QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Janssens, B. Articles by van Roy, F. Search for Related Content PubMed PubMed Citation Articles by Janssens, B. Articles by van Roy, F.

T-Catenin: a novel tissue-specific ß-catenin-binding protein mediating strong cell-cell adhesion

¹ Molecular Cell Biology Unit, Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology (VIB)-Ghent University, B-9000 Ghent, Belgium
² Department of Pathology, University Hospital Antwerp (UZA), B-2650 Edegem, Belgium
³ Department of Radiotherapy, Nuclear Medicine and Experimental Cancerology, University Hospital Ghent, B-9000 Ghent, Belgium

View larger version (60K): [in a new window]   Fig. 1. Sequence comparison between the novel T-catenin and other members of the human -catenin/vinculin family. (A) Alignment of the protein sequences by the CLUSTAL W method (Higgins and Sharp, 1989). The location of N-terminal -helices as determined for E-catenin (Pokutta and Weis, 2000) is shown on top of the sequences. Only one of two characteristic proline residues, inducing kinks in the -helices of E-catenin (marked with P below the sequences) is conserved in T-catenin. Other annotated domains of E-catenin are: ß-catenin-binding domains (Pokutta and Weiss, 2000; Huber et al., 1997), the -actinin-binding domain (Nieset et al., 1997), amphiphatic helices possibly responsible for actin binding (Rudiger, 1998). Also indicated is the position of an alternatively spliced insert generally found for N-catenin (Claverie et al., 1993). The sequences aligned here are available from GenBank under accession nos. AF091606 (T-catenin), D14705 (E-catenin) and M94151 (N2-catenin). (B) Schematic protein alignments including amino acid identities (%) between the three main vinculin homology domains (black boxes) (Herrenknecht et al., 1991). As reported before (Janssens et al., 1999), -catulin and vinculin are clearly more distant members of the -catenin/vinculin family. The total size of the depicted proteins is indicated at the right (aa, amino acid residues), whereas residues flanking various domains are indicated by their codon numbers. N, amino-terminus; C, carboxy-terminus.

View larger version (44K): [in a new window]   Fig. 2. Tissue-specific expression patterns of T-catenin. (A) Rapid-scan RT-PCR expression analysis of human T-catenin and E-catenin mRNAs. The specific 743-bp product of the first reaction was visible in heart, testis and skeletal muscle (not shown). After nested PCR, this first product of 743 bp is still visible, whereas the nested PCR product of 630 bp is detectable in the same three samples and a few more (brain, kidney, liver, fetal liver). PCR with E-catenin-specific primers (yielding a 747-bp product) reveals expression in most tissues. PBL, peripheral blood lymphocytes. (B) RT-PCR analysis of E-catenin, T-catenin and N-catenin mRNAs in mouse organs. GAPDH mRNA analysis served as a positive control. (C) Western blot analysis of T-catenin, E-catenin and ß-catenin protein expression in various mouse organs. For detection of T-catenin, polyclonal serum #952 was applied. In brain tissue, the 104-kDa band revealed by anti-E-catenin most likely corresponds to crossreacting N2-catenin protein. (D) Antibody specificity was tested by western blot analysis of HEK-293 cells transfected with GFP-tagged -catenins: a commercial E-catenin antibody (Sigma) crossreacts with N-catenin but not with T-catenin, whereas serum #952 is highly specific for T-catenin.

View larger version (78K): [in a new window]   Fig. 3. Immunolocalization of T-catenin in cryosections of human heart. (A) Double immunofluorescent staining of T-catenin (monoclonal antibody 892_24D2S) and E-catenin (polyclonal antibody) shows colocalization of the -catenin proteins at intercalated discs of cardiomyocytes. (B) Double immunofluorescent staining of T-catenin (polyclonal antibody #952) and N-cadherin (monoclonal antibody) shows colocalization at intercalated discs of cardiomyocytes. (C) Immunohistochemical staining for T-catenin (monoclonal antibody 892_24D2S) or desmin (monoclonal antibody 33) shows that T-catenin is localized at the intercalated discs of cardiomyocytes, while desmin is present also at Z-discs. In the negative control (neg), only secondary antibody was used.

