Sphingolipid topology and membrane protein nanoclusters

Sphingolipids are an essential class of membrane lipids in eukaryotic cells. Due to their high packing density and their affinity for cholesterol, sphingolipids are able to promote bilayer rigidity and impermeability. Apart from its ability to maintain biomembrane integrity, sphingomyelin (SM) is also a major source for ceramide, involved in signal transduction. Thus, by balancing the formation of ceramide and diacylglycerol (DAG), which is known to act as second messenger, SM synthesis and degradation may play a fundamental role in cell growth and survival. In this thesis, we use C. elegans as an animal model which represents, due to its easy manipulation of gene function by mutation and RNAi, an attractive model to study the implications of SM synthase activity on the molecular level in animal physiology. We identify two proteins of the multigenic protein family as SM synthases and one enzyme as a ceramide phophoethanolamine (CPE) synthase. Glycosphingolipids (GSLs), the other class of membrane sphingolipids, contain polar headgroups which consist of one or more carbohydrate moieties. Here, we discuss the technical issues restraining progress in the elucidation of nanoscale localization of GSLs. Complex GSLs, such as gangliosides, fulfill important roles in recognition and signaling at the plasma membrane and are also used as receptors by parasites, bacteria, viruses and toxins. GM1, a monosialoganglioside, has been shown to be involved in the regulation and activation of the epidermal growth factor receptor, and acts as a receptor for cholera toxin. A defect in the degradation of this interesting lipid leads to its accumulation and storage in the lysosomes, displaying a clinical phenotype, Landing disease or GM1 gangliosidosis. Recently, the accumulation of GM1 was found to activate various channels, and it was suggested that GM1 may accumulate at the ER, eventually leading to a perturbation of calcium homeostasis. In this study we show the localization of accumulated GM1 in diseased cells by immune-electron microscopy after freeze substitution, thereby preventing relocation of lipids during labeling procedures. GM1 was indeed mainly localized to lysosomal/endosomal structures and no evidence for significant increase of GM1 content at other cellular organelles was found. Glycosphingolipids, together with SM and cholesterol are thought to play an essential role in the formation of microdomains, called lipid rafts. The lateral organization of biomembranes was proposed to be required for the sorting of lipids and specialized proteins, and has been shown lately to play a fundamental role in signal transduction. In this present work, we combined for the first time EM and homo-FRET analysis, two techniques that have been successfully used to study nanoscale organization at the plasma membrane. Exploiting the complementary ranges of the two techniques, we were able to study the influence of glycosphingolipids on the distribution of different lipid-anchored proteins on the cytosolic side of the plasma membrane. Glycosphingolipids did not affect the diameter of the protein clusters but increased the number of molecules of the proteins per cluster, while at the same time they lowered the level of molecular interaction between these molecules