engleski

Pathways of endosomal recycling of MHC-I proteins

Endosomal recycling is a highly regulated, dynamic and complex cellular process which, in coordination with endocytic uptake and lysosomal degradation, is essential for the structuring of the plasma membrane and membranous organelle composition. Consequently, endocytic recycling has an irreplaceable role in many cellular physiology processes and pathophysiological conditions. We will present our studies on the cellular physiology of recycling routes of Major Histocompatibility Class I (MHC-I) proteins. MHC-I proteins are present in at least two forms, as either fully conformed trimolecular MHC-I complexes of the heavy chain, beta-2-microglobulin and the peptide (full MHC-I) or as partially folded form devoid of the peptide (empty MHC-I). To study their endosomal trafficking we developed internalization and recycling assays based on immunofluorescence analysis by confocal microscopy and flow cytometry. Migration of MHC-I proteins through the endosomal system was analyzed by colocalization with a wide panel of endosomal markers. The physiology of endosomal subsets and domains was studied using rapidly acting chemical inhibitors of endosomal effector proteins and by RNA silencing of small GTPase of Arf and Rab family. MHC-I trafficking was analyzed in untreated cells and virus (murine cytomegalovirus, MCMV) infected cells. Quantitative analysis and kinetic modeling using the in-house developed software for multicompartment analysis displayed the three stages of early endosomal recycling of full MHC-I proteins: rapid, from pre-early endosomes, fast, from early endosome and slow from the juxtanuclear recycling compartment. MCMV manipulates early endosomal recycling domain by rapid downregulation of Rab proteins that regulate recycling (Rab22a, Rab4, and Rab11) and thereby reshape endosomal system to form virion assembly compartment. Empty MHC-I molecules rapidly recycle but are excluded from the fast and the slow recycling route. However, empty MHC-I molecules were found to recycle from late endosomes suggesting the existence of late endosomal recycling domain and the recycling route. Late endosomal recycling domain, which redirects almost a third of empty MHC towards the cell surface, is Rab7 independent, requires late endosomal motility and generation of recycling endocytic carriers. Studies on recycling of MHC-I proteins and kinetic modeling of endosomal trafficking contribute to the further integration of recycling pathways into cellular physiology.

endocytosis; recycling; MHC-I molecules; endosomal markers

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