# Shiga Toxin--A Model for Glycolipid-Dependent and Lectin-Driven Endocytosis
## 3 Formation of Membrane Invaginations and Tubular Endocytic Pits  
Upon binding to Gb3, Shiga toxin does not directly reach through the plasma membrane to interact with the conventional cytosolic clathrin machinery. Yet, Shiga toxin has been seen in clathrin-coated pits [15], even if efficient inhibition of clathrin function causes at most a 35% reduction of Shiga toxin uptake [16; 17]. How this localization into the clathrin pathway is operated remains to be further investigated. A key question here is whether toxin molecules \"fall\" into preexisting pits, or whether pit biogenesis is actually triggered by the toxin. A sizable fraction of Shiga toxin still enters cells under conditions of clathrin pathway inhibition. In fact, protein toxins from plants and bacteria were the first cargo molecules for which clathrin-independent uptake into cells was suggested more than 35 years ago [18; 19]. How membrane bending and endocytic pit formation are operated in these cases has remained a conundrum. Based on the observation that the formation of a Shiga toxin-Gb3 complex is necessary and sufficient to drive the formation of narrow tubular membrane invaginations [9], a molecular hypothesis has recently been suggested for how this might be achieved (Figure 3a). According to this model, the Shiga toxin-Gb3 complex would be endowed with curvature-active properties, i.e., the capacity to deform the membrane without the need of the cytosolic clathrin machinery. The clustering of several toxin molecules,  
Figure 2: Hypothesis on fluctuation force-driven clustering. The represented nanoparticles could be Shiga toxin pentamers. likely favored by the fluctuation forces that were mentioned above, would then lead to the formation of deep and narrow invaginations as a first step towards the formation of tubular endocytic pits. At first sight, this proposal appears counterintuitive. The asymmetric deposition of toxin molecules on the exoplismic leaflet should lead to steric stress that would be expected to favor the buckling of the membrane to the outside of the cell or model membrane to which it binds, thereby generating positive curvature [23]. Obviously, the curvature-active properties of the Shiga toxin-Gb3 complex overcome this steric stress-induced buckling. How is this achieved? Results from molecular dynamics simulations have recently allowed to propose an explanation. It was shown that also in silico, the receptor-binding B-subunit of Shiga toxin (STxB) induces an increment of negative inward-oriented curvature when interacting with a patch of membrane that contains Gb3 receptor molecules [24] (Figure 3b). On average, 13 out of 15 Gb3 binding sites per STxB molecule were  
Figure 3: Lectin-driven construction of tubular endocytic pits. (**a**) Model for Shiga toxin B-subunit (STxB)-driven formation of membrane invaginations. (**b**) Molecular dynamics data on spontaneous curvature induced by STxB. The red and green binding sites represented in the lower part of the panel force the membrane to bend up at the edges of STxB pentamers. (**c**) Overlays of crystal structures of STxB (green, Reference [4]), cholera toxin B-subunit (red, Reference [20]), and VP1 capsid protein from SV40 (blue, Reference [21]). (**d**) GL-Lect hypothesis on the Gal3-driven, glycolipid-dependent formation of endocytic pits. Glycolipids are represented as red dots. Reproduced from [22]. 2015, Nature Publishing Group. occupied. Because of the positioning of 10 of the Gb3 binding pockets at the rim of STxB molecules in a location slightly above the normal plane of the membrane, the latter must bend up to reach these sites, thereby generating an increment of negative, inward-oriented curvature.