1. Division of Biology 114-96 and,
2. Howard Hughes Medical Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA
3. Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543
4. Boulder Laboratory for 3D Electron Microscopy of Cells, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA

Correspondence to: Pamela J. Björkman^1,2 Correspondence and requests for materials should be addressed to P.J.B. (Email: bjorkman@caltech.edu).

Abstract

The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers^{1, 2}, thereby providing the fetus or newborn with humoral immunity before its immune system is fully functional. In newborn rats, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. The pH difference between the apical (pH 6.0–6.5) and basolateral (pH 7.4) sides of intestinal epithelial cells facilitates the efficient unidirectional transport of IgG, because FcRn binds IgG at pH 6.0–6.5 but not at pH 7 or more^{1, 2}. As milk passes through the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum and jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum)^{3, 4, 5, 6}. Here we use electron tomography to make jejunal transcytosis visible directly in space and time, developing new labelling and detection methods to map individual nanogold-labelled Fc within transport vesicles⁷ and simultaneously to characterize these vesicles by immunolabelling. Combining electron tomography with a non-perturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine whether a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moves through networks of entangled tubular and irregular vesicles, only some of which are microtubule-associated, as it migrates to the basolateral surface. New features of transcytosis are elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis through clathrin-coated pits. Markers for early, late and recycling endosomes each labelled vesicles in different and overlapping morphological classes, revealing spatial complexity in endo-lysosomal trafficking.

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