Light microscopy of normal and tumor tissues

In cryostat sections of frozen tissues as well as in sections through formaldehyde-fixed, paraffin-embedded tissues microwave treated for antigen retrieval, we consistently noticed densely spaced E- as well as N-cadherin–positive reaction sites along the plasma membranes connecting hepatocytes (Fig. 1, A–A′′), including the bile canalicular regions (Fig. 1, B–B′′). In some places, these E- and N-cadherin–positive sites could be resolved as linear arrays of punctate structures indicative of AJs (Fig. 1 B). When these arrays of N-cadherin–positive structures were compared by double-label immunofluorescence microscopy, with reaction sites of antibodies specific for desmosomal molecules, they were clearly different, often appearing in linear alternating arrays of AJs and desmosomes along the canalicular plasma membrane (presenting a direct comparison of N-cadherin with desmoplakin; similar results were also obtained for other desmosomal marker molecules, specifically desmoglein Dsg2 and plakophilin Pkp2; Fig. 1, A–A′′). In contrast, in double-label immunofluorescence microscopy comparisons of N- with E-cadherin, far-reaching colocalization was observed (Fig. 1, B–B′′), and this was also often seen in comparisons of N-cadherin with α-catenin, protein ZO-1, and the armadillo proteins β-catenin, p120, and p0071 (Fig. S1, A and B). Practically identical results were obtained for all five species examined.

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Figure 1.

Identification and localization of the AJ cadherins in mammalian hepatocytes. (A–B′′) Immunofluorescence micrographs of cryostat sections through bovine liver showing double-label reactions of N (A, A′′, B, and B′′; green)- and E-cadherin (B′ and B′′; red), also in comparison with desmosomal junctions (immunolocalizations of desmoplakin are seen as red fluorescent dots in A′ and A′′). A different localization of N-cadherin and the desmosomal protein is seen in the double-label immunofluorescence merged color picture (A′′), whereas colocalization of N- and E-cadherin is recognized as yellow (merge color)-stained AJ structures (B′′; on a phase-contrast background). Bars, 10 µm.

Similar colocalization of E- and N-cadherin in AJ structures was seen in gall bladder epithelium, intra- and extrahepatic bile ducts (Fig. S1, C and D), and pancreatic ducts (Fig. S2), whereas the AJs of the surrounding mesothelium were positive only for E-cadherin (Fig. S1 E shows the absence of N-cadherin in mesothelial cells). Again, essentially identical results were obtained in all five species examined. Colocalization for E- and N-cadherin was also seen in AJ structures of various liver-, gall bladder–, or pancreatic duct–derived tumors as well as in human liver adenomas (Fig. 2), hepato- and cholangiocellular carcinomas, and ductal adenocarcinomas of the pancreas, both in primary and metastatic tumors.

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Figure 2.

Identification of structures containing AJs positive for both E- and N-cadherin in plasma membranes of a human hepatocellular adenoma. Laser-scanning, double-label immunostaining of plasma membranes in a cryostat section through a human hepatocellular adenoma using antibodies specific for E (green)- or N (red)-cadherin (only the merged color picture is presented). Note the colocalization of both cadherins in a high proportion of the tumor cells. Bar, 20 µm.

Microscopy of cell cultures

When monolayer colonies of primary, secondary, and permanent human or rat hepatocyte cultures were examined by double- or triple-label immunofluorescence microscopy, we observed strongly N-cadherin–positive, punctate or even continuously linear-appearing reaction sites in cell–cell contact regions, frequently in perfect colocalization with E-cadherin (Fig. 3, A–B, examples of human cells). Generally, colocalizations of E- and/or N-cadherin were seen with α-catenin, protein ZO-1, and the armadillo proteins β-catenin, p120, and p0071. Colocalizations of E- and N-cadherin in AJs as well as with the aforementioned plaque proteins were also frequent in cultures of hepatocellular tumor cells, including permanently proliferative cell lines (Figs. 3, C–C′′; 4, A–D; and ​and5,5, examples of human liver carcinoma cells of line primary liver carcinoma [PLC]). Such results were also obtained for a series of other human hepatocytic tumor cell lines, such as HepG2, Hep3B, and HuH7, as well as rat hepatocyte and liver carcinoma lines (unpublished data).

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Figure 3.

