Internalization of cholera toxin by different endocytic mechanisms

The mechanism of cholera toxin (CT) internalization has been investigated u sing Caco-2 cells transfected with caveolin to induce formation of caveolae , HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cell s in which antisense mRNA to clathrin heavy chain can be induced. Here we s how that endocytosis and the ability of CT to increase the level of cAMP we re unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-upta ke by less than 20%, suggesting that caveolae do not play a major role in t he uptake. Extraction of cholesterol by methyl-beta -cyclodextrin, which re moves caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% r eduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and n ystatin and filipin had no effect on CT-uptake, indicating major involvemen t of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, w here clathrin-dependent endocytosis is blocked by induction of antisense cl athrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In c onclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different c ell types.