Filipin-dependent Inhibition of Cholera Toxin: Evidence for Toxin Internalization and Activation through Caveolae-like Domains

Abstract

The mechanism by which cholera toxin (CT) is internalized from the plasma membrane before its intracellular reduction and subsequent activation of adenylyl cyclase is not well understood. Ganglioside GM1, the receptor for CT, is predominantly clustered in detergent-insoluble glycolipid rafts and in caveolae, noncoated, cholesterol-rich invaginations on the plasma membrane. In this study, we used filipin, a sterol-binding agent that disrupts caveolae and caveolae-like structures, to explore their role in the internalization and activation of CT in CaCo-2 human intestinal epithelial cells. When toxin internalization was quantified, only 33% of surface-bound toxin was internalized by filipin-treated cells within 1 h compared with 79% in untreated cells. However, CT activation as determined by its reduction to form the A1 peptide and CT activity as measured by cyclic AMP accumulation were inhibited in filipin-treated cells. Another sterol-binding agent, 2-hydroxy-β-cyclodextrin, gave comparable results. The cationic amphiphilic drug chlorpromazine, an inhibitor of clathrin-dependent, receptor-mediated endocytosis, however, affected neither CT internalization, activation, nor activity in contrast to its inhibitory effects on diphtheria toxin cytotoxicity. As filipin did not inhibit the latter, the two drugs appeared to distinguish between caveolae- and coated pit–mediated processes. In addition to its effects in CaCo-2 cells that express low levels of caveolin, filipin also inhibited CT activity in human epidermoid carcinoma A431 and Jurkat T lymphoma cells that are, respectively, rich in or lack caveolin. Thus, filipin inhibition correlated more closely with alterations in the biochemical characteristics of CT-bound membranes due to the interactions of filipin with cholesterol rather than with the expressed levels of caveolin and caveolar structure. Our results indicated that the internalization and activation of CT was dependent on and mediated through cholesterol- and glycolipid-rich microdomains at the plasma membrane rather than through a specific morphological structure and that these glycolipid microdomains have the necessary components required to mediate endocytosis.

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

“…13 Therefore, we also examined the effect of nystatin, a reagent that binds to membrane cholesterol and disrupts internalization through the caveolae. 22,23 Nystatin treatment had no significant effect on the internalization of R8 or STR-R8, suggesting that caveolae formation is not involved in the internalization of these peptides.…”

“…5,20 We also examined the effect of nystatin, a reagent that disrupts internalization via the caveolae. 20,22,23 Cells were preincubated at 41C, in the presence of sucrose, NEM, or nystatin followed by the addition of FITC-labeled R8 or STR-R8 and further incubated for different times as shown under 'Materials and methods'. At the end of the incubation, the cells were fixed and observed by confocal laser microscopy.…”

“…The concentrations and preincubation times for these inhibitors were established by referring to literature references. [18][19][20][21][22][23] The Figure 1 Cellular uptake of R8 and STR-R8 peptides in NIH/3T3 cells. NIH/3T3 cells were incubated with serum-free DMEM medium containing 2 mM FITC-labeled R8 (b), or 1 mM FITC-labeled STR-R8 (c).…”

The Ad5 fiber mediates nonviral gene transfer in the absence of the whole virus, utilizing a novel cell entry pathway

“…29 Growth factors and certain toxins also induce non-clathrin-mediated, pinocytic uptake. 30,31 AAV particles, which utilize HS-GAGs for infection, can be found in both clathrin-coated and noncoated vesicles. 32 The fiber uptake observed here likely explains the mechanism of FPO gene transfer.…”

Membrane Fusion Mediated Intracellular Delivery of Lipid Bilayer Coated Mesoporous Silica Nanoparticles

“…[27,[50][51][52][53][54] In this study, wortmannin (Wor) was used as a micropinocytosis inhibitor as it blocks PI3-kinase, [50,52,55,56] while genistein (Gen) inhibits tyrosine-phosphorylation of Cav 1 and caveolin-dependent endocytosis. [57][58][59] Furthermore, chlorpromazine (Chl) was used as it blocks clathrin-dependent endocytosis, [51,60,61] and microtubule formation was inhibited by nocodazole (Noc). Uptake studies in the presence of these inhibitors give insight in the intracellular trafficking and internalization mechanisms involved in the uptake of the lipid bilayer coated MSNs.…”