# Shiga Toxin--A Model for Glycolipid-Dependent and Lectin-Driven Endocytosis
## 4 Membrane Scission, Targeting to Endosomes, and Retrograde Transport  
Shiga toxin-induced membrane invaginations need to pinch off from the plasma membrane by fusion of the opposing walls of invaginated tubular endocytic pits. The conventional pinchase dynamin affects Shiga toxin uptake, but is not absolutely required [9; 16]. The difficulty with coming to a conclusion for the involvement of dynamin is that feedback loops exist to actin [35], which also contributes to the scission of Shiga toxin-induced membrane invaginations [36]. It has indeed been demonstrated that these invaginations are prone to undergo domain formation, thereby generating domain boundary forces that drive the spontaneous line tension-driven squeezing of the tubule membrane leading to scission [37]. Actin was identified as one of the domain formation triggers [36], likely by directly or indirectly binding to lipids, thereby changing the entropy of the system [38]. Recent findings indicate that yet another scission modality might be operating on Shiga toxin-induced membrane invaginations. The model that is presented in Figure 3a predicts that the signal that is sent by the toxin to the cytosol might be mechanical: the formation of a highly curved membrane domain, which would then be recognized by cellular machinery for further processing (Figure 4). Proteins of the Bin, amphiphysin, and Rvs (BAR) domain family are specialized in such curvature recognition [40], and by screening through a BAR domain protein library, endophilin-A2 (EndoA2) was identified as being functionally localized onto Shiga toxin-induced membrane invaginations in relation to the scission reaction [41]. Furthermore, it was demonstrated for Shiga [41] and cholera toxin [42] that these invaginations are pulled upon by microtubule-based dynein motor activity. Strikingly, if this dynein-mediated pulling force is exerted on tubular invaginations that are scaffolded by EndoA2, membrane scission ensues [41]. This is due to the fact that the EndoA2 scaffold exerts a friction force onto the membrane that limits lipid diffusion [39]. Upon pulling with speeds superior to 50 nm/s (such as that exerted by the dynein motor), the lack in lipid supply leads to tube thinning, and then to scission. Such pulling force or friction-driven scission may operate more generally in processes of clathrin and caveolin-independent endocytosis that are often little reliant on dynamin [43]. It might appear surprising that several scission modalities operate on Shiga toxin-induced membrane invaginations: dynamin [9], actin [36], and EndoA2/dynein [41]. It could be demonstrated that when interfering with each of these individually or in combination, the effects on scission appear to be roughly additive [41], suggesting that the overall probability of scission is the result of individual contributions. Once formed, Shiga toxin-containing carriers are targeted to the endosomal system. This is favored by association with microtubules [44], which might be initiated right at the plasma membrane, as discussed above. Furthermore, the vSNARE proteins VAMP2, VAMP3, and VAMP8 are recruited to Shiga toxin-containing endocytic carriers for their targeting to the endosomal membrane system [45]. Figure 4: Friction-driven scission. See text for details. Reproduced from [39]. 2017, Cell Press. From endosomes, Shiga toxin is transported via the retrograde route to the TGN, the Golgi apparatus, and then on to the endoplasmic reticulum, from where the catalytic A-subunit is translocated to the cytosol [46]. The study of Shiga toxin has allowed to propose the existence of a trafficking interface between early endosomes and the TGN [47], which has since become a hotspot of membrane biology research.