Cholera toxin internalization and intoxication

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Abstract
Our paper focused on whether both CT internalization and activation are mediated by caveolae or by detergent-insoluble glycolipid-enriched complexes (DIGs) (also known as lipid rafts) in cells deficient in caveolin and caveolae. Torgersen et al. primarily were interested in showing caveolae-independent endocytosis of CT. Our approach was to compare CT uptake and action in three cells that have no, low or high levels of caveolin and caveolae, and to use the cholesterol modifiers filipin and β-cyclodextrin (βCD) to selectively inhibit caveolae/DIG-mediated endocytosis. Chlorpromazine (CPZ) and diphtheria toxin (DT) served as inhibitor and probe for clathrin-mediated uptake. One of our cell lines, human intestinal CaCo-2, played a major role in their study and appears to be the source of many of their repetitious complaints. By using anti-CT-A1 antibodies to quantify CT uptake, we found 58% inhibition by filipin. As Torgersen et al. found only 17% inhibition by using a different method, they speculated that our assay may have overestimated CT uptake if the antibodies could not reach CT clustered in the narrow necks connecting caveolae to the cell surface, and if filipin could somehow alter the necks and increase antibody binding. They ignored our second assay in which cells were labeled with rhodamine-conjugated CT-B at 15°C. When warmed at 37°C, there is an extensive redistribution of fluorescence from the plasma membrane to the perinuclear region that is blocked by filipin but not CPZ. Even when Torgersen et al. found filipin to be ineffective on two other cell lines, they failed to show that the filipin was active. Filipin is known to be unstable in solution. This led them to a circular argument: as CT uptake is only slightly inhibited by filipin, it must not be via caveolae/DIGs. Thus when they found that βCD inhibits CT internalization in CaCo-2 cells by 43% (similar to our 39%), they concluded that the uptake is clathrin-dependent based on the weak effect of filipin and cited studies showing that βCD also blocks the latter pathway. Surprisingly, two of the four references cited were not relevant. We found that both βCD- and filipin-treated, but not CPZ-treated, CaCo-2 cells remain sensitive to DT. Others have shown that these agents selectively inhibit caveolae/DIG-mediated, but not clathrin-mediated, endocytosis in a variety of cells (Puri et al., 2001; Wolf et al., 2002).