Identification and localization of E- and N-cadherin–containing AJ structures connecting cells of primary cultures of human hepatocytes and hepatocellular carcinoma cells of line PLC. (A–C′′) Laser-scanning immunofluorescence microscopy of E–N-cadherin heterodimer–containing AJs in primary human hepatocytes (A and B) or hepatocellular carcinoma cells of the line PLC (C), freshly replated and allowed to reassociate in small colonies, after reaction with antibodies against N (A and A’’, red; C and C′′, green)- and E (A′ and A′′, green; C′ and C′′, red)-cadherin, showing cell–cell contacts with extensive colocalization (yellow merge color; A′′, B, and C′′) in small punctate AJs or in extended linear structures. Note that some colonies show cell–cell contact regions positive for both cadherins and presenting extensive colocalization (A–B; the right hand four-cell colony in C–C′′), whereas other—often directly adjacent—cell colonies present junctions that are positive only for one of the two cadherins (here, N-cadherin on the left of C–C′′). Note also the additional occurrence of small whisker- or dot-shaped structures at the cell surface, which are N- or E-cadherin positive or show already a merged color (yellow). Bars: (A′′ and B) 10 µm; (C′′) 20 µm.

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Figure 4.

Marked regional differences of E- and/or N-cadherin–positive AJ structures in cell cultures of hepatocellular carcinoma PLC cells. (A–D) Laser-scanning double-label immunofluorescence microscopy of reactions with antibodies to N (A, C, and D, green; B, red)- and E (A, C, and D, red; B, green)-cadherin in parts of the same PLC cell colonies, be they small (A and B) or large and near confluent (C and D). (A) Note that in the cell colony the upper portion is positive for N-cadherin only (green), whereas most of the cell–cell contact regions in the lower part of the colony are positive for both cadherins (yellow merge color). (B) Another frequent aspect of N- and E-cadherin colocalization. Close localization presents regions of merged localization (yellow) as well as regions positive for N-cadherin (red) or E-cadherin (green), the latter occurring either in small dots or whiskers or in separate, often quite extended thin zonula-like lines. (C) Dense-grown monolayer cell culture region presenting purely E-cadherin–positive (red circumscribed cells in the bottom left and the top right) or purely N-cadherin–positive (green cell–cell contacts) cells with only some plasma membrane contact regions that show E- and N-cadherin–positive (yellow) merged color cell membranes. (D) More advanced state of E–N heterodimer formation (yellow merge color) in a cell colony (green, N-cadherin; red, E-cadherin), shown at higher magnification and allowing to resolve numerous individual AJs (punctate structures). Bars, 20 µm.

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Figure 5.

Advanced state of formation of densely spaced N-cadherin–containing AJs in a culture of human hepatocellular carcinoma cells. Double-label laser-scanning confocal immunofluorescence microscopy of a reformed PLC monolayer as visualized after reaction with antibodies against N-cadherin (red) located in the numerous, very densely spaced puncta adharentia, which in many regions suggest a continuous AJ structure in a fully developed cytoskeletal system (keratin fibril bundles are shown after reaction with antibodies to keratins 8 and 18; green). Bar, 20 µm.

In addition, however, and more frequently in freshly trypsinized and replated cell cultures, we noticed small structures appearing as dots, short beaded chains, or whiskers at cell–cell contact sites, on free cell surfaces or on cytoplasmic vesicles, which could appear as positive for either only N-cadherin or E-cadherin or for both (Figs. 3, A–C′′; and 4, A–D). Not infrequently, such small cadherin-positive structures were also positive for one of the aforementioned plaque proteins (unpublished data). And most startlingly, we repeatedly noted cell colonies in which all cell–cell contact regions were densely packed with AJs intensely stained for both E- and N-cadherin, whereas adjacent colonies were characterized by cell–cell contact structures positive for only one of the two cadherins (Fig. 3, C–C′′). And such heterogeneities could also frequently be seen in the same colony, be it small or large (Fig. 4, A–D). The general impression was that in a given cell–cell contact region, one of the two cadherins started AJ formation, whereas the second cadherin appeared somewhat later to associate to heterocomplexes and -structures (Fig. 4, A–D).

