Apical endocytosis in rat hepatocytes in situ involves clathrin, traverses a subapical compartment, and leads to lysosomes  Christoph Rahner, *, Bruno Stieger, ‡, Lukas Landmann, *  Gastroenterology  Volume 119, Issue 6, Pages 1692-1707 (December 2000) DOI: 10.1053/gast.2000.20233 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Examples for adequate (A) and poor (B) retrograde infusion of endocytic marker. The panels show confocal fluorescence images superimposed on pictures taken in differential interference contrast. (A) Dextran-TxR (red signal) that has been infused for 10 minutes through the common bile duct is confined to the biliary space (C, canaliculi) with no signal in the blood space (S, sinusoids) or Kupffer cells (K). (B) Inadequate preparations according to the same protocol show marker in the canaliculi (C) and after penetration of the hepatocellular tight junctions and dissipation in the blood compartment in sinusoidal Kupffer (K) and endothelial (E) cells. Bar = 5 μm. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Surface renderings of confocal image stacks that were obtained from 10-μm cryostat sections of livers infused retrogradely with a fluid-phase marker (Dx-TxR; A and B) or with a marker associated predominantly with the PM (3 MAbs with canalicular domain specificity [3xcan]; see Materials and Methods; G, H). After short infusion times (A, 5 minutes; G, 10 minutes), marker was confined to the canalicular lumen and associated predominantly with the apical PM. After longer time intervals it showed an additional punctate distribution pattern that spread with time from the subapical (pericanalicular) cytoplasm to the perinuclear area (B, 10 minutes; H, 15 minutes). Number and size of marker-positive structures were increased with prolonged infusion times. Distribution patterns of the 2 markers did not differ except for a temporal delay in internalization of 3xcan MAbs. Bar = 5 μm. Electron micrographs of livers infused retrogradely with HRP for various time intervals show that this fluid-phase marker is initially found in 60–100 nm tubulovesicular structures close to the apical PM (C, 5 minutes). MVBs (D) and cup-shaped vesicles (150–200 nm, E) in the apical and perinuclear area became labeled with retardation (10 minutes) but well before lysosomes (F, 15 minutes). Bar = 500 nm. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Singular confocal image from rat liver infused retrogradely with Dx-TxR (red) for 8 minutes. Semithin cryosections (0.5–1 μm) were probed with ricin-fluorescein isothiocyanate (green) and 3xcan MAb (blue). The apical PM and pericanalicular vesicles displayed overlap of all 3 probes (white) indicating biochemical similarity of their membranes. C, canaliculus; N, nucleus; S, sinusoids. Bar = 5 μm. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Subcellular distribution of clathrin: 0.5–1 μm cryosections from retrogradely infused livers were stained for clathrin by indirect fluorescence. (A) To minimize false-positive signal a stack of confocal optical sections was deconvoluted by the Imaris Huygens module operating in the maximum likelihood estimation mode and visualized as MIP. Clathrin (Cy5, represented in false color as green) was expressed in a number of locations including the basolateral PM, the perinuclear and pericanalicular area. Colocalization of endocytic marker (red, Dx-TxR, 10 minutes) and immunolabel for clathrin (green) is highlighted in yellow and localized mostly in close proximity to the canalicular PM. (B) The same stack of unprocessed confocal images in MIP mode. C, canaliculus; N, nucleus; S, sinusoid. Bar = 5 μm. (C) Immuno-EM shows the association of immunogold (arrows) with the clearly visible coat of pits (top) and vesicles (bottom) in or close to the apical PM. Bar = 500 nm. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Endocytic marker (red, Dx-TxR, 10 minutes) shows overlap (yellow) with compartments displaying immunoreactivity for cathepsin D (green, Cy2). This hydrolytic enzyme is expressed in lysosomes and late endosomes. Its partial colocalization with dextran indicates that lysosomes are accessible from the apical PM. (A) Deconvoluted image stack in MIP. (B) Unprocessed images show topographic features: C, canaliculus; N, nucleus. Bar = 5 μm. (C) Immuno-EM shows the association of immunogold (arrows) with lysosomes (top) and some (bottom) but not all MVBs. Bar = 500 nm. conds, sections were examined in a Philips CM10 electron microscope. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Endocytic marker (green, 3xcan, Cy2, 15 minutes) shows overlap (yellow) with compartments immunoreacting with Abs against rab7 (red, Cy3). This small GTPase is associated with late endosomes and its subcellular distribution was confined to perinuclear and subapical areas. Colocalization indicates accessibility of late endosomes for the apical endocytic pathway. (A) MIP of deconvoluted confocal images. (B) Unprocessed images allow orientation: C, canaliculus; N, nuclei; S, sinusoids. Bar = 5 μm. (C) Immunogold was associated with MVBs at the EM level. Bar = 500 nm. