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.

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.generalized polarization value ͉ giant unilamellar vesicle ͉ membrane organization ͉ lipid sorting ͉ lipid raft T he lipid raft hypothesis postulates that selective interactions among sphingolipids, cholesterol, and membrane proteins contribute to lateral membrane heterogeneity (1). A tenet of the model is that small, dynamic cholesterol-sphingolipid-enriched assemblies can be induced to coalesce into larger, more stable structures through clustering of domain components (2). Although experimental data support cholesterol-dependent nano-scale membrane heterogeneity (3-8) and selective domain formation upon raft cross-linking (9-12), the mechanisms that govern such associations in cell membranes remain unclear.On the molecular level, a key feature that is thought to contribute to raft assembly is the propensity of cholesterol to pack tightly with saturated acyl chains of lipids causing them to adopt an extended conformation (13,14). In multi-component model membranes (n Ͼ 2), this interaction can lead to microscopically separate fluid membrane phases: the liquid-ordered (Lo) phase, enriched in saturated (sphingo-)lipids and cholesterol in a highly condensed state, and the liquid-disordered (Ld) phase, enriched in unsaturated glycerophospholipid in a disordered state (15)(16)(17).Several features of the Lo phase in model membranes correspond to the predicted properties of lipid rafts in cel...

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.

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.The membranes that surround the various organelles of eukaryotic cells have distinct lipid compositions. For example, the concentration of sphingolipids and sterols increases along the secretory pathway, being lowest in the endoplasmic reticulum and highest at the plasma membrane (1-3). The major sorting station for vesicular transport of proteins and lipids within the cell is the trans-Golgi network (4). Here, clusters of sphingolipids and sterols as well as proteins have been proposed to be involved in the formation of secretory vesicles (SVs) 3 (5, 6). These clusters, called lipid rafts, were proposed to form by the preferential interaction between lipids containing saturated acyl chains, especially (glyco-) sphingolipids and sterols, and by intermolecular hydrogen bonds between (glyco-) sphingolipids. As compared with bulk cellular membranes, lipid rafts are characterized by a higher acyl chain order and tight packing of lipids (7). Protein-free model membranes have been widely used to study the self-associative properties of sphingolipids and sterols, which are believed to be responsible for lipid raft formation in vivo (8,9). In model membranes with a lipid composition similar to that of detergent-resistant membranes (DRMs) from mammalian cells, the preferential interaction between sphingolipids and sterols is manifested as the coexistence of two fluid membrane phases, which can be observed microscopically in giant unilamellar vesicles (GUVs) (10 -13). More specifically, model membranes produced from equimolar mixtures of sphingomyelin (SM), phosphatidylcholine (PC), and cholesterol show domains in the liquid-disordered (Ld) state that are enriched in PC coexisting with a liquid-ordered (Lo) phase rich in SM and cholesterol, the latter being a defining component of the Lo phase (14, 15). The Lo phase is characterized by a higher acyl chain (...

Background: Living cells maintain a fluid membrane at their surface. Results: Bacteria and eukaryotes display comparable surface order. Transmembrane proteins order cell membranes in the absence of sterol (Bacteria) and disorder in its presence (Eukarya). Conclusion: Bidirectional ordering may provide a means to achieve similar barrier properties despite compositional differences. Significance: Nature may use different protein/lipid combinations to standardize cell surface order.