Finally, in extended, dense-grown monolayer cultures of hepatocytes or hepatocellular tumor cells, characterized by fully developed AJ systems and cytoskeletal filament bundles of the actin microfilament and the keratin intermediate-sized filament (IF) category, we noted high frequencies of AJs positive for both E- and N-cadherin interspersed with desmosomes. Fig. 5 presents an example of an extended, dense-grown PLC cell monolayer with a fully developed keratin IF bundle system, in which apparently all the densely spaced AJs contain N-cadherin in addition to their typical E-cadherin.

Electron microscopy

In the liver tissues of all five mammalian species examined, the hepatocytes were connected not only by gap and tight junctions, desmosomes, and the canalicular zonula adherens but also by numerous rather small, near-isodiametric (30–100 nm) AJs of the puncta adharentia category (Fig. 6, A and B, arrows). Most of these AJs showed rather small, electron-dense cytoplasmic plaques and were interspersed with mostly larger but less frequent typical desmosomes and small junction-free regions (Fig. 6, B–D). Although the plaques of the desmosomes were generally associated with laterally abutting bundles of keratin IFs, the plaques of the punctate AJs were only occasionally resolved to anchor microfilament bundle structures. In systematic evaluations of bovine liver cell structures, almost half (43%) of the lateral contact areas between hepatocytes was occupied by AJs.

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Figure 6.

Electron microscopy of ultrathin sections through bovine liver tissue, showing cell–cell contact regions with high densities of small AJs of the puncta adharentia type that contain E- as well as N-cadherin. (A and B) Electron micrographs of ultrathin sections showing lateral contact regions of hepatocytes with small but frequent—and for the most part closely spaced—puncta adharentia (denoted by arrows), in comparison with desmosomes (Des) and gap junctions (GJ). G, glycogen aggregate. (C and D) Immunoelectron microscopy of ultrathin sections obtained from cryostat sections and reacted with antibodies specific for E (C)- or N (D)-cadherin shows that most, if not all, puncta adharentia contain both E-cadherin (denoted by arrows; here, the specific epitope is located on the extracellular portion of the molecule) as well as N-cadherin (denoted by arrows; the inset in D also shows a desmosome on the left and an intensely labeled punctum adherens on the right). Bars, 0.5 µm.

Immunoelectron microscopy revealed that by far most, if not all, of these numerous punctate AJs contained not only E-cadherin (Fig. 6 C) but also N-cadherin (Fig. 6 D). Moreover, specific antibodies and preparation techniques allowed us to localize the glycosylated extracellular domains of these cadherins in the intercellular bridges connecting these AJs (Fig. 6 C). These immunolocalization results corresponded with reactions of the specific cytoplasmic plaques positive for the armadillo proteins β-catenin, p120, and p0071. Immunoelectron microscopic control reactions also showed the specific immunolabeling of the adjacent desmosomes with antibodies to desmoplakin, plakophilin Pkp2, and desmoglein Dsg2 (unpublished data).

Biochemical analyses

In gel electrophoretic analyses of the proteins of total cells, of cytoskeletal residues, or of plasma membrane fractions enriched in bile canalicular structures (for electron microscopy of such fractions see Franke et al., 1979), N-cadherin was a major protein in liver tissues as well as in cultures of hepatocyte-derived, normal, or malignantly transformed cells (Fig. 7 A presents a series of results). As expected, N-cadherin was also a major protein in cell culture lines of mesenchymal origin, including human hepatic stellate cells of line LI-90 or human glioma cells of line hG-U333 but was totally absent in diverse other epithelial tissues and carcinomas, including several endoderm-derived cell lines, such as human colon carcinoma CaCo-2 cells (Fig. 7 A). Liver epithelial cells contained comparable amounts of E-cadherin, which was also found as a major component in CaCo-2 and other colon and colon carcinoma cells but was absent from LI-90 and hG-U333 cell cultures (Fig. 7 A). Remarkably, in most of the total cell lysates or cytoskeletal protein preparations from hepatocyte origin, the silver staining and immunoblot reactions indicated near-isostoichiometric amounts of N- and E-cadherin (Fig. 7 A, lanes 2, 4, 5, and 7–10).

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Figure 7.

