Submitted on April 15, 2004
Accepted on July 28, 2004
Trafficking of cholera toxin-ganglioside GM1 complex into the Golgi and the induction of toxicity depend on the actin cytoskeleton
Kamran Badizadegan1*, Heidi E Wheeler2, Yukako Fujinaga3, and Wayne I Lencer4
1 Department of Pathology, Massachusetts General Hospital, Boston, MA, USA; Department of Pathology, Harvard Medical School, Boston, MA, USA; GI Cell Biology, Children's Hospital Boston, Boston, MA, USA
2 GI Cell Biology, Children's Hospital Boston, Boston, MA, USA
3 Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan; GI Cell Biology, Children's Hospital Boston, Boston, MA, USA
4 GI Cell Biology, Children's Hospital Boston, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA; Harvard Digestive Diseases Center, Boston, MA, USA
* To whom correspondence should be addressed. E-mail: kbadizadegan{at}partners.org.
Intestinal epithelial lipid rafts contain ganglioside GM1 that is the receptor for cholera toxin (CT). The ganglioside binds CT at the plasma membrane (PM) and carries the toxin through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER). In the ER, a portion of the toxin unfolds and translocates to the cytosol to activate adenylyl cyclase. Activation of the cyclase leads to an increase in intracellular cAMP, which results in apical chloride secretion. Here, we find that an intact actin cytoskeleton is necessary for the efficient transport of CT to the Golgi and for subsequent activation of the adenylyl cyclase. CT bound to GM1 on the cell membrane fractionates with a heterogeneous population of lipid rafts, a portion of which is enriched in actin and other cytoskeletal proteins. In this actin-rich fraction of lipid rafts, CT and actin co-localize on the same membrane microdomains, suggesting a possible functional association. Depolymerization or stabilization of actin filaments interferes with transport of CT from the PM to the Golgi, and reduces the levels of cAMP generated in the cytosol. Depletion of membrane cholesterol, which also inhibits CT trafficking to the TGN, causes displacement of actin from the lipid rafts, while CT remains stably raft-associated. Based on these observations, we propose that the CT-GM1 complex is associated with the actin cytoskeleton via the lipid rafts, and that the actin cytoskeleton plays a role in trafficking of CT from the PM to the Golgi/ER and the subsequent activation of the adenylyl cyclase.
