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First published online 29 May 2007
doi: 10.1242/jcs.004713

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A unique and specific interaction between T-catenin and plakophilin-2 in the area composita, the mixed-type junctional structure of cardiac intercalated discs

Steven Goossens^1,2,*, Barbara Janssens^1,2,*,, Stefan Bonné^1,2,, Riet De Rycke^1,2, Filip Braet^1,2,¶, Jolanda van Hengel^1,2 and Frans van Roy^1,2,**

¹ Department for Molecular Biomedical Research, VIB, Ghent University, B-9052 Ghent, Belgium
² Department of Molecular Biology, Ghent University, B-9052 Ghent, Belgium

View larger version (115K): [in this window] [in a new window]   Fig. 1. Delineation of the domains mediating mutual interactions between T-catenin and plakophilins (PKP3 and PKP2). Yeast two-hybrid analysis was performed on various deletion mutants of (A) T-catenin (yellow), (B) PKP3 (blue) and (C) PKP2 (purple). We also used a chimeric construct in which the amino-terminal half of T-catenin was replaced with that of E-catenin (green; row A.6). Each open reading frame (T-catenin, PKP3 and PKP2) was fused to the GAL4-DNA binding domain (DBD, grey boxes) or to the GAL4-activating domain (AD, white boxes). The narrowed down interacting domains are depicted at the bottom in red. Amino acid residue (AA) numbers indicate the positions of the corresponding domains. VH, vinculin homology domain; AMD, adhesion modulation domain; N, amino terminus; C, carboxyl terminus.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Co-immunoprecipitation confirms the unique and specific interaction between T-catenin and PKP2 both in vitro and in vivo. (A) Myc-tagged T-catenin (114 kDa, left panel), Myc-tagged E-catenin (111 kDa, right panel) and E-tagged full-length human PKP2 (100 kDa) were produced by coupled transcription-translation in vitro. Proteins immunoprecipitated with anti-Myc or anti-E-tag antibodies were analyzed by western blot. Interaction between T-catenin and PKP2 is clearly observed (left panel; asterisk), but no interaction is seen between E-catenin and PKP2 (right panel; arrowhead). (B) Endogenous PKP2 was co-immunoprecipitated with T-catenin from mouse heart lysate. T-catenin was specifically immunoprecipitated with polyclonal antibody 952, followed by western blot analysis using a PKP2-specific monoclonal antibody.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Rat monoclonal antibody 1159_12A4S4 specifically detects T-catenin (right panel). Antibody specificity was tested by western blot analysis of HEK-293 cells transfected with constructs expressing GFP-tagged mouse -catenins, as indicated above the lanes. Anti-GFP antibody was used to detect all fusion proteins as a positive control for transfection efficiency (left panel).

View larger version (59K): [in this window] [in a new window]   Fig. 4. Immunohistochemical staining identifies colocalization of T-catenin and PKP2 at the intercalated discs of adult mouse heart. Tissue was fixed in paraformaldehyde, embedded in paraffin, and sections were stained using specific antibodies followed by histochemical detection. (A) Counterstaining with Hematoxylin and Eosin, (B) conventional immunofluorescence and (C) confocal laser scanning microscopy. Overlay images of red and green fluorescence are shown in the third column. In B, the rightmost panel shows double fluorescence combined with differential interference contrast microscopy (DIC). In C, maximum intensity projections (MIPs) of 29 z-sections of 0.25 µm were made (rightmost panel).

View larger version (95K): [in this window] [in a new window]   Fig. 5. Immuno-EM identifies colocalization of T-catenin and PKP2 at adhering junctions of the intercalated discs of cardiomyocytes. (A) Single labeling with silver amplification of T-catenin and PKP2 at fascia adhaerens-like junctions (left panel) and desmosome-like junctions (right panel). Whereas PKP2 is detected in both junction types, T-catenin is confined to the fascia adhaerens-like junction type, which we named `hybrid adhering junctions'. (B) Consecutive double labeling at the mixed-type junctional structure (area composita): detection of T-catenin by silver amplification (large dots) was followed by PKP2 labeling (10-nm gold particles, small dots, arrows). (C) Detection of intermediate filaments at the area composita by consecutive double labeling: T-catenin detection by silver amplification (big dots, indicated by arrowheads) was followed by desmin labeling (10-nm gold particles, small dots). (D) Schematic representation of the number of gold particles counted at the site of hybrid adhering junctions (left, black bars) and desmosome-like junctions (right, black bars) in comparison to the cytoplasmic background label (grey bars) after immunogold labeling of several cadherin/catenin-associated proteins (T-catenin, E-catenin and β-catenin) and several desmosomal proteins (PKP2, desmoglein and desmoplakin). The classic cadherin-catenin complex is found only at the hybrid adhering junctions, whereas desmosomal proteins are detected at both the desmosome-like and the hybrid adhering junctions of the intercalated disc.

View larger version (206K): [in this window] [in a new window]   Fig. 6. Models for cadherin-based cell-cell junctions in epithelia (left) and in heart (right panel). In the heart, T-catenin recruits desmosomal proteins to hybrid adhering junctions (top drawings), thereby forming, together with desmosomes (see below), an area composita, which is an enforced, mixed-type junctional structure attached to both the actin cytoskeleton and the intermediate filaments. By contrast, epithelial tissues do not express T-catenin, and therefore their adherens junctions are not attached to the intermediate filaments. The composition of typical desmosomes (drawings at the bottom) is largely similar in the two tissue types. Although desmosomes are shown here as containing simultaneously all three types of plakophilins as well as heterophilically binding desmosomal cadherins, we are not aware of evidence for such high complexity in individual desmosomes.

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