Immunoblot identification of E- and N-cadherin in the gel-electrophoretically separated polypeptides and protein complexes of whole-cell lysates, sucrose density centrifugation, and IP fractions of mammalian liver tissue and cultured hepatocellular tumor cells. (A) On SDS-PAGE separation (8% gel) of total proteins, E-cadherin (E-cad) and N-cadherin (N-cad) are detected as distinct bands of ~135 kD in the following human cells and tissues: hepatocellular carcinoma cells of the line primary liver carcinoma (PLC; lane 2), human hepatoblastoma-derived cells of the line HepG2 (lane 3), human hepatocellular carcinoma cells of the lines Hep3B (lane 4) and HuH7 (lane 5); primary cultures of human hepatocytes (phH; lane 6), human liver tissue (hLiver; lane 7), and a solid human hepatocellular carcinoma (HCC; lane 8). Different detergent-soluble fractions from bovine liver (bLiver; RIPA or Triton buffers; lanes 9 and 10, respectively) have also been analyzed. E-cadherin is not detected in cells of human hepatic stellate cells of line LI-90 (lane 1) and of the human glioma cell line hG-U333 (lane 11) analyzed in parallel but is a major component in cultured cells of the human colon carcinoma line CaCo-2 (lane 12). N-cadherin is detected in all hepatocyte and hepatocyte-derived cells as well as in human hepatic stellate cells of line LI-90 and in the positive controls of human glioma cells (hG-U333) but not in CaCo-2 cells. (B) Sucrose density centrifugation (5–30%) of particles obtained by solubilization of total lysates of PLC cells, followed by fractionation (fraction numbers are given), SDS-PAGE of the fractions obtained, and immunoblotting with antibodies against E-cadherin (top) and N-cadherin (bottom). Note that for both cadherins, the maximum peak is near that of catalase (the positions of proteins analyzed in parallel are given at the top margin: BSA [B] with 4.3 S, catalase [C] with 11.5 S, and thyroglobulin [T] with 16.5 S). (C) Immunoblots of proteins contained in the peak fractions 5–8 of the gradient shown in A and obtained by IP with antibodies to E-cadherin (lane E) or N-cadherin (lane N). Apparent molecular masses according to SDS-PAGE mobility are 135 kD (N-cadherin) and 124 kD (E-cadherin). Molecular masses of reference proteins examined in parallel are indicated on the left. White lines indicate that intervening lanes have been spliced out.

When particles, proteins, and protein complexes obtained from liver tissue or from cultured hepatocyte-derived cells after mild nondenaturing detergent treatments were analyzed by gel filtration chromatography or sucrose gradient centrifugations, the majority of both E- and N-cadherin was recovered in particles of ~11.5 S and with apparent mean molecular masses of 220–240 kD, which is indicative of heterodimers (Fig. 7 B). When these fractions were subjected to immunoprecipitations (IPs) with antibodies specific for E- or N-cadherin, the peak fractions indeed contained both cadherins in major amounts suggestive of an isostoichiometric ratio (Fig. 7 C).

To test for a possible close neighborhood of the E- and N-cadherin molecules in the cell cultures and tissues positive for both E- and N-cadherin, we exposed preparations of cell fractions, enriched cell particles, or total cytoskeletal residues to various protocols of chemical cross-linking with a wide range of bond bridge distances. Even with the shortest bond distance examined (2.05 Å), i.e., in a reaction with Cu²⁺-phenanthroline to allow the oxidative formation of disulfide bridge bonds, remarkable proportions of both E- and N-cadherin were recovered in covalently linked heterodimer complexes of E- and N-cadherin (Fig. 8, A and B). By exposure to reducing agents, usually DTT or mercaptoethanol, these disulfide bonds could be completely cleaved. Similar covalent heterodimeric complexes were also obtained with other disulfide cross-linkers or with reagents connecting other amino acid residues, including bis(sulfosuccinimidyl)suberate (BS³) as the longest distance (>11 Å) cross-linker applied, which in addition, allowed to obtain even larger cross-link products, up to at least the size of double heterodimers (Fig. 8 C).

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Figure 8.

