The mystery of membrane organization: composition, regulation and roles of lipid rafts

Abstract

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.

Figure 1. General overview of lateral heterogeneity in the plasma membrane

a | Lipid raft domains are small, highly dynamic and transient plasma membrane entities enriched in saturated phosho-, sphingo- and glycolipids, cholesterol, lipidated proteins and glycosylphosphatidylinositol (GPI)-anchored proteins. Enrichment in these hydrophobic components endows lipid rafts with distinct physical properties, including increased lipid packing and order, and decreased fluidity. In addition to membrane components, cortical actin plays an active role in domain maintenance and remodelling. Further, membrane lipids are asymmetrically distributed in the inner and outer leaflets, and this may further impact membrane organization. b | It is likely that membrane organization is not binary (i.e. highly specified raft and non-raft regions), but rather consists of various raft-like and non-raft domains with distinct compositions and properties.

Figure 2. Tools to study membrane domain organization, composition and function

a | In principle, membrane domains can be pure lipid clusters, but in most physiologically-relevant cases, also involve proteins, including glycosylphosphatidylinositol (GPI)-anchored protein clusters or clusters of Ras proteins. These domains can be purely lipid-driven entities, such as domains established through liquid–liquid phase separation in model membranes. They can also be induced by clustering agents, such as cholera toxin which binds to monosialotetrahexosylganglioside (GM1) or by antibodies recognizing surface receptors b | Tools that are commonly used to investigate membrane domains. These include various model membranes (such as synthetic giant unilamellar vesicles (GUVs) and cell-derived giant plasma membrane vesicles (GPMVs)); detergent resistance assays, wherein raft-like membrane regions persist as detergent-resistant membranes (DRMs), whereas non-raft components are fully solubilized; single molecule imaging to evaluate the diffusion of membrane molecules; fluorescence spectroscopy (such as Förster resonance energy transfer (FRET)), and mass spectrometry. c | Also various probes can be used to study raft domains. Domain selective probes partition selectively to one of the domains while domain sensitive probes partition to both domains and change their photophysical behaviour (for instance absorbance and emission spectra) depending on the nature of the surrounding lipid environment. d | Treatments that interfere with cholesterol or sphingolipid levels have been used to disturb rafts in cells and can shed light on the cellular functions of these domains.

Figure 3. Area coverage of membrane domain and domain size

a | Models of membranes with varying raft coverage. Total raft coverage in a given membrane may vary broadly, ranging from small isolated domains to percolating raft phases of increasing size. The specific organizational state depends on a variety of factors, including cell type, specific cellular conditions (e.g. cell cycle), and/or the identity of the membrane (e.g. plasma membrane versus intracellular membranes). b | Another mode of modulation of membrane organization can occur without changing overall raft abundance. For example, the size and/or lifetime of individual domains may be influenced by cellular processes such as endo- and exo-cytosis, lipid metabolism, etc. In addition, binding of clustering agents (antibodies and toxins) to their receptors can promote the formation of large membrane domains.

Figure 4. Regulation of membrane domains

a | Lipid–lipid interactions, in particular interactions between cholesterol and sphingolipids (but also between other relatively saturated lipids) are the defining feature of lipid-driven ordered domain formation. The preferential interaction between sphingolipids and sterols is due to the saturation of sphingolipid hydrophobic tails, but also hydrogen bonding between these lipid species. The amide of the sphingolipid backbone can both donate and accept a hydrogen bond, and these hydrogen bonds are within the interfacial region of the membrane, where the relative paucity of water increases the relative stability of these bonds. b | Some proteins harbour lipid binding domains, interacting with cholesterol or sphingolipids, and these lipid–protein interactions may determine the affinity of proteins for ordered lipid domains. c | Lipidated proteins, modified by the attachment of a saturated acyl chain (such as palmitoyl moieties), are recruited to raft domains, but may also nucleate and recruit membrane domains if they are intergrated into a relatively static protein scaffold. d | Hydrophobic interactions can contribute to membrane domain organization and composition. In particular, proteins possessing transmembrane domains (TMD) of different lengths prefer different lipid environments to protect their hydrophobic TMDs from exposure to the aqueous surrounding. For example, proteins with long TMDs were found to associate with domains harbouring long chain saturated lipids (top). When there is a mismatch between the length of the TMD and the local lipid environment in which the protein resides, protein–protein interaction might be favoured instead, leading to local protein concentration (bottom). e | The immobilization of inner leaflet lipids containing long saturated acyl chains by actin clusters results in the engagement of long acyl chain containing lipid-anchored proteins (such as glycosylphosphatidylinositol (GPI)-anchored proteins) located in the outer leaflet, which, in the presence of cholesterol, induces their active clustering. This results in locally ordered, transbilayer nanodomains, which are dynamic owing to the dynamics of actin clusters, and may form even in conditions that do not favour liquid–liquid phase separation of lipids or other supporting interactions.

Figure 5. Cellular functions of lipid rafts

a | Mechanisms by which membrane domains can potentially regulate bioactivity of their associated components. Rafts can concentrate certain molecules resulting in the establishment of functional catalytic platforms. For example, enzyme and substrates can be brought together to increase their encounter probability and thereby trigger reactions (e.g. signal transduction). A related possibility is that distinct physicochemical environments provided by lipid rafts directly impact protein conformation, thereby regulating bioactivity. b | Examples of physiological functions of membrane domains. Kinases of the SRC family are enriched in raft domains owing to their palmitoylation, whereas transmembrane phosphatases are generally excluded. This segregation has been found to be important for immune signalling, where raft associated SRC kinases are involved in regulating the phosphorylation state, and hence the signal transduction activity, of various immune receptors (including the T-cell receptor and the IgE receptor) (left). Many pathogens and their products (such as bacterial toxins) selectively bind membrane rafts owing to the presence of their specific receptors, such as glycoshpingolipids (GSLs; for the cholera toxin) or CD4 (for the human immunodeficiency virus (HIV)) in these domains, thereby gaining access to their host cells. Virus budding is also thought to occur preferentially at raft-like domains. Although the mechanism behind this selective budding is not yet clear, viral proteins such as Gag proteins of HIV are believed to be sensitive to membrane fluidity, and to associate with cholesterol-enriched domains.