Recently, there has been considerable controversy surrounding lipid rafts contained in the plasma membrane. It has been suggested that these microdomains enriched in cholesterol and sphingolipids could play an important role in many cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry. However, rafts have proven difficult to visualize in living cells. Most of the evidence for their existence relies on indirect, and sometimes even inadequate, methods such as detergent extraction. Direct studies of the distribution of putative raft components in living cells have not yet reached a consensus on the size, or even the presence, of these microdomains, and hence it seems that a definitive proof of raft existence has yet to be obtained.
As is the case in every highly disputed field, the number of models, counter models, explanations, and theories at the heart of the lipid raft controversy has lead to a great deal of confusion. In this review, we will attempt to provide a summary of the different models of membrane microdomains, including lipid rafts, as well as the methods used in their study, and their short falls. Furthermore, we will try to describe some promising new investigative methods that should be able to shed a great amount of light on some of the most controversial aspects of lipid raft research, mainly, whether they actually do exist.

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Sphingolipids are important constituents of biological membranes. Ceramide, the major metabolite of this family, is involved in many cellular processes, ranging from differentiation to senescence and apoptosis. Ceramide is an amphipathic molecule with a small head group that allows it to be more promiscuous within membranes than other lipids. Ceramide has a strong ability to change the physical properties of membranes through the formation of ceramide-rich domains, whose physical and morphological characteristics can be studied by a variety of biophysical techniques. While the existence of lipid domains is widely accepted, data from the literature is not consistent concerning many of their properties. We now discuss the biophysical and biological significance of two types of membrane domains (lipid rafts and ceramide-domains). In addition, we discuss other properties of ceramide, such as its ability to permeabilize the outer membrane of mitochondria. Finally, we attempt to integrate these various issues from biochemical and biophysical perspectives.

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Early notions on membrane lipid domains were derived mainly from experimental models based on artificial membranes and from studies on membrane fractions prepared by density gradient centrifugation of a cell lysate obtained by treating cells with detergents under controlled conditions. These studies introduced the biological concept that Golgi apparatus is capable to sort proteins and to send them to the plasma membrane through “rafts”, membrane lipid domains highly enriched in glycosphingolipids, sphingomyelin, ceramide and cholesterol. This concept has evolved and has been expanded in the last 10 years, and now several experimental approaches with the potential for observing these domains in intact cells are available. Here we critically discuss the necessity of discriminate between what is present on a cell membrane and what we can prepare from cell membranes in a laboratory tube.

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Raft domains have been proposed to work as a platform where raftophilic signaling molecules assemble and interact for efficient signal transduction. However, this raft hypothesis has been difficult to prove. Our recent single-molecule tracking experiments revealed that cytoplasmic signaling molecules were frequently, but very transiently recruited to the rafts formed on demand by the clusters of raftophilic glycosylphosphatidylinositol (GPI)-anchored receptors (e.g., CD59) that were generated after the engagement of the receptors by the binding of extracellular signaling molecules. All of the cytoplasmic signaling molecules examined thus far, Gαi2, Lyn, and PLCγ, exhibited short residency times of ~200 ms within the CD59-cluster rafts. This recruitment period of each individual signaling molecule was short, compared with the periods of overall bulk activation of these molecules by a factor of 4,000. Argument has been advanced that the analogue bulk signal, which lasts for over several thousands seconds, is generated by the superposition of the short-lived, digital-like individual signals, which last for a fraction of a second.

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Gangliosides have been shown to modulate various growth factor receptors such as epidermal growth factor receptor (EGFR), insulin receptor (IR), platelet derived growth factor receptor (PDGFR), neural growth factor receptor (NGFR) and fibroblast growth factor receptor (FGFR). Although traditional data obtained by exogenous addition of gangliosides into culture medium suggested that gangliosides are involved in various cellular functions as coordinators of multiple receptor functions, the molecular mechanisms underlying these phenomena were remained unknown. Recent studies show that the presence of membrane microdomains (lipid raft) highly enriched in cholesterol and glycosphingolipids (GSL), but lacking in phospholipids. Within the past decade, data have emerged from many laboratories implicating these lipid microdomains as critical for proper compartmentalization of growth factor signaling. In this review, we will summarize these observations and discuss a new concept ganglioside-mediated regulation of growth factor receptors in microdomains. How gangliosides and growth factor receptors are related to diseases? Here, we present evidence of a new pathological feature of insulin resistance in adipocytes caused by dissociation of IR-caveolin-1 complex by ganglioside GM3 in microdomains.

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Since their discovery, lipid rafts have been implicated in several cellular functions, including protein transport in polarized cells and signal transduction. Also in multidrug resistance lipid rafts may be important with regard to the localization of ATP-binding cassette (ABC) transporters in these membrane domains. This specific localization may support the activity of these ABC transporters as drug efflux pumps and this raises important questions regarding the dependence on lipid raft constituents. In this respect, two lipid classes immediately come into play, as both sphingolipids and sterols are generally assumed to be important in the generation and maintenance of lipid rafts. Apart from lateral interactions with lipids in the membrane bilayer, one can also envision that transverse interactions with respect to the membrane bilayer play a role in positioning and function of ABC transporters. Indeed, some evidence exists for a role of the actin cytoskeleton in stabilizing the position of ABC transporters in a certain membrane area. ABC transporters may be directly linked to the actin cytoskeleton, or indirectly via lipid rafts. In this review, we will evaluate whether ABC transporters are dependent on a particular membrane environment for their function and which of the lipid raft constituents appear to be essential for this dependency.

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