Composition of Rafts
Although their existence has been debated, the presence of specific microdomains into the biological membranes is now a largely accepted concept (106). According to this concept, the microdomains have been named “rafts” because they can be seen as floating device within the “fluid mosaic” lipid sea of the Singer and Nicholson model (108). In model membranes made of a pure phospholipid-sphingolipid mixture, sphingolipids tend to pack together in microdomains separate from phospholipids because the former have long, largely saturated acyl chains. In the presence of cholesterol, the sphingolipids are organized in microdomains or rafts in the ordered rigid liquid crystal state (Lo), distinct from the disordered fluid liquid phase membranes (Lc) of the surrounding phospholipids (16, 62). The tight packing organization of lipid rafts confers their resistance to some detergents, such as the nonionic detergent Triton X-100 (TX-100) at low temperature, and allows their purification from low-density fractions after flotation in a sucrose gradient. In cell plasma membranes, a similar organization of lipids is likely to occur, and after solubilization in TX-100 at 4°C, membrane microdomains rich in cholesterol and sphingolipids can be similarly isolated by flotation. These microdomains were given various names such as detergent-resistant membrane (17, 38), detergent insoluble glycolipid-enriched complex (48), or Triton-insoluble floating fraction (48) and are now called membrane rafts. The detergent resistance of rafts is critically dependent on the presence of cholesterol.
In addition to biochemical fractionation, several lines of evidence support the in vivo existence of rafts (30, 38, 55). Their in vivo size has been estimated to be between 25 and 700 nm by using fluorescence resonance energy transfer microscopy and single-molecule-tracking microscopy (55, 94, 104, 116). Many, but not all, proteins anchored to the membrane by a lipid moiety associate with membrane rafts. They include the glycosylphosphatidylinositol (GPI)-anchored proteins, which are located on the extracellular leaflet, and palmitoylated or doubly acetylated proteins, which are enriched in the inner cytoplasmic leaflet. However, geranylated proteins are excluded from rafts (see references 48, 105, and 107 for reviews).
Several data point to the existence of different subsets of rafts depending on the combinatorial association of different sphingolipid species with cholesterol and protein contents (72, 104). One particular membrane raft subset is caveolae. Present in many mammalian cells except lymphocytes and neurons, caveolae are 50- to 70-nm plasma membrane invaginations which are surrounded by a striated coat made of the 22-kDa caveolins tightly bound to cholesterol (77). Likewise, some bona fide membrane rafts are soluble in 1% TX-100 yet insoluble in a lower concentration of TX-100 or in other nonionic detergents, e.g., Brij or Lubrol (98).
Several techniques to study and characterize membrane rafts have been described in the literature. The simplest one is to collect the pellet from a cell extract solubilized with 1% TX-100 at 4°C and centrifuged at 10,000 × g. This technique is not reliable because only the “heaviest” raft structures, which are contaminated with unsoluble material such as protein aggregates, are collected. The classical biochemical experiment involves flotation on a density (sucrose or iodixanol) gradient, with the cell extract being loaded at the bottom of the gradient. The quality of the separation should be checked using bona fide raft and nonraft markers. The alternative approach is to study the colocalization of a protein with a raft marker by microscopic examination (confocal microscopy, fluoresence resonance energy transfer microscopy, electron microscopy, etc.). However, in most cases, rafts are visualized only after clustering of one of the raft components. In any case, disruption of the membrane rafts by treatment of the cells with a cholesterol chelator (methyl-β-cyclodextrin) or a cholesterol-sequestring agent should correlate with the loss of the association of a protein with raft markers.
