Abstract
The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers, 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. 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). 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 vesicles 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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🧪 Sample Preparation
🔬 Cell Lines
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🏛️ Research Organizations (ROR)
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📋 Methods
SUMMARY Uptake of Au-Fc and Au-dextran 1.4nm monomaleimido-Nanogold was attached to reduced cysteine(s) in the rat Fc hinge, and sulfo-NHS-Nanogold was attached to dextran primary amines as described 7 . Labeled molecules were purified by FcRn affinity chromatography and/or gel filtration chromatography. For steady-state and immunolabeling experiments, 11- to 13-day suckling rats were fed 3×100μl of Au-Fc or Au-dextran (~3μM), after which the small intestine was harvested for chemical or cryofixation. For kinetic experiments, ligated intestines were incubated with 3μM Au-Fc (pulse experiments), followed by unlabeled Fc or IgG (pulse/chase experiments).
Gold enlargement procedures
Intestinal segments were chemically fixed, treated with GoldEnhance–EM 2113 (Nanoprobes, Inc.), and fixed/stained with OsO 4 and uranyl acetate as described 7 . Traditional pre-embedding enhancement protocols are incompatible with FSF, therefore we developed the methods for enlarging small gold clusters in the cold organic solvents used during FSF 7 , 10 . After HPF of intestinal segments, we used a three-step enlarging protocol involving silver enhancement to slightly enlarge (≤8nm) the Nanogold 10 ; coating the silver shell with gold to make it impervious to osmium; and enlargement to 10-16nm using gold enhancement 7 . Samples were then treated with OsO 4 and uranyl acetate and warmed to room temperature.
Microscopy and 3D modeling
HPF/FSF or chemically-fixed samples were infiltrated with resin, polymerized, cut into 120-200nm sections, and examined using an FEI T12 transmission electron microscope operating at 120 kV. For tomography, dual axis tilt series were collected at 6500x from 120-200nm sections at 700nm underfocus. Tomograms were computed for each tilt axis using enlarged golds as alignment markers, and then aligned and combined to form a dual-axis tomogram using IMOD 28 . Unless otherwise indicated, tomographic slices were 1.6nm. Tomographic reconstructions were interpreted and modeled by manual segmentation using IMOD 28 . Immunolabeling Immunolabeling was done on gold-enhanced intestinal samples using a modification of the Tokuyasu method for immunolabeling cryosections 29 .
Show full methods section
SUMMARY Uptake of Au-Fc and Au-dextran 1.4nm monomaleimido-Nanogold was attached to reduced cysteine(s) in the rat Fc hinge, and sulfo-NHS-Nanogold was attached to dextran primary amines as described 7 . Labeled molecules were purified by FcRn affinity chromatography and/or gel filtration chromatography. For steady-state and immunolabeling experiments, 11- to 13-day suckling rats were fed 3×100μl of Au-Fc or Au-dextran (~3μM), after which the small intestine was harvested for chemical or cryofixation. For kinetic experiments, ligated intestines were incubated with 3μM Au-Fc (pulse experiments), followed by unlabeled Fc or IgG (pulse/chase experiments).
Gold enlargement procedures
Intestinal segments were chemically fixed, treated with GoldEnhance–EM 2113 (Nanoprobes, Inc.), and fixed/stained with OsO 4 and uranyl acetate as described 7 . Traditional pre-embedding enhancement protocols are incompatible with FSF, therefore we developed the methods for enlarging small gold clusters in the cold organic solvents used during FSF 7 , 10 . After HPF of intestinal segments, we used a three-step enlarging protocol involving silver enhancement to slightly enlarge (≤8nm) the Nanogold 10 ; coating the silver shell with gold to make it impervious to osmium; and enlargement to 10-16nm using gold enhancement 7 . Samples were then treated with OsO 4 and uranyl acetate and warmed to room temperature.
Microscopy and 3D modeling
HPF/FSF or chemically-fixed samples were infiltrated with resin, polymerized, cut into 120-200nm sections, and examined using an FEI T12 transmission electron microscope operating at 120 kV. For tomography, dual axis tilt series were collected at 6500x from 120-200nm sections at 700nm underfocus. Tomograms were computed for each tilt axis using enlarged golds as alignment markers, and then aligned and combined to form a dual-axis tomogram using IMOD 28 . Unless otherwise indicated, tomographic slices were 1.6nm. Tomographic reconstructions were interpreted and modeled by manual segmentation using IMOD 28 . Immunolabeling Immunolabeling was done on gold-enhanced intestinal samples using a modification of the Tokuyasu method for immunolabeling cryosections 29 .
Gold enlargement procedures
Intestinal segments were chemically fixed, treated with GoldEnhance–EM 2113 (Nanoprobes, Inc.), and fixed/stained with OsO 4 and uranyl acetate as described 7 . Traditional pre-embedding enhancement protocols are incompatible with FSF, therefore we developed the methods for enlarging small gold clusters in the cold organic solvents used during FSF 7 , 10 . After HPF of intestinal segments, we used a three-step enlarging protocol involving silver enhancement to slightly enlarge (≤8nm) the Nanogold 10 ; coating the silver shell with gold to make it impervious to osmium; and enlargement to 10-16nm using gold enhancement 7 . Samples were then treated with OsO 4 and uranyl acetate and warmed to room temperature.
📊 Figures
Figure 1
Au-Fc uptake in intestinal cells
Bar=2u03bcm (b); 300nm (c); 200nm (d-g). (a) Chemically-fixed/gold-enhanced intestinal samples from the duodenum (D), jejunum (J) and ileum (I) of Au-Fc-fed, Au-dextran-fed, and control rats ( Supplem...
Figure 2
Gold-containing tubular vesicles from jejunal Regions 1-2
Bar=100nm (a-d,e) or 200nm (f-h). c is chemically-fixed; others are HPF/FSF. White arrowheads: RTVs; white arrows: Au-Fc in RTVs; red arrowheads: ITVs; red arrows: Au-Fc in ITVs. Colours for segmented...
Figure 3
Gold-containing irregular vesicles in jejunal Regions 2-3
Bar=100nm. Tomographic slices (a-c) and models (d-e; coloured as in Fig. 2 ). (a-c) INTVs (asterisk in a). ITV (red arrows in a). (d-e) Segmented models, coloured as in Fig.2 . (d) ITV. (e) The cellul...
Figure 4
Gold-containing MVBs in jejunal cells
Bar=200nm. Tomographic slices (a-e) and model (f; coloured as in Fig.2 ). White arrows: Au-Fc on MVB main and inner vesicle membranes; red arrow: Au-Fc in a spherical vesicle; red arrowheads: fibrous ...
Figure 5
Jejunal LIS and schematic pathways
Bar =200nm (a,c-g) or 50nm (b). White arrows: Au-Fc in LIS; red arrows: Au-Fc in irregular vesicles. (a) LIS and vicinity as projection (left) and segmented model (right; coloured as in Fig.2 ). (b) H...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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