Lipid raft model

The raft hypothesis, as it was proposed by Simons and Ikonen (1997), claims that lipids and proteins are not distributed randomly within membranes, but that interactions between differ-ent lipids result in domain formation. Main constitudiffer-ents of raft domains are sphingolipids and cholesterol. The model proposes that sphingolipids associate laterally with one another and

1.2. Lipid rafts 35

are kept together by weak interactions between the carbohydrate heads of glycosphingolipids.

Cholesterol stabilises these interactions by filling the spaces within the hydrophobic plane of the membrane, which results from glycosphingolipid carbohydrate heads occupying a larger area in the plane than their mainly saturated fatty acids. These interactions result in an assembly with closely packed lipids, which “floats” in the membrane consisting of loosely packed glycero-phospholipids with mainly unsaturated, kinked fatty acids. The originally proposed function of lipid rafts was in the apical delivery of proteins in polarised epithelial cells. This function was thought to be fullfilled by specific inclusion or exclusion of proteins from these domains. Gly-cosylphosphatidylinositol (GPI)-anchored proteins, doubly acylated proteins of the Src-family as well as some transmembrane proteins were known to preferentially associate with lipid rafts.

Much of the raft hypothesis is based on studies with model membranes, which consist of lipid bilayers with known chemical composition. Depending on the lipid composition, membranes undergo phase transition at a specific temperature. At low temperatures the membrane is in a solid ordered (So), or gel phase, which is characterised by a highly ordered structure with acyl side chains in the straighttransconformation, tightly packed which results in a low lateral mobility of the lipids. As temperature increases, the ordered phase switches to a liquid dis-ordered (Ld) phase where acyl side chains are in the kinkedcis conformation and lipids have high lateral mobility. Sufficiently high cholesterol concentrations can promote a third phase, which is characterised by highly ordered acyl side chains resembling of the S^o phase, but also by a high lateral mobility of the lipids similar to that in the Ld phase. This phase is therefore designated liquid ordered (Lo) phase. While the existence of Ldphase and So phases depends on the temperature and both phases cannot co-exist, it has been shown for model membranes with appropriate mixtures of sphingomyelines, unsaturated phospholipids and cholesterol that Ldphases can co-exist with Lo phases. This enables domain formation driven by lipid-lipid in-teractions, as it is hypothesised for lipid rafts. Lipid rafts are postulated to be the cell membrane equivalent to the Lo phase of model membranes. But although the co-existence of different phases within model membranes is commonly accepted, it could not be shown unambiguously that this phase co-existence is also found in cell membranes (for reviews on model membranes refer to Simons and Vaz (2004); Edidin (2003b); London (2005)).

Another basis for the raft hypothesis was the finding that membranes, especially in the presence of cholesterol, are not completely solubilised by anionic detergents (mostly Triton-X-100) at 4^◦C. Due to their high lipid proportion, these detergent resistant membranes (DRM) float to

36 1. Introduction

low densities in linear sucrose gradients. Proteins may also be associated with DRMs (Brown and Rose, 1992). Those detergent insoluble membranes have been found to be enriched in gly-cosphingolipids and cholesterol (therefore an alternative name for DRM is detergent-insoluble glycolipid-enriched complexes (DIG)) (Simons and Ikonen, 1997), and extraction of cholesterol prior to solubilisation resulted in solubilsation of otherwise DRM-associated proteins (Edidin, 2003b). The lipid composition and the dependence on cholesterol for detergent resistance led to the assumption that DRMs are isolated lipid rafts. This conclusion has been a matter of debate (Lichtenberg et al., 2005; Munro, 2003). Nevertheless, DRM association of proteins is still a commonly used method to show lipid raft association of proteins.

Based on the raft concept a lot of experiments have been made investigating the detergent resis-tance of many proteins and thus a potential association of proteins with rafts. Functional assays determined how cellular processes are perturbed upon cholesterol depletion. Many studies set out to investigate lipid phase behaviour in model membranes. From all these investigations, the original raft model has been modified to a more sophisticated model.

Lipid rafts are now proposed to be highly dynamic assemblies of tightly packed sphingolipids and cholesterol within the exoplasmic leaflet of the membrane and are variable in size (Rajen-dran and Simons, 2005). Small rafts can cluster upon stimuli, thereby forming larger domains, bringing proteins formerly separated in different rafts in close contact and excluding non-raft proteins. Thus, they exert their function by compartmentalising the membrane into different domains (Pike, 2006). Selective interaction of several proteins is facilitated by inclusion or exclusion of proteins (Rajendran and Simons, 2005). In contrast to the original hypothesis the emphasis in current models is no longer on lipid-lipid interactions being the main forces keep-ing together lipid rafts, but that lipid-protein or even protein-protein interactions may be equally important forces (Hancock, 2006; Edidin, 2003b; Munro, 2003). Additionally, it has been hy-pothesised recently that rafts do not pre-exist in a stable form but that they form spontaneously and degrade in a steady state (Hancock, 2006).

The probably most recent definition of rafts was made at the “Keystone Symposium on Lipid Rafts and Cell Function” and is the following: “Membrane rafts are small (10-200 nm), het-erogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions.” (Pike, 2006).

1.2. Lipid rafts 37

The originally proposed function of rafts, the apical delivery of proteins, has been greatly ex-panded (Rajendran and Simons, 2005). Besides a role for apical transport of proteins (this is discussed in more detail in 1.3.2), general transport actions are mediated via lipid rafts. The most important function of rafts appears to be regulation within signal transduction. Rafts ap-pear to regulate signal transduction by clustering proteins necessary for a signal transduction cascade and by excluding inhibitors (Simons and Toomre, 2000). Many important complex sig-naling events, such as during T cell signalling, have a raft involvement (Kabouridis, 2006).

Endocytosis can also be mediated via lipid rafts. Besides poorly characterised lipid raft struc-tures which seem to be involved in endocytosis, there are well characterised strucstruc-tures spe-cialised in endocytosis designated caveolae. These structures show all biochemical character-istics of lipid rafts and are additionally enriched in the cholesterol binding protein caveolin-1.

Caveolin-1 reduces caveolae invagination at the plasma membrane, thereby regulating endocy-tosis. Invagination is an active process induced by signaling cascades. Endocytosis via caveolae is cholesterol and dynamin dependent (Nabi and Le, 2003; Smart et al., 1999; Pelkmans, 2005).

Many pathogens also exploit lipid raft functions (reviewed in Manes et al. (2003)). Interactions of viruses with lipid rafts will be described in more detailed in 1.2.3.

Most of the concepts of lipid rafts are based on studies investigating the detergent resistance of proteins, the effect of cholesterol depletion on cellular functions, or on results obtained from model membranes. All these methods are yielding results which cannot be interpretated unam-biguously with respect to the raft hypothesis. Additionally the composition of the inner leaflet of raft domains is still unclear, making adoption of results from model membranes even more critical. In cell membranes, the size or even the mere existence of rafts could still not be ambigu-ously proven, not even with more recent methods such as fluorescence resonance energy transfer (FRET), single particle trafficking, or atomic force microscopy (AFM). Therefore, the raft con-cept is still under debate (Munro, 2003). Nevertheless, the raft model remains a powerful tool to explain many cellular processes and Edidin (2003b) aptly described it: “Despite great reser-vations about the interpretation of “classical” operational definitions of lipid raft components and functions, we are left with cells stubbornly insisting that lipids, lipid-anchored proteins, and acylated cytoplasmic signaling proteins are selectively trafficked and associated, and left with viruses that selectively sample host lipids to enrich their envelopes with sphingolipids and cholesterol.”

38 1. Introduction