
Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are multilamellar, lipid-rich membranes produced by oligodendrocytes in the central nervous system. To act as an insulator, myelin has to form a stable and firm membrane structure. In this study, we have analyzed the biophysical properties of myelin membranes prepared from wild-type mice and from mouse mutants that are unable to form stable myelin. Using C-Laurdan and fluorescence correlation spectroscopy, we find that lipids are tightly organized and highly ordered in myelin isolated from wild-type mice, but not from shiverer and ceramide synthase 2 null mice. Furthermore, only myelin lipids from wild-type mice laterally segregate into physically distinct lipid phases in giant unilamellar vesicles in a process that requires very long chain glycosphingolipids. Taken together, our findings suggest that oligodendrocytes exploit the potential of lipids to self-segregate to generate a highly ordered membrane for electrical insulation of axons.
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Lipídeos de Membrana/metabolismo , Modelos Biológicos , Bainha de Mielina/metabolismo , Animais , Difusão , Ácidos Graxos/análise , Lipídeos de Membrana/química , Membranas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes Neurológicos , Esfingolipídeos/metabolismo , Extratos de TecidosRESUMO
The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol-enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.
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Colesterol/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Fusão de Membrana/fisiologia , Proteínas SNARE/metabolismo , Animais , Autofagia , Western Blotting , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endocitose/fisiologia , Receptores ErbB/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Técnicas Imunoenzimáticas , Imunoprecipitação , Doenças por Armazenamento dos Lisossomos/patologia , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Camundongos , Fosfolipídeos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The lipid raft concept proposes that biological membranes have the potential to form functional domains based on a selective interaction between sphingolipids and sterols. These domains seem to be involved in signal transduction and vesicular sorting of proteins and lipids. Although there is biochemical evidence for lipid raft-dependent protein and lipid sorting in the yeast Saccharomyces cerevisiae, direct evidence for an interaction between yeast sphingolipids and the yeast sterol ergosterol, resulting in membrane domain formation, is lacking. Here we show that model membranes formed from yeast total lipid extracts possess an inherent self-organization potential resulting in liquid-disordered-liquid-ordered phase coexistence at physiologically relevant temperature. Analyses of lipid extracts from mutants defective in sphingolipid metabolism as well as reconstitution of purified yeast lipids in model membranes of defined composition suggest that membrane domain formation depends on specific interactions between yeast sphingolipids and ergosterol. Taken together, these results provide a mechanistic explanation for lipid raft-dependent lipid and protein sorting in yeast.
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Ergosterol/química , Microdomínios da Membrana/química , Membranas Artificiais , Saccharomyces cerevisiae/química , Esfingolipídeos/química , Ergosterol/biossíntese , Ergosterol/genética , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/biossíntese , Esfingolipídeos/genéticaRESUMO
Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165-3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective.
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Membrana Celular/química , Lipídeos/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , 2-Naftilamina/metabolismo , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Lauratos/química , Lauratos/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Modelos Moleculares , Modelos Teóricos , Espectrometria de FluorescênciaRESUMO
Biological membranes are not structurally passive solvents of amphipathic proteins and lipids. Rather, it appears their constituents have evolved intrinsic characteristics that make homogeneous distribution of components unlikely. As a case in point, the concept of lipid rafts has received considerable attention from biologists and biophysicists since the formalization of the hypothesis more than 10 years ago. Today, it is clear that sphingolipid and cholesterol can self-associate into micron-scaled phases in model membranes and that these lipids are involved in the formation of highly dynamic nanoscale heterogeneity in the plasma membrane of living cells. However, it remains unclear whether these entities are manifestations of the same principle. A powerful means by which the molecular organization of rafts can be assessed is through analysis of their functionalized condition. Raft heterogeneity can be activated to coalesce and laterally reorganize/stabilize bioactivity in cell membranes. Evaluation of this property suggests that functional raft heterogeneity arises through principles of lipid-driven phase segregation coupled to additional chemical specificities, probably involving proteins.
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Membrana Celular , Microdomínios da Membrana , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Detergentes/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Esfingolipídeos/química , Esfingolipídeos/metabolismoRESUMO
The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.