engleski

Chasing recycled MHC class I molecules. 4th European Congress of Immunology

After endocytic uptake, membrane proteins can be directed either to the degradation or can be recycled back to the cell surface. Recycling route is best studied for transferrin receptor (TfR) but it is still questionable whether basic mechanisms established for TfR can be simply replicated for other membrane proteins. In this study, we tested several recycling assays that are based on labeling of cell surface proteins with high-affinity monoclonal antibodies and their detection after recycling by immunofluorescent analysis. These assays were tested on TfR and fully conformed MHC-I molecules. Additionally, these assays were extended by testing also peptide-empty MHC-I proteins that do not recycle from the early endosomal system and thereby, allow quantitative assessment of the endocytic rate of internalized proteins. Considering that endocytic rate for MHC-I molecules cannot be directly measured, we developed the mathematical model, the first-order dynamics compartment model. We showed that two most frequently used protocols, which are based on the detection of recycled proteins at the cell surface, are not useful for characterization of recycling of rapidly re-internalizing proteins (TfR). However, these protocols could be used for analysis of recycling of molecules which have lower endocytic rate (MHC-I). In contrast, the third protocol, based on secondary antibody catching of mAb-labeled recycling receptor can detect both recycled TfRs and MHC-I proteins with similar efficiency. These protocols enabled us to calculate that MHC-I proteins recycle from the early endosomal system with lower recycling efficiency (~40%) than TfR (~90%) although this two molecules follow similar endosomal route

MHC-I molecules; recycling; endocytosis

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