Preferences help
enabled [disable] Abstract
Number of results
2007 | 54 | 4 | 757-767
Article title

Distinct role of clathrin-mediated endocytosis in the functional uptake of cholera toxin

Title variants
Languages of publication
The involvement of the clathrin-mediated endocytic internalization route in the uptake of cholera toxin (CT) was investigated using different cell lines, including the human intestinal Caco-2 and T84 cell lines, green monkey Vero cells, SH-SY5Y neuroblastoma cells and Madin-Darby canine kidney cells. Suppression of the clathrin-mediated endocytic pathway by classical biochemical procedures, like intracellular acidification and potassium depletion, inhibited cholera toxin uptake by up to about 50% as well as its ability to raise intracellular levels of cAMP. Also prior exposure of these cell types to the cationic amphiphilic drug chlorpromazine reduced the functional uptake of cholera toxin, even to a greater extent. These effects were dose- and cell type-dependent, suggesting an involvement of clathrin-mediated endocytosis in the functional uptake of cholera toxin. For a more straightforward approach to study the role of the clathrin-mediated uptake in the internalization of cholera toxin, a Caco-2eps- cell line was exploited. These Caco-2eps- cells constitutively suppress the expression of epsin, an essential accessory protein of clathrin-mediated endocytosis, thereby selectively blocking this internalization route. CT uptake was found to be reduced by over 60% in Caco-2eps- paralleled by a diminished ability of CT to raise the level of cAMP. The data presented suggest that the clathrin-mediated uptake route fulfils an important role in the functional internalization of cholera toxin in several cell types.
Physical description
  • UA-Laboratory of Human Biochemistry, University of Antwerp, Antwerp, Belgium
  • UA-Laboratory of Human Biochemistry, University of Antwerp, Antwerp, Belgium
  • UA-Laboratory of Human Biochemistry, University of Antwerp, Antwerp, Belgium
  • Brech A, Kjeken R, Synnes M, Berg T, Roos N, Prydz K (1998) Endocytosed ricin and asialoorosomucoid follow different intracellular pathways in hepatocytes. Biochim Biophys Acta 1373: 195-208.
  • Carver LA, Schnitzer JE (2003) Caveolae: mining little caves for new cancer targets. Nat Rev Cancer 3: 571-581.
  • Damke H, Baba T, Warnock DE, Schmid SL (1994) Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol 127: 915-934.
  • de Haan L, Hirst R (2004) Cholera toxin: a paradigm for multi-functional engagement of cellular mechanisms. Mol Membr Biol 21: 77-92.
  • De Wolf MJS (2000) A dipeptide metalloendoprotease substrate completely blocks the response of cells in culture to cholera toxin. J Biol Chem 275: 30240-30247.
  • Ehrlich M, Boll W, Van Oijen A, Hariharan R, Chandran K, Nibert ML, Kirchhausen T (2004) Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118: 591-605.
  • Fishman PH, Orlandi PA (2003) Cholera toxin internalization and intoxication. J Cell Sci 116: 431-432.
  • Ford MG, Mills IG, Peter BJ, Vallis Y, Praefcke GJ, Evans PR, McMahon HT (2002) Curvature of clathrin-coated pits driven by epsin. Nature 419: 361-366.
  • Fra AM, Williamson E, Simons K, Parton RG (1994) Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae. J Biol Chem 269: 30745-30748.
  • Fraker PJ, Speck JC Jr (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem Biophys Res Commun 80: 849-857.
  • Harder T, Simons K (1997) Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains. Curr Opin Cell Biol 9: 534-542.
  • Ilangumaran S, Hoessli DC (1998) Effect of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane. Biochem J 335: 433-440.
  • Jobling MG, Holmes RK (2000) Identification of motifs in cholera toxin A1 polypeptide that are required for its interaction with human ADP-ribosylation factor 6 in a bacterial two-hybrid system. Proc Natl Acad Sci USA 97: 14662-14667.
  • Larkin JM, Brown MS, Goldstein JL, Anderson RG (1983) Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell 33: 273-285.
