Abstract
Protein delivery across polarized epithelia is controlled by receptor-mediated transcytosis. Many studies have examined basolateral-to-apical trafficking of polymeric IgA (pIgA) by the polymeric immunoglobulin receptor (pIgR). Less is known about apical-to-basolateral transcytosis, the direction the neonatal Fc receptor (FcRn) transports maternal IgGs across intestinal epithelia. To compare apical-to-basolateral and basolateral-to-apical transcytosis, we co-expressed FcRn and pIgR in Madin-Darby canine kidney (MDCK) cells and used pulse-chase experiments with confocal microscopy to examine transport of apically applied IgG Fcγ and basolaterally applied pIgA. Fcγ and pIgA trafficking routes were initially separate but intermixed at later chase times. Fcγ was first localized near the apical surface, but became more equally distributed across the cell, consistent with concomitant transcytosis and recycling. By contrast, pIgA transport was strongly unidirectional: pIgA shifted from near the basolateral surface to an apical location with increasing time. Some Fcγ and pIgA fluorescence colocalized in early (EEA1-positive), recycling (Rab11a-positive), and transferrin (Tf)-positive common/basolateral recycling endosomes. Fcγ became more enriched in Tf-positive endosomes with time, whereas pIgA was sorted from these compartments. Live-cell imaging revealed that vesicles containing Fcγ or pIgA shared similar mobility characteristics and were equivalently affected by depolymerizing microtubules, indicating that both trafficking routes depended to roughly the same extent on intact microtubules.
Highlights
Epithelia represent a fundamental type of multi-cellular organization, lining numerous internal and external surfaces of the body and forming a selective barrier between the lumen of an organ and the underlying tissue
The receptor is expressed on the basolateral surface of epithelial cells, where it binds to polymeric immunoglobulins secreted by local plasma cells [4,5,6]. polymeric immunoglobulin receptor (pIgR)–polymeric IgA (pIgA) complexes are internalized from the basolateral surface via clathrin-coated pits and delivered to basolateral early endosomes [7]
We extended these studies to compare the transcytosis mediated by FcRn, a bidirectionally transcytosing receptor, with the trafficking of pIgR, a unidirectional, basolateral-toapical transcytosing receptor
Summary
Epithelia represent a fundamental type of multi-cellular organization, lining numerous internal and external surfaces of the body and forming a selective barrier between the lumen of an organ and the underlying tissue. Less is known about receptors that transport ligands in the apical-to-basolateral direction One such receptor is the neonatal Fc receptor (FcRn), a class I MHC-related protein that transports maternal IgG across epithelial cell barriers to provide immunity to fetal or newborn www.traffic.dk 1205 mammals [17,18]. Much of what is known about transcytotic pathways comes from studies of pIgR-mediated transcytosis of pIgA in Madin–Darby canine kidney (MDCK) cells [12,33,34,35] This cultured cell line forms a well-polarized epithelial monolayer when grown on permeable filters [1]. A limited number of recent studies have begun to elucidate mechanistic aspects of FcRn-mediated transcytosis using RNA interference technology [42] and electron tomography [49]
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