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Journal of Cell Science 114, 3309-3321 (2001)
© 2001 The Company of Biologists Limited

RESEARCH ARTICLE

Brefeldin A rapidly disrupts plasma membrane polarity by blocking polar sorting in common endosomes of MDCK cells

Exing Wang1, Janice G. Pennington1, James R. Goldenring2, Walter Hunziker3 and Kenneth W. Dunn1,*

1 Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, IN 46202, USA
2 Departments of Medicine, Surgery and Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia and the Augusta VA Medical Center, Augusta, GA 30912, USA
3 Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609

*Author for correspondence (e-mail: kwdunn{at}iupui.edu)

Accepted May 28, 2001

Recent studies showing thorough intermixing of apical and basolateral endosomes have demonstrated that endocytic sorting is critical to maintaining the plasma membrane polarity of epithelial cells. Our studies of living, polarized cells show that disrupting endocytosis with brefeldin-A rapidly destroys the polarity of transferrin receptors in MDCK cells while having no effect on tight junctions. Brefeldin-A treatment induces tubulation of endosomes, but the sequential compartments and transport steps of the transcytotic pathway remain intact. Transferrin is sorted from LDL, but is then missorted from common endosomes to the apical recycling endosome, as identified by its nearly neutral pH, and association with GFP chimeras of Rabs 11a and 25. From the apical recycling endosome, transferrin is then directed to the apical plasma membrane. These data are consistent with a model in which polarized sorting of basolateral membrane proteins occurs via a brefeldin-A-sensitive process of segregation into basolateral recycling vesicles. Although disruption of polar sorting correlates with dissociation of {gamma}-adaptin from endosomes, {gamma}-adaptin does not appear to be specifically involved in sorting into recycling vesicles, as we find it associated with the transcytotic pathway, and particularly to the post-sorting transcytotic apical recycling endosome.

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Key words: Endocytosis, Polarity, Transferrin, Epithelium, MDCK cell


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© The Company of Biologists Ltd 2001