Definition of distinct compartments in polarized Madin-Darby Canine Kidney(MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling

Previous studies of fibroblasts have demonstrated that recycling of endocyt ic receptors occurs through a default mechanism of membrane-volume sorting. Epithelial cells require an additional level of polar membrane sorting. bu t there are conflicting models of polar sorting, some suggesting that it oc curs in early endosomes, others suggesting it occurs in a specialized apica l recycling endosome (ARE). The relationship between endocytic sorting to t he lysosomal. recycling and transcytotic pathways in polarized cells was ad dressed by characterizing the endocytic itineraries of LDL, transferrin (Tf ) and IgA. respectively, in polarized Madin-Darby canine kidney (MDCK) cell s. Quantitative analyses of 3-dimensional images of living and fixed polari zed cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from Lot. into apical and medial recycling endosomes, in a manner consiste nt with default sorting of membrane from volume. While Tf is recycled to th e basolateral membrane from recycling endosomes, IgA is sorted to the ARE p rior to apical delivery. Quantifications of the efficiency of sorting of Ig A from Tf between the recycling endosomes and the ARE match biochemical mea surements of transepithelial protein transport, indicating that all polar s orting occurs in this step. Unlike fibroblasts, rab11 is not associated wit h Tf recycling compartments in either polarized or glass-grown MDCK cells, rather it is associated with the compartments to which IgA is directed afte r sorting from Tf. These results complicate a suggested homology between th e ARE and the fibroblast perinuclear recycling compartment and provide a fr amework that justifies previous conflicting models of polarized sorting.