engleski

ENDOCYTIC PATHWAY(S) OF MHC CLASS I MOLECULES AND CHOLERA TOXIN B SUBUNIT ARE INTERSECTED

Problem The aim of our study was to compare the endocytotic pathway and intracellular localization of MHC class I (MHC-I) molecules (Kd and Dd) with cholera toxin B subunit (CTB) on Balb3T3 cell line. Materials and methods In order to investigate the internalization of CTB and MHC-I molecules we used different chemical inhibitors known to impair clathrin-mediated endocytosis (chlorpromazine) and endocytosis mediated by caveolae (filipin and β -cyclodextrin). Cells were analyzed by flow cytometry and confocal microscopy. To dissect intracellular pathway of CTB and MHC-I we used markers of subcellular compartments. Kinetics of degradation of investigated molecules was followed by flow cytometry and WB analysis. Results CTB was localized in Golgi apparatus (GA) following 30-45 minutes of incubation at 37 °C. However, the majority of CTB was found in the Lamp-1 compartments after 75 minutes. Filipin and nocodazol (the agens that disturbs microtubules) prevented transport of CTB from plasma membrane to GA while chlorpromazine, cytochalazin D (the agens that disturbs actin microfilaments) as well as inhibitors of vesicular acidification (monensin, bafilomycin A1 and ammonium chloride) had not this effect. It is important to mention that monensin did not prevent transport to GA, but it prevented degradation of CTB. Although, it is important to notice that filipin prevented internalization while nocodazol inhibited only vesicular transport to GA. Chlorpromazine had no effect on intermalization of MHC-I molecules, while filipin had only partial effect. Kd and Dd molecules were starting to colocalize with CTB not before 20 minutes and with transferrin (Tf) 10-15 minutes following internalization. Conclusion Kinetics of internalization and degradation of MHC- I molecules is slower then that of CTB. Although MHC- I molecules and CTB do not colocalize to each other at the early time points of the experiment, they are found in the same compartments after 20 minutes. We presume that CTB and MHC-I molecules are not internalized by the same mechanism but their pathways are intersected afterwards.

cholera toxin; endocytosis

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