For steady-state experiments, Au-Fc was fed to neonatal rats, rather than incubated with excised intestines, which causes morphological changes5

For steady-state experiments, Au-Fc was fed to neonatal rats, rather than incubated with excised intestines, which causes morphological changes5. tomography to directly visualize jejunal transcytosis in space and time, developing new labeling and detection methods to map individual nanogold-labeled Fc within transport vesicles7 and to simultaneously characterize these vesicles by immunolabeling. Combining electron tomography with a non-perturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine if a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moved through networks of entangled tubular and irregular vesicles, only some of which were microtubule-associated, as it migrated to the basolateral surface. New features of transcytosis were elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis via clathrin-coated pits. Markers for early, Glyoxalase I inhibitor free base late, and recycling endosomes each labeled vesicles in different Glyoxalase I inhibitor free base and overlapping morphological classes, revealing unexpected spatial complexity in endo-lysosomal trafficking. To prevent ligand misdirection caused by a bulky label, we covalently attached small (1.4nm) Nanogold to IgG-Fc (Au-Fc) at a site distant from where FcRn binds7. To avoid attaching >1 ligand/gold, which could artifactually prolong release through avidity, we used monofunctional Nanogold and purified Au-Fc by sizing and FcRn-affinity chromatography7. Glyoxalase I inhibitor free base For steady-state experiments, Au-Fc was fed to neonatal rats, rather than incubated with excised intestines, which causes morphological changes5. The concentration of ingested Au-Fc was approximately equal to IgG in rat milk, because previously-used higher concentrations5 saturated FcRn, resulting in degradation of excess IgG8. Intestinal samples were prepared for electron tomography by high pressure freezing, freeze-substitution fixation (HPF/FSF), the most accurate method for preserving dynamic trafficking events and ultrastructure9, and we developed methods to enlarge endocytosed Nanogold during FSF7,10. Internal controls verified that enlarged gold accurately marked transported Au-Fc: (i) Gold was in physiologically-relevant locations (apical surface, tubulovesicular compartments in proximal cells; inside degradative compartments in distal cells), but not in nuclei, mitochondria, the ER or Golgi (Fig.?(Fig.11-?-5;5; Supplementary Fig.S1-S7); (ii) 98% of particles in proximal cells were 6-7nm from a membrane (Supplementary Table S1), consistent with Au-Fc bound to FcRn; Glyoxalase I inhibitor free base and (iii) Au-Fc, but not Au-dextran, was enhanced in proximal (FcRn-positive) cells, whereas both were enhanced in distal (FcRn-negative) cells, reflecting receptor-mediated and fluid-phase uptake in the proximal and distal intestine, respectively (Fig.1a; Supplementary Fig.S7). Open in a separate window Figure 1 Au-Fc uptake in intestinal cellsBar=2m (b); 300nm (c); 200nm (d-g). (a) Chemically-fixed/gold-enhanced intestinal Glyoxalase I inhibitor free base samples from the duodenum (D), jejunum (J) and ileum (I) of Au-Fc-fed, Au-dextran-fed, and control rats (Supplementary Fig.S7 shows corresponding projections). (b-c) Projections of jejunal sections showing Regions 1-3 (b; 180nm section) or 1 (c; 150nm section). (See also Supplementary Fig.S3-S6.) TJ: tight junction; NU: nucleus; M: mitochondrion. White arrows: Au-Fc in RTVs. (d-g) Tomograhic slices showing Au-Fc in Region 1 on (d) microvilli, (e) coated pit and apical irregular vesicle, (f) RTVs, apical irregular vesicles, (g) RTV proximal to a coated pit. (h-i) Histograms constructed using the immunolabeling data in Supplementary Table S2. Ap-CP: apical coated pits/coated vesicles; Ap-IV: apical irregular vesicles; Bl-CP/CV: basolateral coated pits/coated vesicles; LYS: lysosomes; SmV: 30-40nm vesicles related to lysosomes. (h) Percent of each marker found in each structure. (i) Percent of each structure that was positive for each marker. Open in a separate window Figure 5 Jejunal LIS and schematic pathwaysBar =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) Hexagonal (left) and heptagonal (right) clathrin lattices (chemically-fixed samples). (c-g) Au-Fc in coated pits. (c and d are projections.) (h) Schematic of FcRn-mediated transcytosis. TJ: tight junctions; NU: nucleus; green: apical membrane; cyan: basolateral membrane; red dashes: clathrin; pink straws: microtubules; gold spheres: Au-Fc; black arrows: early trafficking steps; gray arrows: later trafficking steps; green arrow: endocytosis of FcRn-containing coated pit after ligand release; cyan arrow: excess Au-Fc transferred to late endosome (rarely observed). Multiple arrows indicate parallel pathways. Bmp1 Vesicles shown using schematic versions (Supplementary Fig.S8) in a lower denseness and larger size than observed. Amounts beside schematic vesicles make reference to endosomal markers (1=EEA1, 5=Rab5, 7=Rab7, 9=Rab9, 11=Rab11); the font size demonstrates relative levels of label in each kind of area (discover Fig.1i). A lot more than 50 tomograms, each ~1.8 m3, had been recorded from jejunal cells from Au-Fc-fed neonatal rats (steady-state tests) (Supplementary Desk S1). For kinetic evaluation, ligated intestinal lumens had been incubated with Au-Fc (>50 pulse or pulse/run after tomograms or projections) (Supplementary Desk S3). We described three jejunal.