View larger version (73K): [in a new window]   Fig. 4. Immunolocalization of T-catenin in cryosections of human testis. (A) Double immunofluorescent staining of T-catenin (monoclonal antibody 892_24D2S) and E-catenin (polyclonal antibody) shows differential localization of these two related proteins. The T-catenin is present in peritubular cells, clearly separated from E-catenin, which is present in cells within the seminiferous tubules. (B) Immunohistochemical staining of consecutive sections for T-catenin (monoclonal antibody 892_24D2S) and desmin (monoclonal antibody 33) demonstrates that T-catenin is localized in desmin-expressing peritubular myoid cells. In the negative control (neg), only secondary antibody was used.

View larger version (46K): [in a new window]   Fig. 5. Confirmation of T-catenin/ß-catenin interactions by co-immunoprecipitation (IP). (A) IP from HEK-293 cells transfected with a plasmid encoding Myc-tagged T-catenin. In the western blots (left), which serve as controls for efficient transfection, T-catenin was detected by monoclonal antibody 892_24D2S and ß-catenin by a polyclonal antibody. The IP results (right) were obtained either with monoclonal anti-ß-catenin antibody or with monoclonal anti-Myc antibody. SDS-PAGE was followed by western blotting. A mixture of both antibodies was then used to probe this blot. In mock transfected cells, only ß-catenin was detected as expected (data not shown). (B) IP from mouse tissues, performed with polyclonal antibody #952, specific for T-catenin, and a polyclonal antibody specific for ß-catenin (Sigma). After western blotting of total lysates (left) and coimmunoprecipitates (right), T-catenin and ß-catenin were detected by use of the same antibodies.

View larger version (51K): [in a new window]   Fig. 6. Transient overexpression of T-catenin in -catenin-negative HCT-8/R1 colon carcinoma cells restores cadherin/catenin-mediated cell-cell adhesion. At 10 hours after transfection with pE/L-GFP-haTctn plasmid and simultaneous infection with A36R vaccinia virus, opposing cells expressing GFP-T-catenin show increased fluorescence at their common cell-cell contacts (A). This results in recruitment of ß-catenin and E-cadherin to the same intercellular contact sites (B).

View larger version (97K): [in a new window]   Fig. 7. Relocalization of multiple cell-cell adhesion components in stably transfected colon cancer cells, expressing Myc-tagged T-catenin. The -catenin-negative parental HCT-8/R1 cells (left panels) were compared to the cloned transfectant HCT-8/R1/T31 (right panels). Cells were stained for the Myc tag (exogenous T-catenin), for E-cadherin, desmoglein-2 or ZO-1 antigens.

View larger version (24K): [in a new window]   Fig. 8. Fast aggregation of -catenin-negative HCT-8/R1 colon cancer cells is restored upon stable transfection with T-catenin cDNA. After preparation of single-cell suspensions, cell aggregation was measured by determination of the volume distribution (%) in function of the particle diameter (µm) at the starting point (t0) and after 30 minutes (t30). HCT-8/R1, HCT-8/E11R1 and HCT-8/E8 cells were all obtained by subcloning HCT-8 cells, but only HCT-8/E8 cells express endogenous E-catenin. HRpCN2 is a cloned transfectant of HCT-8/E11R1 cells expressing exogenous N-catenin (van Hengel et al., 1997); HCT-8/R1/T31 is a cloned transfectant of HCT-8/R1 cells expressing exogenous T-catenin. MB2 is a monoclonal E-cadherin blocking antibody.

View larger version (51K): [in a new window]   Fig. 9. Slow aggregation, compaction and decompaction assays on various related HCT-8 colon cancer cells. (A) Representative immunofluorescent staining for -catenins in the cell lines used: no expression in untransfected HCT-8/R1 cells (R1), strong expression of E-catenin in untransfected HCT-8/E8 cells (E8), and moderate expression of exogenous T-catenin in transfectant HCT-8/R1/T31 (T31). (B) After slow aggregation of single-cell suspensions for 24 hours on semi-solid agar, images of representative cultures were taken. No aggregation is seen in cultures of HCT-8/R1 cells. Cells expressing exogenous T-catenin form compacted large aggregates, similar to those of cells expressing endogenous E-catenin. (C-D) Compaction-decompaction assays. (C) After Gyrotory shaking of suspended cell cultures for 3 days, cell aggregates were similar to the results obtained in (B). (D) Small spheroids of HCT-8/R1 cells were dissociated by repeated pipetting, whereas no decompaction could be seen of larger spheroids of HCT-8/E8 and HCT-8/R1/T31 cells. (E) Quantification of compaction/decompaction by volume distribution in function of the particle diameter: unbroken line, before compaction; dashed line, compaction; dotted line, decompaction after pipetting.