## 2 Cellular Pathways Exploited by Toxins
### Endocytic Mechanisms and Recycling  
The protein toxin rich binds to a variety of molecules with terminal galactose. As described in the current review, the toxin has provided us with important information about several cellular mechanisms. More than 40 years ago, we found that ricin could easily be labeled with radioactive iodine by an enzymatic method (lactoperoxidase), which preserved its binding affinity to the cell surface, as well as its toxicity [28]. This contrasted with labeling using strong oxidizing conditions, which reduced the activity of ricin. Furthermore, ricin can easily be removed from the cell surface with addition of lactose, thereby facilitating its use in studies of endocytosis. Early studies revealed that there was not any strict temperature cut-off for uptake of ricin [29] and, furthermore, that endocytosis of fluid phase and ricin is dependent on cell density [30; 31; 32]. Cell density was later shown to affect the lipid composition of cells [33; 34]. Furthermore, these early studies revealed that endocytic uptake is not a one-way street, as ricin that was internalized could come out of the cell again in an intact form [29; 35]. The same was the case for ricin B-chain, showing that the recycling was unrelated to the toxicity of ricin. Since these initial studies of ricin recycling, knowledge about different recycling mechanisms and the molecules involved has exploded, and a review about the complexity of these processes was recently published by [PERSON] et al. [36]. Clathrin-independent uptake of a toxin was first suggested for cholera toxin, which could be observed in caveolae by EM [37], and subsequently inside cells in endocytic vesicles. It was suggested that these organelles, which have a characteristic appearance as flask-shaped invaginations with a diameter of 60-80 nm [38], were responsible for uptake of the toxin. Since cholera toxin can induce a transmembrane Ca\({}^{2+}\)-flux and kinase activation [39; 40; 41] it possible that the toxin in itself may affect the pinching off of caveolae. Figure 3: An overview of endocytic mechanisms in a non-polarized cell (**A**) and a polarized cell (**B**). We have indicated some pathways such as clathrin-dependent endocytosis, caveolae (now regarded to be quite stable structures), Cdc42/GRAF1, and others. It should be noted that clathrin-independent uptake is regulated in different ways on the apical and basolateral side as described in the text. It should be noted that in MDCK cells all caveolae are on the basolateral side. Reprinted from an open-access review [27]. Also, at that time, it was not excluded that the toxin was stuck in this location and, at least to some extent, rapidly taken up by other mechanisms such as clathrin-dependent endocytosis. Later studies revealed that cholera toxin, in fact, can be internalized by different mechanisms, and both clathrin-dependent and clathrin- and dynamin-independent uptake mechanisms can be involved [42; 43; 44]. Uptake from caveolae is normally not a very efficient process [45]. However, caveolae can pinch off, and the content is then transferred to early endosomes. It was initially published that the content from caveolae ended up in neutral structures called \"caveosomes,\" and from there, the content was transported to the endoplasmic reticulum [46]. However, the caveosomes were later found to be artefacts [47; 48], and the authors suggested that the term should no longer be used. In spite of this, researchers continue to describe that they may target, for instance, nanoparticles to \"caveosomes\" to avoid lysosomal degradation [49]. Although a virus such as SV40 may cause signaling and entry through the caveolae [50], this virus can also enter cells by other endocytic mechanisms [51]. Interestingly, it has been demonstrated that caveolin can stabilize certain ligands at the cell surface. This is the case, for instance, for autocrine mobility factor (AMF) and cholera toxin [52].