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Rat livers were infused retrogradely with 3xcan (green, Cy2, apical pathway) and anterogradely with HRP (red, TxR, basolateral pathway) (see Materials and Methods). Semithin sections were probed with antibodies (blue, Cy5) against rab7 (A and B) and cathepsin D (C and D). A and D show MIP of deconvoluted image stacks, B and C stacks of unprocessed images. C, canaliculus; N, nucleus; S, sinusoid. Overlap of basolateral marker and immunolabel (magenta) indicates late endosomes and lysosomes that are accessible for the basolateral endocytic pathway, whereas overlap of canalicular marker with immunoreactivity (cyan) demonstrates an association of the apical endocytic route with these organelles. Colocalization of apical and basolateral marker demonstrates a merger of the 2 pathways, a substantial part of which is localized to late endosomes and lysosomes, respectively (white). Minor proportions of overlap are found in pericanalicular structures free of lysosomal markers thus indicating SAC (yellow). Bar = 5 μm. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Overlap of marker for apical endocytosis with endolyn-78 and rab11. Dx-TxR (red) was infused retrogradely for 5 minutes, and semithin sections were probed with antibodies recognizing endolyn-78 (green, Cy2) and rab11 (blue, Cy5). There is some overlap of endolyn-78 and rab11 (cyan) as well as of dextran with endolyn-78 (yellow) and rab11 (magenta). Overlap of all 3 probes (white) indicates a compartment that is accessible from the apical PM and immunoreacts with endolyn-78 and rab11. (A) Stack of deconvoluted images in MIP. Bar = 5 μm. (B) Same stack of unprocessed images. C, canaliculus; N, nucleus; S, sinusoid. (C) Immunolabel (arrows) for endolyn-78 (top) is found predominantly in MVBs (left) and lysosomes (right) as well as with vesicles and tubules in all hepatocellular areas. Immunogold recognizing rab11 (bottom) is associated with subapical membrane-like structures. Bar = 500 nm. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 Overlap of marker for apical endocytosis with endolyn-78 and pIgA-R. Dx-TxR (red) was infused retrogradely for 10 minutes and semithin sections were probed with antibodies recognizing endolyn-78 (green, Cy2) and pIgA-R (blue, Cy5). There is some overlap of dextran with endolyn-78 (yellow) the size and distribution of which match those shown in Figure 8A. Colocalization of endolyn-78 and pIgA-R (cyan) includes some perinuclear vesicles but is concentrated in pericanalicular structures some of which show overlap with dextran (white) indicating a compartment that is accessible from the apical PM and immunopositive for endolyn-78 as well as pIgA-R. Overlap of dextran with pIgA-R (magenta) is confined to the canalicular PM. (A) MIP of deconvoluted images. Bar = 5 μm. (B) Same stack of unprocessed images. C, canaliculus; N, nucleus. (C) Immunolabel for pIgA-R is found predominantly in small subapical vesicles (arrows). Additional immunogold is associated with MVBs. Bar = 500 nm. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 10 Overlap of marker for apical endocytosis and pIgA-R with clathrin (A, B) and cathepsin D (C, D), respectively. Dx-TxR (red) was infused retrogradely for 5 (A, B) or 10 (C, D) minutes and semithin sections were probed with antibodies against clathrin (A, B, blue) or cathepsin D (C, D, green). Colocalization (white) of pIgA-R and Dx with both proteins at or below the canalicular PM indicates reinternalization and lysosomal degradation of apical PM proteins. (A, D) Stacks of deconvoluted images in MIP, Bar = 5 m. (B, C) Stacks of unprocessed images. C, canaliculus; N, nucleus; S, sinusoid. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 11 Summary of vesicular pathways associated with SAC. Our data show that SAC is an intermediate sorting station in the apical endocytic pathway (1) leading via late endosomes to the lysosomal compartment (2). In addition, SAC is intersected by the transcytotic pathway (3). A SAC-to-apical pathway (4), which is the final leg of the transcytotic route, may be deduced from the known pathways of pIgA-R and newly synthesized apical PM proteins.21 The sorting function of SAC is demonstrated by the biosynthetic pathway of endolyn-78 that is shipped transcytotically to SAC and then delivered to the lysosomal compartment.21 The strategic location of SAC makes it a key candidate for mediating membrane retrieval and reinsertion (5), a mechanism that is very likely to control secretory capacity of the apical PM. C, canaliculus. Gastroenterology 2000 119, 1692-1707DOI: (10.1053/gast.2000.20233) Copyright © 2000 American Gastroenterological Association Terms and Conditions