engleski

Cholera toxin uses multiple trafficking pathways from plasma membrane to Golgi

Problem The aim of our study was to characteriz endocytotic pathway(s) and intracellular trafficking of cholera toxin binding subunit (CTB) in murine fibroblasts (Balb3T3) and human epithelial cells (HeLa). Materials and methods In order to investigate the endocytotic pathway and intracellular trafficking of CTB, we used flow cytometry and immunoflurescence confocal microscopy . To disect entry and intracellular pathway a panel of chemical inhibitors of endocytosis and vesicular transport was used. Furthermore, the intracellular localization of CTB was determinated by using markers of subcellular compartments. Kinetics of degradation was followed by flow cytometry and WB analysis. Results Following 30 minutes of incubation at 37 °C CTB was found in Golgi apparatus (GA) on HeLa cell line and after 45 minutes on Balb3T3 cell line. The majority of CTB was colocalizated with marker of lysosomes after 75 minutes on Balb3T3 cell line but not on HeLa cell line. On both cell lines CTB was colocalizated with caveolin-1 and transferrin but following different kinetics while CTB is colocalizated with early endosomes only on HeLa cell line. Filipin (an inhibitor of caveolar endocytosis) and chlorpromazine (an inhibitor of clathrin endocytosis) only partially prevented transport of CTB from plasma membrane to GA. Cytochalazin D (the agens that disturbs actin microfilaments) had no effect. Nocodazol (the agens that disturbs microtubules) on both cell lines inhibited only vesicular transport to GA but had not effect on internalization of CTB.. Inhibitors of vesicular acidification (monensin, bafilomycin A1 and ammonium chloride) inhibited CTB transport to GA only on HeLa cell line. Importantly, monensin did not prevent transport to GA on Balb3T3, but it prevented transport to lysosomes and degradation of CTB. Conclusion From these results we can conclude that CTB enters host cells via multiple routes (clathrin-, caveolar-, and lipid raft dependent endocytic pathway) but it remains plausible that a particular cell type might use one pathway in preference to others. On both investigated cell lines CTB reaches GA but kinetics is slower on Balb3T3 cell line. Also we observed that early endosomal compartment is involved in its intracellular trafficking only on HeLa cell line.

cholera toxin; vesicular transport

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