Identification and characterization of cross-link products of E- and N-cadherin contained in PLC cells and enriched after IP. (A and B) Total cytoskeletal proteins of PLC cells taken up in RIPA buffer were reacted with cross-link (CL) reagents (in this case oxidative disulfide bridge formation in the presence of Cu²⁺-phenanthroline) and subjected to IP with antibodies to E-cadherin (E-cad; A) or N-cadherin (N-cad; B). The pelleted immunoprecipitates were then analyzed by SDS-PAGE under nonreducing conditions, and polypeptides were identified by immunoblotting using antibodies to E (lane E)- or N (lane N)-cadherin. Spn, remaining supernatant; pellet, material contained in the final sediment. Note that in both kinds of immunoprecipitates the occurrence of a large proportion of E- and N-cadherin in a common polypeptide with an apparent molecular mass of ~230 kD (molecular mass markers analyzed in parallel are indicated on the left margin). Note also the formation of some proteolytic breakdown products in the specific immunoprecipitates. (C) Cross-link products of higher molecular masses up to ~450 and >600 kD can be obtained with the very long distance (>11 Å) lysine–lysine cross-link reagent BS³. Positions of major monomeric and cross-linked molecules are marked by dots. White lines indicate that intervening lanes have been spliced out.

Molecular interference with the formation and stability of E–N heterodimers and AJs

To examine the stability and the dynamics of the E–N heterodimer complexes, we exposed monolayer cultures of cells derived from human hepatocytes and hepatocellular carcinomas to conditions in which the one or the other or both of the two cadherins were drastically reduced or even depleted. In siRNA experiments, Fig. S3, for example, presents the situation at day 3 of exposure of hepatocellular carcinoma cells of line PLC to siRNAs interfering with E-cadherin, N-cadherin, or with both. When the level of E-cadherin was reduced, the level of N-cadherin was only slightly affected, and the reverse was found in experiments interfering with mRNA encoding N-cadherin. In striking contrast, interfering with mRNAs for both cadherins (Fig. S3, lane 3) led to very dramatic reductions of both E- and N-cadherin. Interestingly, in such experiments, the levels of the armadillo proteins (Fig. S3, β-catenin) were only moderately reduced. When the times of exposure to media containing these siRNAs were prolonged for a further 1 d, only very minor residues of the specific cadherins were detected.

In such mRNA interference experiments using siRNA for E- or N-cadherin or mixtures of both, the state of cell–cell adhesion was also routinely examined by immunofluorescence microscopy in combination with phase or interference contrast optics (Fig. 9 presents results obtained at the end of day 3). When one of the two cadherins was markedly reduced or even appeared completely depleted, the monolayer organization remained—by and large—in close cell–cell contact mediated by “normal” homodimer cadherin complexes (Fig. 9, A–H). Control experiments revealed the presence of desmosomes, tight junction, and gap junction connections in such cell colonies.

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Figure 9.

Changes of structure and composition of AJs upon treatment of monolayer cultures of human hepatocyte-derived (PLC) cells with siRNAs interfering with mRNAs encoding different cadherins. (A–N) Laser-scanning double-label immunofluorescence microscopy of monolayer cultures of PLC cells treated with siRNA interfering with mRNA encoding E-cadherin (B–D) or N-cadherin (E–H) or with both siRNAs (I–M), in comparison with control cultures without siRNA added (A) or treated with siRNA interfering with nuclear lamins A and C (N). Results of treatments for 72 h, with one change of siRNA-containing medium after 24 h, are shown. (A) Densely grown PLC cells immunostained here for N-cadherin (red) and plaque protein ZO-1 (green). Note the extended cell–cell contact regions stained for one of the two markers or in yellow merged color. (B–D) Cells treated with siRNA interfering with E-cadherin show exclusively the N-cadherin reaction in their cell–cell contact regions (B and C, green), whereas only occasionally, some indistinct E-cadherin (red) fluorescence is seen in the perinuclear cytoplasm (C; e.g., cell on the bottom right). All cell–cell contact regions are also positive for all AJ plaque markers as shown, for example, for protein ZO-1 (D, green). (E–H) Upon treatment with siRNA interfering with mRNA encoding N-cadherin, cell–cell contact regions are intensely stained for E-cadherin (green), whereas only very little and faint residual N-cadherin (E–G, red), mostly in endocytotic vesicles, can still be seen in the cytoplasm (F and G). The AJ plaque proteins are continually present (G; e.g., protein ZO-1 is shown in green). The cells are obviously still kept in contact by AJs based on E-cadherin alone (H, red), which is closely associated with the AJ plaque proteins (green; protein ZO-1 in H), as indicated by yellow merge color. (I–M) When the cell cultures are exposed to both, i.e., siRNA interfering with E-cadherin mRNA and siRNA interfering with N-cadherin mRNA, some remaining cell colonies, although mostly smaller, are still recognized that are not demonstrably positive for E- or N-cadherin but in which extended AJ-type contacts are seen that are still positive for AJ plaque proteins, such as protein ZO-1 (green). In contrast, some residual cadherins (E-cadherin is shown in red immunofluorescence) are demonstrable only deep in the cytoplasm, often recognizable as distinct dotlike endocytotic vesicle structures or still in association with the extended AJ plaque-derived cytoplasmic bands positive for protein ZO-1 (green bands in K, L, and M) and other plaque proteins (not depicted; for details of the formation and organization of such zonula adherens–like AJ plaque-derived structures see Kartenbeck et al., 1982). (N) Control cell culture treated in parallel with siRNAs to lamins A/C but reacted with antibodies for E-cadherin (red) and nuclear lamins A/C (green) to demonstrate the stability of the cell monolayer cultures in such experiments. The cells are connected by normal-looking extended AJ structures positive for E-cadherin. Positivity of the latter structures for N-cadherin has also been seen in parallel experiments. In contrast, no nuclear lamina decorated by lamin A/C immunofluorescence is seen. Bars, 10 µm.

