## 2 Cellular Pathways Exploited by Toxins
### Endocytic Mechanisms and Recycling
It should be noted that a new role for caveolae has recently been characterized: Caveolae can provide additional membrane upon mechanical stress and reform in an ATP-dependent manner [38; 53]. During the 1980s, rich played an important role in showing evidence for the existence of clathrin-independent endocytosis. When cells are exposed to a hypotonic shock and potassium-depletion, some cell types lose the clathrin-coat at the cell membrane [54]. After exposure of cells to such a treatment, rich could still end up in an intracellular compartment unavailable to antibodies, and subsequently intoxate cells [55]. Similarly, when the cytosol was acidified--a condition that blocks pinching off of clathrin-coated pits--ricin was endocytosed in several different cell types [56]. However, early estimates suggested that clathrin-coated pits could account for all uptake into the cells [57]. At that time, a number of arguments were used to raise doubts about the existence of clathrin-independent uptake. For instance, when clathrin is released from the membrane after potassium depletion, is there still a sufficient uptake mechanism to preserve endocytosis from the same structures? When you remove the clathrin coat or acidify the cytosol to block pinching off clathrin-coated pits, do you then induce alternative mechanisms? Even experiments with unperturbed cells, which showed that Concanavalin A and transferrin ended up in separate vesicles soon after uptake, did not seem to help [58]. It was not until the Schmid-lab showed that fluid phase uptake continued even when clathrin-dependent uptake was blocked by the expression of a dominant negative mutant of dynamin [59; 60] that the resistance against existence of clathrin-independent endocytosis seemed to disappear. Thus, a change in opinion in the field can take several years. The ability of cells to regulate endocytic pathways in response to changes in other uptake mechanisms is a challenge when studying such mechanisms. Fluid uptake continues at a similar rate after a block in clathrin-dependent endocytosis due to compensatory mechanisms [59; 60], and later studies have revealed a co-regulation of caveolar and Cdc42-dependent fluid phase endocytosis by phosphocaveolin-1 [61]. Upon reduction of phosphocaveolin-1, there is an increase in the Cdc42 dependent uptake and vice versa. Cavin has also been reported to downregulate the Cdc42-dependent pathway [62]. Certainly, later studies have demonstrated the complexity of uptake mechanisms [26; 27; 63]. It should be noted that clathrin-independent endocytosis in polarized cells is subjected to a differential regulation on the apical versus basolateral side. When ricin was used to study uptake from the two poles in polarized MDCK cells, the apical endocytosis was upregulated by protein kinase A, protein kinase C, cyclooxygenase, and the inhibition of calmodulin. Under the same conditions, there was no effect on the basolateral uptake (for review, see [27]). Caveolae are known to be dependent on cholesterol, and removal of this lipid or addition of filipin, which binds to cholesterol, rapidly destroys these structures [64; 65]. However, experiments with ricin and addition of methyl-\(\beta\)-cyclodextrintrim, which extracts cholesterol from the cell membrane, showed that also macropinocytosis is sensitive to a reduction of this lipid [66]. Furthermore, invagination of clathrin-coated pits is inhibited by the addition of methyl-\(\beta\)-cyclodextrintrin [67; 68]. Even under such conditions, ricin can still be endocytosed by other mechanisms (for review, see [27]). In addition, the literature shows that the inhibition of uptake of a certain ligand by reduction of cholesterol is taken as a proof for uptake of that ligand by caveolae. When reporting that the uptake of rather large nanoparticles (far larger than the diameter of caveolae) occurs by this mechanism due to a reduction of the uptake after addition of methyl-\(\beta\)-cyclodextrintrin, caution should be exercised before drawing any conclusions.