Flotillas of lipid rafts fore and aft.

T effectively combat invading pathogens, immune cells must rapidly switch from roughly spherical resting cells to polarized migratory ones, which then move in a directed fashion to the site of infection. The dramatic metamorphosis of leukocytes into polarized cells and their subsequent migration are two of the most fascinating phenomena in cell biology. Polarization and migration require the spatial and temporal control of signal transduction molecules so that substrate attachment and membrane extension occur at the cell front, while detachment and membrane retraction happen at the rear. How do cells coordinate signaling molecules to perform contrasting functions at opposite poles? It has long been appreciated that there is polarization in the protein machinery involved in cell migration. However, it is becoming evident that lipids are also distributed nonuniformly, and the distribution of lipids is an important factor for directional migration. In a paper in this issue of PNAS, Gómez-Moutón et al. (1) provide further evidence for the importance of specialized lipid domains in establishing and maintaining the polarity of motile cells. In particular, they show that both the leading edge and the uropod of polarized T lymphocytes are enriched in lipid components that partition into raft-like lipid domains. An interesting finding is that the distribution of certain lipid raft components differs at the two poles. Thus, ganglioside GM3 is enriched at the leading edge, whereas GM1 is concentrated at the uropod. Treatments such as cholesterol depletion, which disrupts plasma membrane organization, prevent the development of a polarized morphology and cell migration. Although it is now understood that lipids are distributed nonrandomly in the plasma membrane and that this has important consequences for cell signaling and other functions (2–4), the precise nature of these lipid inhomogeneities (microdomains) remains somewhat enigmatic—partly because the lipid microdomains are apparently in a size range (10–300 nm) that is below the resolution of optical microscopy. In the current view of the plasma membrane, certain lipids and proteins assemble into dynamic, sub-mm-sized lateral organizations that function to facilitate signal transduction events (2, 4, 5). Regions of the plasma membrane that are enriched in sphingolipids and cholesterol are thought to exist in a liquid-ordered phase (6, 7) that confers detergent resistance to these structures (8) and allows for their ready isolation by f lotation on sucrose density gradients (9). One model (3) is that signaling molecules are recruited to these small ‘‘rafts’’ from a largely liquiddisordered membrane. Because the plasma membrane contains up to 50 mole % cholesterol and also a very high amount of sphingomyelin in the outer leaflet, it might be expected that a high fraction of the lipids in the plasma membrane are resistant to extraction by cold nonionic detergents (10, 11). In fact, when cells are treated with cold Triton X-100 and the residual membranes are imaged by fluorescence microscopy and electron microscopy (11), most of the area of the cell remains covered by detergent-resistant membrane. In addition to cholesterol and sphingomyelin, glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins are in the detergent-resistant lipid pools. In contrast to the lipids, most transmembrane proteins that are not linked to the cytoskeleton are solubilized by cold nonionic detergent treatment, but a small subset of proteins are insoluble. The fact that only a small fraction of membrane proteins are detergent resistant may give rise to the mistaken impression that rafts are a small fraction of the plasma membrane. A second oversimplification is that there are just two types of membrane domains: rafts and non-rafts. The report by Gómez-Moutón et al. (1) on rafts in polarized T cells adds to mounting evidence dispelling both of these misconceptions. It was shown recently that rafts could be subdivided based on their susceptibility to solubilization in nonionic detergents (12); a subset of raft proteins in an epithelial cell line was found to be resistant to solubilization by the detergent Lubrol, but susceptible to solubilization by TritonX-100. Additionally, in epithelial cells, approximately half of CD44containing lipid rafts f loated to the lowdensity fraction of sucrose gradients, whereas the other half of CD44-containing lipid rafts only f loated after disruption of the actin cytoskeleton (13). These findings imply the existence of two kinds of CD44-containing lipid rafts, which are distinguished by their association to the actin cytoskeleton or not. Now, Gómez-Moutón et al. show that the leading edge and the uropod of T cells contain raft domains with different compositions. Images of polarized T cells, f luorescently labeled for markers of each type of raft domain, show that GM3enriched rafts localize to the cell front (lamella), whereas GM1-enriched rafts localize to the cell rear (uropod; ref. 1). Together, these raft domains constitute a significant portion of the total cell surface, in line with previous reports demonstrating that a large fraction ('40– 70%) of the plasma membrane is in detergent-resistant (i.e., liquid-ordered) structures (10, 11). Clearly, a binary model of the plasma membrane, in which rafts comprise a very minor fraction of the total cell surface, cannot accommodate these findings. Rather, the plasma membrane may more closely resemble a dense assembly of rafts of different types. Various signals may recruit certain types of rafts into larger assemblies (f lotillas; Fig. 1). These larger assemblies are easily seen by optical microscopy. Gómez-Moutón et al. (1) demonstrate that the association of proteins with distinct membrane domains dictates their