Drastic changes, however, were noted in the cell culture colonies treated with both siRNAs, i.e., after drastic reduction of both E- and N-cadherin. Here, only some residual, irregularly shaped cell colonies were seen, which presented the AJ plaque proteins still in continuous zonula adherens–like structures extending along the remaining cell–cell contact regions (Fig. 9, I and J) or—in a more advanced state of AJ disintegration—as long curvilinear bands containing the plaque proteins, which were translocated deeper into the cytoplasm (Fig. 9, J–M, protein ZO-1). As these structures of detached plaques resembled, in both light and electron microscopy, very much the cytoplasmic AJ belt retraction aggregates known from exposures of epithelial cell cultures to media of reduced Ca²⁺ contents (e.g., Kartenbeck et al., 1982), we also performed such experiments with low Ca²⁺ media or with 2 mM EGTA and obtained very similar results (unpublished data). Remarkably, in such cell colonies, the appearance of AJ plaque retraction belts was accompanied by both the retention of some E- or N-cadherin at the plasma membrane amino acids well as the formation of cadherin-containing structures in the cytoplasm (Fig. 9, J–M), which on electron microscopic examination, were revealed as clusters of vesicles (compare Kartenbeck et al., 1982; Le et al., 1999; Akhtar and Hotchin, 2001; de Beco et al., 2009; Hong et al., 2010).

Antibodies and reagents

Primary antibodies used in this paper are listed in Table S1. Secondary antibodies used were Alexa Fluor 488– and 594–coupled mouse and rabbit antibodies (MoBiTec) as well as the specific Cy3- and Cy5-coupled mouse, rabbit, or guinea pig antibodies (Dianova). For immunoblot analysis, horseradish peroxidase–conjugated secondary antibodies were applied (Dianova).

Immunocytochemistry

Tissue cryosections were air dried for ≥1 h and then fixed in acetone for 10 min at −20°C. Liver, gall bladder, and pancreas tissue specimens were, in most cases, preincubated with PBS containing 0.2% Triton X-100 for 5 min followed by exposure to primary antibodies in PBS for 30 min each, two washes in PBS for 5–10 min each, and exposed to the specific secondary antibodies for 30 min. After two 5–10-min washes in PBS, the cell preparations were rinsed in distilled water, fixed for 5 min in ethanol, and mounted in Fluoromount G (Biozol Diagnostica).

The cell suspensions were centrifuged at 50 g for 10 min, and the cell pellets were resuspended in ice-cold buffer. An aliquot of the cell preparations was separated for cell counts and viability analyses (phase-contrast microscopy and trypan blue exclusion test). For all experiments, suspensions with >85% of viable hepatocytes were used. The nonhepatocyte fraction, including leukocytes and nonparenchymal liver cells, in the cell suspension was <5% (microscopic analyses). Plating efficacy of the hepatocytes on collagen-coated cell-culture dish surfaces exceeded 80% in all preparations. Primary and secondary rat hepatocytes were isolated and grown on coverslips essentially as previously described for liver and other cell cultures (e.g., De Smet et al., 1998; Peters et al., 2010; Franke and Rickelt, 2011).

