## 2 Glycosphingolipids  
The mole percentage of glycosphingolipids (GSLs) in biological membranes varies from very low numbers in the nuclear envelop and ER to double digit numbers at the plasma membrane of epithelial cells [34]. GSLs are essential for life from lower organisms such as the fruit fly _D. melanogaster_[35] and the earthworm, _C. elegans_[36], to mammals [37]. Figure 1: Endocytic pit construction according to the GL–Lect hypothesis. The sugar binding protein galectin-3 (Gal3) is monomeric in solution. It binds to complex carbohydrates of plasma membrane glycoproteins (glycoslated cargo). This leads to Gal3 oligomerization, thereby enabling its capacity to interact with glycosphingolipids (GSLs) in a way such as to induce narrow membrane bending and the clathrin-independent formation of endocytic pits into which the glycosylated cargoes are recruited. From these pit, endocytic carriers are then generated for the cellular uptake of the glycosylated cargoes (not shown). Reproduced with permission from Ref. [16]. They have been ascribed a multitude of functions (e.g., receptor signaling, cell adhesion, membrane trafficking), but molecular mechanisms of action remain poorly explored. A striking feature of GSLs is their capacity to organize into domains that have been termed lipid rafts [38]. This lateral \"connectivity\" results from unique features, such as long acyl chains and the capacity to form hydrogen bonds. How raft connectivity is translated into biological functions has mostly remained enigmatic. The raft hypothesis itself has evolved since its initial formulation. While phase separation with raft fabric occurs in passive systems such as liposomes, active cell membranes are understood as non-equilibrium systems. In this context, the construction of raft-type assemblies is thought to be driven by appropriate triggers (e.g., GSL-binding lectins), which thereby transform the \"raftophilic\" potential of GSLs into defined cell biological outcomes [39; 40]. Hundreds of different GSL species have been described that are made by chains of biosynthesis enzymes [41] (Figure 2). It is thereby virtually impossible to \"mutate\" the expression of a specific GSL species in the complex cellular environment without affecting the others. Chemical synthesis has provided a means to address this difficulty, which will be exemplified here in the context of the GL-Lect hypothesis. Figure 2: GSL biosynthesis pathways. Left panel: ceramide (Cer) can be acylated (acyl-Cer), phosphorylated (Cer-1-phosphate), or conveyed to the TGN for the synthesis of sphingomyelin (SM). Alternatively, Cer is glycosylated for the synthesis of the GSL precursors, glucosylceramide (GlcCer) or galactosylceramide (GalCer) along the secretory pathway. GalCer is then processed for the production of sulfatides. Right panel: GlcCer is galactosylated to lactosylceramide (LacCer), which serves as a common precursor for the different GSL series: globo (red), ganglio (green), asialo (blue) and lacto (purple). Glycosphingolipid-synthetizing enzymes catalyzing the major synthetic reactions are shown in dark orange. Reproduced with permission from The Journal of Cell Science, Ref. [41]. The bacterial Shiga toxin and the closely related verotoxins from enterhoemorrhagic _E. coli_ strains are a threat to human health by inhibiting protein biosynthesis in target cells [42; 43]. It is the non-toxic homopentameric B-subunit, termed STxB, which drives the clathrin-independent biogenesis of tubular endocytic pits from which the toxins are taken up according to the GL-Lect mechanism (Reference [14], reviewed in Reference [24]). STxB indeed binds to the GSL globotriaosylceramide (Gb3) not only as the cellular toxin receptor, but also to cluster and induce narrow membrane bending. How is this achieved? Synthetic approaches have allowed addressing this question. In solution, STxB homopentamers do not cluster, while they very efficiently do so on membranes.