  • Ma Z, Lim LY (2003) Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles. Pharm Res 20: 1812-1819.
  • Massol RH, Larsen JE, Fujinaga Y, Lencer WI, Kirchhausen T (2004) Cholera toxin toxicity does not require functional Arf6- and dynamin-dependent endocytic pathways. Mol Biol Cell 15: 3631-3641.
  • Moya M, Dautry-Varsat A, Goud B, Louvard D, Boquet P (1985) Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin. J Cell Biol 101: 548-559.
  • Nichols BJ (2003) GM1-containing lipid rafts are depleted within clathrin-coated pits. Curr Biol 13: 686-690.
  • Orlandi PA, Fishman PH (1998) Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains. J Cell Biol 141: 905-915.
  • Parton RG (1996) Caveolae and caveolins. Curr Opin Cell Biol 8: 542-548.
  • Pearse B (1982) Coated vesicles from human placenta carry ferritin, transferrin, and immunoglobulin G. Proc Natl Acad Sci 79: 451-455.
  • Rink TJ, Montecucco C, Hesketh TR, Tsien RY (1980) Lymphocyte membrane potential assessed with fluorescent probes. Biochim Biophys Acta 595: 15-30.
  • Rodal SK, Scretting G, Garret O, Vilhardt F, van Deurs B, Sandvig K (1999) Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell 10: 961-974.
  • Sanders RB, Bekairi AM, Abulaban FS, Yochim JM (1986) Uterine adenylate cyclase in the rat: responses to a decidual-inducing stimulus. Biol Reprod 35: 100-105.
  • Sandvig K, Olsnes S, Petersen OW, van Deurs B (1987) Acidification of the cytosol inhibits endocytosis from coated pits. J Cell Biol 105: 679-689.
  • Schnitzer JE, Oh P, Pinney E, Allard J (1994) Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J Cell Biol 127: 1217-1232.
  • Shogomori H, Futerman AH (2001) Cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalised via raft-independent mechanism. J Biol Chem 276: 9182-9188.
  • Sofer A, Futerman AH (1995) Cationic amphiphilic drugs inhibit the internalization of cholera toxin to the Golgi apparatus and the subsequent elevation of cyclic AMP. J Biol Chem 270: 12117-12122.
  • Sorkin A, Carpenter G (1991) Dimerization of internalised epidermal growth factor receptors. J Biol Chem 266: 23453-23460.
  • Thomas JA, Buchsbaum RN, Zimniak A, Racker E (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18: 2210-2218.
  • Thomsen P, Roepstorff K, Stahlhut M, van Deurs B (2002) Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol Biol Cell 13: 238-250.
  • Torgersen ML, Skretting G, van Deurs B, Sandvig K (2001) Internalization of cholera toxin by different endocytic mechanisms. J Cell Sci 114: 3737-3747.
  • Tran D, Carpentier JL, Sawano F, Gorden P, Orsi L (1987) Ligands internalised through coated or noncoated invaginations follow a common intracellular pathway. Proc Natl Acad Sci 84: 7957-7961.
  • Vanden Broeck D, De Wolf MJS (2006) Selective blocking of clathrin-mediated endocytosis by RNA interference: epsin as target protein. Biotechniques 41: 475-484.
  • Vanden Broeck D, De Wolf MJS (2007) Vibrio cholerae: cholera toxin. Int J Biochem Cell Biol 39: 1771-1775.
  • Van Heyningen WE, Carpenter CC, Pierce NF, Greenough WB (1971) Deactivation of cholera toxin by ganglioside. J Infect Dis 124: 415-418.
  • Wang LH, Rothberg KG, Anderson RG (1993) Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol 123: 1107-1117.
  • WHO (2006) Cholera 2005. Wkly Epidemiol Rec 81: 297-308.
  • Wolf AA, Fujinaga Y, Lencer WI (2002) Uncoupling of the cholera toxin GM1 ganglioside-receptor complex from endocytosis, retrograde Golgi trafficking, and down-stream signal transduction by depletion of membrane cholesterol. J Biol Chem 277: 16249-16256.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.