engleski

Comparison of MHC class I molecules and Cholera toxin B subunit endocytic pathway(s)

Problem The aim of our study was to compare the endocytotic pathway and intracellular localization of MHC class I molecules (Kd and Dd) with cholera toxin B subunit (ChtxB) on murine fibroblasts. Materials and methods In order to investigate the endocytotic pathway and intracellular trafficking of ChtxB and MHC class I molecules we used flow cytometry and immunoflurescence microscopy and to disect their intracellular pathway a panel of chemical inhibitors of endocytosis and vesicular transport was used. Furthermore, the intracellular localization of those molecules was determinated by using markers of subcellular compartments. Kinetics of degradation of investigated molecules was followed by flow cytometry and WB analysis. Results ChtxB was localized in Golgi compartments following 30-45 minutes of incubation at 37 °C. However, the majority of ChtxB was found in the Lamp-1 acid compartments after 75 minutes. Filipin (an inhibitor of caveolar endocytosis) and nocodazol (the agens that disturbs microtubules) prevented transport of CTB from plasma membrane to Golgi while chlorpromazine (an inhibitor of clathrin endocytosis), 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 Golgi apparatus (GA), but it prevented degradation of CtxB. Although, it is important to notice that filipin prevented internalization while nocodazol inhibited only vesicular transport to GA. Kd and Dd molecules were starting to colocalize with ChtxB not before 20 minutes and with transferin (Tf) 10-15 min following internalization. Conclusion Kinetics of internalization and degradation of MHC class I molecules is slower then that of CtxB. Although MHC class I molecules and CtxB 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 CtxB and MHC class I molecules are not internalized by the same mechanism but their pathways are intersected afterwards.

cholera toxin; vesicular transport; MHC class I molecules; endocytosis

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