Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism.
We have evaluated transcytotic routes in MDCK cells for their ability to generate a polarized surface distribution of trafficking proteins by following the intracellular sorting of transferrin receptors (TRs). We find that the selective basolateral expression of TRs is maintained in the face of extensive trafficking between the apical and basolateral surfaces. Biochemical studies of receptors loaded with tracer under conditions approaching steady state indicate that TRs internalized from the two surfaces are extensively colocalized within MDCK cells and that both populations of receptors are selectively delivered to the basolateral surface. Tailless TRs in which the cytoplasmic domain has been deleted display an unpolarized cell surface distribution and recycle in an unpolarized fashion. We show by EM that wild-type receptors internalized from each surface are colocalized within endosomal elements distributed throughout the cytoplasm. By preloading endosomal elements directly accessible from the basolateral surface with transferrin (Tf)-HRP, we show that apically internalized TRs rapidly enter the same compartment. We also show that both transcytosing (apically internalized) and recycling (basolaterally internalized) TRs are delivered to the basolateral border by a distinctive subset of exocytotic, 60-nm-diam vesicles. Together, the biochemical and morphological data show that apical and basolateral endosomes of MDCK cells are interconnected and contain a signal-dependent polarized sorting mechanism. We propose a dynamic model of polarized sorting in MDCK cells in which a single endosome-based, signal-dependent sorting step is sufficient to maintain the polarized phenotype.
Odorizzi
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G
Department of Cancer Biology, Salk Institute for Biological Studies, San Diego, California 92186-5800, USA.
Pearse
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A
Domingo
D
D
Trowbridge
I S
IS
Hopkins
C R
CR
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses; D001692:Biological Transport; D002451:Cell Compartmentation / Q000502:physiology*; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology*; D003593:Cytoplasm / Q000737:chemistry; D004285:Dogs; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry / Q000502:physiology*; D006735:Horseradish Peroxidase / Q000235:genetics; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D011990:Receptors, Transferrin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011991:Receptors, Virus / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000502:physiology
D011990:Receptors, Transferrin; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D006735:Horseradish Peroxidase