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First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00815

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A novel cell-cell junction system: the cortex adhaerens mosaic of lens fiber cells

¹ Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
² Protein Analysis Facility, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
³ Structural Analysis Group, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
⁴ Institute for Physiological Chemistry, Medical Faculty, University of Halle, Magdeburger Strasse 18, 06097 Halle/Saale, Germany

View larger version (41K): [in a new window]   Fig. 1. SDS-PAGE and immunoblot analysis of junctional proteins of the cytoskeletal fractions from bovine lens fiber cells, including cortical regions. Proteins of water-insoluble fractions were separated by 8% PAGE and probed with antibodies to the following proteins: lanes 1 and 2, N-cadherin (N-cad); lanes 3 and 4, cadherin-11 (Cad-11); lanes 5 and 6, E-cadherin (E-cad); lanes 7 and 8, -catenin (-cat); lanes 9 and 10, ß-catenin (ß-cat); lanes 11 and 12, ezrin (81 kDa); lanes 13 and 14, periplakin (190 kDa) (Ppl); lanes 15 and 16, vinculin (Vinc). Two lanes are shown for each probe, the left with a lower protein load and the right with a higher protein load. Molecular weights of reference proteins examined in parallel are given on the left margin.

View larger version (23K): [in a new window]   Fig. 2. SDS-PAGE and immunoblot detection of the very large (>600 kDa) protein desmoyokin. On SDS-PAGE-separation (5.5% gel) of total proteins, desmoyokin (arrows) is detected in cultured kidney epithelial cells of line MDCK (lanes 1 and 2) as a positive control and in bovine lens fiber cells (lanes 3 and 4). Molecular weights of reference proteins examined in parallel are indicated on the left.

View larger version (57K): [in a new window]   Fig. 4. Molecular complexes identified in junctional plaques present in cytoskeletal fractions from lens fibers, as identified by immunoprecipitations followed by SDS-PAGE (6%), immunoblotting and MALDI-MS. (a) Coomassie-Blue-stained proteins present in immunoprecipitates from water-insoluble, 1%-Triton-X-100-soluble fractions from bovine lens fiber cells. (Lane 1) Major proteins of the supernatant used for immunoprecipitation are indicated by symbols on the left margin (from top): [spectrin/fodrin, filensin, and ezrin (81 kDa). (Lane 2) Immunoprecipitate obtained with the monoclonal ezrin antibody 3C12, showing from top: desmoyokin (>600 kDa), periplakin (190 kDa), [periaxin (two bands of 170 kDa and 150 kDa) and ezrin. (Lane 3) Immunoprecipitation with rabbit antibodies against desmoyokin (IP Dy), showing (from top): desmoyokin, periplakin and [periaxin. (Lane 4) Immunoprecipitate obtained with guinea pig antibodies against desmoyokin (IP Dy), showing (from top): desmoyokin, periplakin, together with and guinea pig immunoglobulin chains. (Lane 5) Immunoprecipitate obtained with guinea pig antibodies against periplakin (IP Ppl): desmoyokin and periplakin, together with and guinea pig immunoglobulin chains. (b) Immunoprecipitate obtained with ezrin antibody [as in (a), lane 2] was subjected to SDS-PAGE and probed with antibodies against periplakin (lane 1; Ppl), desmoyokin (lane 2; Dy), spectrin/fodrin (lane 3; Spec), showing the presence of these proteins in the immunoprecipitated ezrin complexes. Controls of proteins bound to blank Sepharose and to Sepharose coated with antibodies to desmoplakins or desmocollins were negative. Molecular weight markers are indicated on the left.

View larger version (47K): [in a new window]   Fig. 3. Identification of the N-cadherin-associated protein complexes in lens fiber plasma membranes, demonstrated by immunoprecipitation, SDS-PAGE (8%) and immunoblotting. (Lane 1) Coomassie-Blue-stained major polypeptides of the immunoprecipitate obtained from water-insoluble, 1%-Triton X-100-soluble fractions from Sepharose-bound antibodies against N-cadherin. (Lanes 2-7) Proteins of immunoprecipitates subjected to SDS-PAGE were probed by immunoblotting with antibodies against N-cadherin (lane 2; N-cad), cadherin-11 (lane 3; Cad-11), -catenin (lane 4; -cat), ß-catenin (lane 5; ß-cat), plakoglobin (lane 6; PG) and p120ctn (lane 7; p120). Antibodies against E-cadherin showed no immunoreactivity; similarly controls using Sepharose-bound uncoated or coated with antibodies against desmoplakins or desmocollins were negative. Molecular weight markers are indicated on the left.

View larger version (74K): [in a new window]   Fig. 6. Double-label immunofluorescence microscopy of bovine lens fiber cells stained for different cytoskeletal and junctional proteins. Actin (A, red) shows far-reaching colocalization with ezrin (green, notice the yellow reaction in A', merged picture). Almost the entire cell cortex is also positive for plectin (B, green), shown in contrast to the glycoprotein phakinin (B, red). By contrast, in the plane shown in C-C'', antibodies to N-cadherin (C, red; N-cad) intensely decorate the narrow sides, but seem to be excluded from large parts of the lateral membranes, specifically the punctate reaction sites corresponding to gap junctions, which in turn are positive for protein ZO-1 (C'', merged picture). Scale bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 5. Immunolocalization of adherens junction proteins in cryosections through near-cortical regions of bovine lens fiber cells. Immunofluorescence microscopy showing the hexagonal appearance of the fiber cells, with lateral long sides and apical short sides, after double immunolabeling with antibodies against N-cadherin (A,B,D, red; N-cad) in combination with either -catenin (A', green; -cat) or cadherin-11 (B', green; Cad-11), with antibodies against cadherin-11 in combination with ß-catenin (C', green; ß-cat), or with a combination of antibodies to N-cadherin and ezrin (D', green) observed by confocal laser scanning microscopy. Notice the far-reaching colocalizations of N-cadherin and -catenin (A'', merged picture), cadherin-11 (B'', merge) and – shown here indirectly – with ß-catenin (C'', merge), whereas N-cadherin localized close to ezrin on the short sides, often indicative of partial colocalization (D-D''; for higher resolution see, however, Fig. 8). Notice also the weak, if not absent, reaction of N-cadherin on the long sides. Scale bars, 10 µm.

View larger version (95K): [in a new window]   Fig. 7. Double-label immunofluorescence microscopy of desmoyokin (AHNAK protein), periplakin, ezrin and actin in cortical fiber cells of bovine lens. Desmoyokin (A, red, Dy) occurs along the entire membrane but is enriched on the short sides, showing far-reaching colocalization with actin (A', green; A'' merged picture). Similarly, periplakin (B, red) shows extensive colocalization with ezrin (B', green; B'', merged picture). Scale bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 8. Higher magnification of double-label immunofluorescence microscopy (merged images) showing N-cadherin (red) in combination with ß-catenin (A, green; ß-cat) and ezrin (B, green) on the short sides of bovine lens fiber cells. Notice the practically complete colocalization of N-cadherin with catenin (yellow), different from the only partial colocalization with ezrin, which for the most part seems to occur in patches (green) alternating with N-cadherin-positive (red) patches. Scale bar, 10 µm.