## 2 Glycosphingolipids
Based on coarse grained dissipative particle dynamics simulations, it was suggested that this occurred through the suppression of thermally excited protrusion fluctuations of membrane lipids between two tightly membrane associated STxB molecules that are at a distance of less than 10 nm from each other [44] (Figure 3). Such fluctuation forces as a source for membrane-mediated clustering had not been suggested before. How should this be tested experimentally? As the first step in this direction, Gb3 molecules were synthesized in which the ceramide part was separated from the globotriose sugar by flexible PEG linkers of increasing length. In dissipative particle dynamics simulations it was shown that with increasing linker length, the suppression of thermally excited protrusion fluctuations became progressively weaker [44]. Under these conditions, clustering was also progressively lost. Satisfyingly, the same trend was observed when STxB clustering was now measured by fluorescence correlation spectroscopy on giant unilarmellar vesicles (GUVs) that were made with the corresponding PEG linker types of Gb3 molecules. Furthermore, STxB endocytosis was strongly reduced in cells that were reconstituted with PEG linker Gb3 species, even when the surface density of STxB matched that of cells that were reconstituted with natural Gb3 (Reference [44]). These findings were consistent with the proposal of membrane fluctuation forces as a novel modality to induce heterogeneity in the lateral distribution of GSL-binding proteins in correlated fluids such as biological membranes [45]. The bacterial cholera toxin from _V. cholerae_ has a very similar overall structure to Shiga toxin and enters cells by GL-Lect endocytosis via clathrin-independent endocytic carriers [22]. Its B-subunit, CTXB, interacts with the GSL GM1 as the cellular toxin receptor. Using synthetic GM1 species from Reference [46] it was very recently shown that CTXB induced GM1 co-clustering with the GPI-anchored protein CD59 only when the acyl chain of GM1 was fully saturated [47]. Thus, different ceramide species of GM1 dictate their co-assembly with CD59 into CTXB-induced nanodomains, which likely underlies endogenous sorting processes driven by cellular galectin. Figure 3: Hypothesis on fluctuation force-driven clustering. Thermally excited fluctuations are suppressed by membrane inclusions (yellow) of nanometric size like STxB, not only at the sites of binding to the membrane, but also for the membrane patch between the two nanoparticles, if they are separated only by a distance of their own size. This perturbation results in an attractive force whose strength is expected to be similar to that of shielded electrostatics or van der Waals interactions. Reproduced with permission from Toxins, Ref. [24]. Synthetic GSL species were also instrumental to propose hypotheses on how the plasma membrane is bent by STxB [14] or CTxB [48] to narrow radii. Using the synthetic PEG linker Gb3 species from Reference [44], it was shown by grazing incidence x-ray diffraction that STxB induced lipid compression in a manner that depended on its mechanical coupling onto Langmuir trough monolayers [49]. This finding was surprising as it is commonly assumed that membranes are difficult to compress. When transposed to bilayered biological membranes, STxB-induced lipid compression would lead to an asymmetric compressive stress and corresponding inward-bound negative curvature (Figure 4), as needed for the construction of uptake sites at the plasma membrane. This asymmetric compressive stress mechanism would be expected to synergize with negative curvature induction that results from the specific geometry of GSL binding sites on STxB [50] and CTxB [51; 52].