Trafficking of cholera toxin-ganglioside G_M1 complex into Golgi and induction of toxicity depend on actin cytoskeleton

Abstract

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 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 colocalize 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. On the basis of 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 adenylyl cyclase.

Bacterial protein toxins and lipids: role in toxin targeting and activity

“…Moreover, the addition of GM1 to cells expressing low levels of GM1 enhances caveolae/raftdependent endocytosis (Pang et al, 2003). Lipid rafts containing CT-GM1 complexes are associated with the actin cytoskeleton which is involved in the intracellular trafficking from plasma membrane to the Golgi and ER (Badizadegan et al, 2004). Cholesterol might also be involved in the sorting of CT-GM1 complexes, since agents, such as β-cyclodextrin and filipin, which immobilize or sequester cholesterol and, thus, disrupt lipid microdomains, prevent the transport of CT to the Golgi (Orlandi and Fishman, 1998;Wolf et al, 1998).…”

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: Roles of NADPH oxidase, mitochondria, and lipid rafts

“…A biochemical fractionation of cells by floating sucrose gradient was used as previously described for DRM analysis [14,26]. Briefly, cells grown in 15 cm dishes were treated as described above to obtain SLO-HK, Chol-HK and HK (4-5 × 10 6 cells per plate).…”

“…In this procedure, proteins associated with DRM, which include lipid rafts, are found in a more buoyant fraction than detergent-solubilized proteins [14]. Proteins in cell lysates were separated on a 5-35-40% sucrose-DEB discontinuous gradient and DRM are expected at the 5-35% interface [14,26]. After the centrifugation, 1 ml fractions were immunoblotted using antibodies against flotillin-2 and caveolin-1, proteins associated with lipid rafts when Triton X-100 is used [14,37,38].…”

Gangliosides Are Important for the Preservation of the Structure and Organization of RBL-2H3 Mast Cells

“…The connection between lipid raft proteins and actin filaments can affect the lateral distribution and mobility of these membrane proteins (Rodgers and Glaser 1993;Lenne et al 2006). The presence of actin and other cytoskeletal proteins in lipid raft fractions has been known since the earliest description of these structures (Jordan and Rodgers 2003;Badizadegan et al 2004), and proteomic analyses have documented the presence of numerous cytoskeletal proteins in these fractions (Nebl et al 2002). Furthermore, cholesterol depletion, which is often regarded as a functional test of dependence on lipid rafts, is associated with loss of phosphatidylinositol 4,5-biphosphate from the plasma membrane and a global reorganization of the actin cytoskeleton (Kwik et al 2003), suggesting a possible role for actin in the organization and/or function of lipid rafts.…”