Lipid rafts--composition, characterization, and controversies.

In this issue of The Journal of Nutrition, an article reporting on the effect of (n-3) PUFA on the composition of lipid rafts in tumor cells is published, to our knowledge, for the first time (1). The authors report that the enrichment of lipid rafts of MDAMB-231 breast cancer cells with (n-3) PUFA is probably linked to modification of cell signaling processes leading to cell death. The results of this study further our understanding of the mechanism by which different types of fatty acid can influence tumor cell growth. So, what are lipid rafts, and why does this article represent an important step forward in our knowledge? Lipid rafts are regions of membranes with a distinct, characteristic structural composition and that appear to act as platforms to colocalize proteins involved in intracellular signaling pathways. The organization of membranes into such microdomains recognizes that, far from being randomly arranged, lipids may actually be highly organized within different parts of the membrane, and that this organization influences the way that membrane proteins are distributed (2). Rafts are particularly rich in sphingolipids and cholesterol, and the side chains of the phospholipids present are usually highly enriched in saturated fatty acids compared with the surrounding nonraft regions of the membrane. Enrichment of phospholipids with saturated fatty acids allows for the close packing of lipids within rafts because 1) sphingolipids also contain saturated fatty acid side chains, and 2) cholesterol and saturated fatty acids are able to pack closely. As a result of the presence of cholesterol and saturated fatty acids, lipid rafts are more ordered and less fluid than the surrounding membrane. Cytoplasmic proteins that are covalently modified by saturated fatty acids (palmitoyl or myristoyl moieties) and cell surface proteins that are attached via a glycosyl phosphatidylinositol anchor are highly concentrated within lipid rafts. Many proteins involved in signal transduction, such as Src family kinases, G proteins, growth factor receptors, mitogen-activated protein kinase (MAPK), and protein kinase C are predominantly found in lipid rafts, which appear to act as signaling platforms by bringing together (i.e., colocalizing) various signaling components, facilitating their interaction (3). Despite a large body of work, doubts still persist regarding the existence and nature of lipid rafts (4–7). These doubts have arisen mainly because of limitations in the methods available to isolate and to study rafts. Lipid rafts have been defined largely according to their insolubility in nonionic detergents such as Triton X-100, which probably relates to their structural characteristics, and they have frequently been termed detergentresistant membranes on this basis. Detergent-resistant membranes have traditionally been isolated by density gradient flotation of cellular material that has been homogenized in the presence of Triton X-100. Flotillin and glycosyl phosphatidylinositol– anchored proteins are used as raft markers, and the material obtained in this way is rich in cholesterol and sphingolipids. However, variation in experimental conditions can have a major impact on exactly what is obtained in the detergent-resistant fraction. For example, detergents other than Triton X-100 can be used, and the nature of the material that is isolated depends upon the detergent used and its concentration. Thus, different methods yield different membrane fractions, and so the nature of the membrane material (rafts) studied by different investigators varies. Clearly, direct visualization of rafts, for example by microscopy, would resolve some of the uncertainties about their existence and structure, but fluorescence microscopy studies have tended to produce mixed results (4). Membrane microdomains have been studied extensively with respect to T-lymphocyte responses to activation (8–10). Furthermore, T cells are the only cell type in which the effects of PUFA on the structure and function of detergent-resistant membranes have been studied to any great extent. PUFA have long been known to alter T cell functional responses (11), and research over the last few years suggests that raft disruption underlies the mechanism of action of PUFA upon T cells (12–17). Schley et al. (1) report the effect of (n-3) PUFA on the composition of detergent-resistant membranes (which they term lipid rafts) in the MDA-MB-231 human breast cancer cell line. The study by Schley et al. (1) appears to be the first examination of the influence of fatty acids on detergent-resistant membranes in tumor cells and furthers our understanding of the mechanism by which different types of fatty acid can influence tumor cell growth. The cells were grown in a medium supplemented with linoleic acid or the long-chain (n-3) PUFA, eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA), or combinations of linoleic acid and long-chain (n-3) PUFA. The (n-3) PUFA were shown to decrease cell numbers, as previously observed by others with this cell line. Detergent-resistant membranes were 3 Abbreviations used: DHA, docosahexaenoic acid; EGF, epidermal growth factor; EPA, eicosapentaenoic acid; MAPK, mitogen-activated protein kinase. * To whom correspondence should be addressed. E-mail: pcc@soton.ac. uk.