## 2 Cellular Pathways Exploited by Toxins
### Endocytic Mechanisms and Recycling
Shiga toxin, which binds to the neutral glycosphingolipid Gb3, has taught us several lessons in cell biology. The toxin can be endocytosed by different endocytic mechanisms, and it can clearly affect its own uptake (for reviews, see [69; 70; 71; 72]). It was the first toxin shown to be able to aggregate in clathrin-coated pits [73] in spite of binding to a lipid that does not seem to interact with surface proteins. Later studies applying different methods to interfere with clathrin-dependent endocytosis, including the expression of a dominant negative mutant of dynamin and inducible expression of antisense RNA to clathrin heavy chain, have supported this finding [74; 75]. Furthermore, the expression of both dominant negative mutants of epsilon and eps15 reduced Shiga toxin uptake [43]. The mechanism behind the toxin aggregation in clathrin-coated pits is still unknown. Although Gb3 does not transverse the membrane, the toxin is able to induce activation of kinases [76; 77; 78] and phosphorylation of clathrin [76]. Shiga toxin is also able to increase the number of clathrin-coated pits, and it can affect the lifetime of these structures [79]. Furthermore, it should be noted that Shiga toxin uptake is increased at high toxin concentrations in several cell lines. This increase is dependent on the A-subunit and seems to occur through clathrin-coated pits, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain [80]. Although Shiga toxin can be endocytosed from clathrin-coated pits, the fraction using this uptake mechanism is likely to be cell-type dependent. Upon reduction of accumulation of Shiga toxin in clathrin-coated pits, the toxin may more easily be taken up by other mechanisms. In addition, since interfering with clathrin-dependent uptake also may upregulate other mechanisms, the significance of clathrin-dependent uptake is likely to be underestimated. There are still several questions to answer when it comes to uptake of Shiga toxin from clathrin-coated pits: Does this occur mainly from coats with or without AP2 [81]? Which other proteins or protein-modifications associated with clathrin-coated pits could be required for this process? Is the interaction between the outer membrane leaflet, which contains Gb3, and the internal membrane lipids such as phosphatidylserine (PS) or phosphatidyltisolphosphates (PIPs) required? Are specific lipid species important for this process? We recently published a review describing how certain species of PS can play a role for interaction with cytosolic proteins [82]. Recently, PS was found to be important for the formation of clathrin-coated vesicles [83]. The complex formation and disappearance of PIPs during formation of clathrin-coated vesicles might similarly be involved [23]. Thus, further studies using Shiga toxin in this connection are likely to provide more information on cell biology. Shiga toxin was many years ago found to be able to induce invaginations at the cell surface [84], and it was shown to be taken up from such structures in a dynamin-, cholesterol-, and energy-dependent manner. Later on, it was shown that the process is dependent on endophilin A2 and actin and somewhat related to the endocytic pathway called FEME [26], although the latter is dependent on growth factor activation. Shiga toxin uptake has, on the other hand, been found to be dependent on toxin-induced curvature changes, a process studied by [PERSON] and coworkers within recent years [69].