Immunofluorescence microscopy

Immunofluorescence microscopy of cell cultures and of sections through formalin-fixed, paraffin-embedded tissues was performed as previously described (e.g., Straub et al., 2003, 2008; Rickelt et al., 2009). Therefore, cells grown on glass coverslips were briefly rinsed in PBS and fixed for 5 min either with 2% formaldehyde in PBS at RT or in methanol (5 min) followed by acetone (20 s), both at −20°C. The samples were then briefly air dried and rehydrated in PBS just before the immunostaining procedure. After permeabilization in 0.2% Triton X-100 for 5 min, cells were washed several times in PBS. Primary antibodies were applied for 1 h at RT followed by three washes in PBS (5 min each), an incubation with secondary antibodies (30 min at RT), washing with PBS (3× 5 min), a short rinse in distilled water, and finally, dehydration in 100% ethanol (1 min). After air drying, specimens were mounted with Fluoromount G. Epifluorescence was documented with a photomicroscope (Axiophot; Carl Zeiss) equipped with a camera (AxioCam HR; Carl Zeiss) as well as Plan Neofluar 63×/1.25 NA oil and Plan Neofluar 40×/1.30 NA oil objectives. For confocal laser-scanning immunofluorescence microscopy, a microscope (LSM 510 Meta; Carl Zeiss) equipped with Plan Apochromat 63×/1.40 NA oil and Plan-Neofluar 40×/1.30 NA oil objectives was used. AxioVision Release 4.6.3.0 and LSM Image browser 3.2.0.115 software (both obtained from Carl Zeiss) was used for image processing, and Photoshop CS3 (Adobe) was used for final figure preparation.

Electron and immunoelectron microscopy

For ultrathin section experiments, liver, gall bladder, or pancreas tissue pieces of human, bovine, porcine, or rodent origin were fixed with 2.5% glutaraldehyde in 50 mM sodium cacodylate buffer, pH 7.2, containing 50 mM KCl and 2.5 mM MgCl₂, for 30 min, washed three times in the same buffer, postfixed with 2% OsO₄ in 50 mM cacodylate buffer for 2 h, and repeatedly washed in this buffer. Thereafter, the tissue blocks were stained overnight with 0.5% uranyl acetate in water, washed, dehydrated, and embedded in Epon. Ultrathin sections were poststained with lead (Franke et al., 1979).

For immunoelectron microscopy, ~5-µm-thick cryostat sections were fixed with 2% formaldehyde in PBS for 10 min, rinsed three times for 5 min each with PBS containing 50 mM NH₄Cl to quench residual-free aldehyde groups, and then treated with 0.1% saponin in PBS for 5 min followed again by several PBS washes. Primary antibodies were applied for 1 or 2 h followed by three washes with PBS for 5 min. As secondary antibodies, goat anti–mouse or anti–guinea pig immunoglobulins coupled to particles (Nanogold; Biotrend) were applied for 2 h or longer. Specimens were then washed with PBS, briefly fixed with 2.5% glutaraldehyde (5, 10, or 15 min), and rinsed with cacodylate buffer followed by rinses with Hepes buffer, pH 5.8, containing 200 mM sucrose. The primary gold particles bound were then silver enhanced using a silver enhancement kit (HQ Silver; Biotrend) for 3–6 min. Enhancement was stopped with 50 mM Hepes buffer containing 250 mM sodium thiosulfate, and specimens were washed with distilled water and postfixed with 2% OsO₄ for 30 min, dehydrated, and embedded in Epon. Ultrathin sections were prepared with an ultramicrotome (Reichert Jung; Leica). Electron micrographs were taken at 80 kV using an electron microscope (EM 910; Carl Zeiss; compare Rickelt et al., 2009).

Fractionations of cell junction components

Small tissue blocks were taken up in SDS sample buffer (250 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 125 mM DTT) or IP buffer (see Gel electrophoresis, IPs, and immunoblotting) and homogenized with a homogenizer (Dounce or Potter-Elvehjem; Braun). To obtain total cell lysate proteins, tissues or cultured cells were directly homogenized in SDS sample buffer for particle fractionations, and the proteins of the pelleted material of the fractions of interest were analyzed as described in the following paragraph. For preparations of cytoskeletal fractions from confluent cell cultures, cells were washed with ice-cold PBS, incubated in low salt buffer containing 10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1% Triton X-100, and 5 mM EDTA for 2–3 min on ice. Then, the cells were incubated in high salt buffer (as before but with 1.5 M KCl) for 20 min. After brief homogenization and centrifugation, the pellet obtained was washed several times with PBS (cytoskeletal fraction).

Gel electrophoresis, IPs, and immunoblotting

SDS-PAGE was essentially as previously described (Achtstaetter et al., 1986; Schäfer et al., 1994; Rickelt et al., 2009). For IP, liver tissue pieces or loose pellets of cultured cells were taken up in Triton X-100–containing IP buffer (Triton-IP buffer: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA or 0.5 mM CaCl₂, 1% Triton X-100, and 1 mM DTT plus protease inhibitors), in radioimmunoprecipitation assay (RIPA) IP buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA or 0.5 mM CaCl₂, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM DTT, and protease inhibitors), or in Empigen-containing IP buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA or 0.5 mM CaCl₂, 0.1 or 0.5% Empigen, 1 mM DTT, and protease inhibitors) and centrifuged at 16,100 g in a laboratory centrifuge (5414; Eppendorf) for 15 min at 4°C. The supernatant obtained was then precleared with protein G– or A–coupled magnetic beads (Dynal Dynabeads; Invitrogen) for several hours. In parallel, protein A– and/or protein G–coated magnetic beads were reacted with the specific antibodies or with unrelated antibodies (mouse myeloma IgG1; Invitrogen) in 50 mM Tris-HCl, pH 7.5, at 4°C for several hours. The precleared supernatant was then reacted with the antibody-coupled beads overnight in 4°C and washed intensely. The pellets obtained were solubilized in 20–40 µl SDS sample buffer, and the immunoprecipitates were compared by SDS-PAGE with the proteins contained in pellets and the supernatants of the preclearing steps before and after IP.

Chemical cross-linking

Chemical cross-linking was performed using the Cu²⁺-phenanthroline reaction to obtain short distance (2.05 Å) cysteine-bridged polypeptide molecules (compare, e.g., Soellner et al., 1985; Kapprell et al., 1987) or for longer cross-bridges with 1–5 mM amine-reactive reagent BS³ (Perbio Science) in PBS, a noncleavable, membrane-impermeable, water-soluble lysine–lysine long distance (>11 Å) cross-linking agent (Sinz, 2003; Leitner et al., 2010). After the reaction, the cells were washed with PBS, and pellets and lysate supernatants were analyzed directly or submitted to IP as described in the preceding paragraph.

siRNA transfections, incubations, and evaluations

The specific siRNA for human E-cadherin (cdh1; L-003877-00) and N-cadherin (cdh2; L-011605-00) as well as control siRNAs (nontargeting control siRNA; ON-TARGETplus SMARTpool; see Pieperhoff et al., 2008) and lamin A/C siRNA (specific for human, mouse, and rat lamin A/C; Pieperhoff et al., 2008) were purchased from Thermo Fisher Scientific. All siRNA transfections were essentially performed according to the manufacturer’s protocols using lipid-based transfection reagents (DharmaFECT no. 1 or 4; Thermo Fisher Scientific; for methods of evaluation see Pieperhoff et al., 2008).

We thank Michaela Hergt, Edeltraut Noffz, and Heiderose Schumacher for cell cultures and Elisabeth Specht-Delius for immunohistochemical work as well as Drs. Judit Boda-Heggemann, Frank-Ulrich Pape, and Oscar Cahyadi for their contributions in some of the initial experiments. Thanks go also to Dr. Thomas Longerich and to Dr. Esther Herpel for providing cryopreserved human tissue samples.

This work was supported by grants of the Ministry of Science, Research and the Arts of Baden-Wuerttemberg (grant 23-7532.22-23-12/1 to B.K. Straub and W.W. Franke), the Federal Ministry for Research and Technology (Standardization for Regenerative Therapy–Mesenchymal Stem Cells grant 01GN0942 to W.W. Franke), and a grant of the Deutsche Krebshilfe (grant 10-2049 to W.W. Franke). B.K. Straub has been supported by a stipend of the Olympia-Morata program of the Medical Faculty of Heidelberg University.