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mARVCF cellular localisation and binding to cadherins is influenced by the cellular context but not by alternative splicing

Institut der Anthropologie und Humangenetik fuer Biologen, Johann-Wolfgang-Goethe-Universitaet Frankfurt, Siesmayerstrasse 70, D-60054 Frankfurt/Main, Germany

View larger version (23K): [in a new window]   Fig. 1. (A) Schematic overview of murine ARVCF with the N-terminal coiled-coil domain and the central armadillo repeat region (GenBank accession no. AJ243418). The positions of the three gene-specific primer sets generating novel isoforms when used in RT-PCR are marked with arrows. (UTR, untranslated region). (B) Schematic drawing of the two N-terminal and four C-terminal splice variants of murine ARVCF obtained by RT-PCR. Exons (E) were postulated by comparing the known mouse and human ARVCF cDNA sequences with the human genomic ARVCF clone (AC005663). Numbers of the exons correspond to the human sequence. (C) Amino acid sequence comparison of the N- and C-terminal isoforms of mARVCF. The asterisks mark the stop codons; the putative PDZ-binding domain in isoform 3/7 is underlined.

View larger version (27K): [in a new window]   Fig. 2. RT-PCR analysis of alternative splicing events in (A) the N- and (B,C) C-terminal region of mARVCF mRNA. The mRNA used for RT-PCR was isolated from differentiating mouse myoblasts (i28), total mouse heart (heart) and CMT-cells derived from mouse colon carcinoma (CMT). (A) Agarose gel electrophoresis of RT-PCR products amplified with a sense primer in the 5'UTR and an antisense primer within exon 6 amplifying both N-terminal isoforms at once. (B) Agarose gel electrophoresis of RT-PCR products amplified with a sense primer in exon 15 and an antisense primer in exon 20 amplifying all four C-terminal isoforms at once. (C) Agarose gel electrophoresis of RT-PCR products amplified with one sense primer in exon 15 and two antisense primer in exon B and exon 19. (D) Agarose gel electrophoresis of RT-PCR products amplified with (1) oligonucleotide primers specific for the armadillo repeat region of ARVCF which is not affected by alternative splicing (upper band) and (2) oligonucleotide primers specific for the housekeeping gene BIP (binding protein) as a standard (lower band). The mRNA used for RT-PCR was isolated from the cell lines indicated above. (FL, full length).

View larger version (40K): [in a new window]   Fig. 3. (A) Western blot analysis of the eight potential isoforms cloned as EGFP-fusion proteins. COS-7 cells were transfected with pEGFP-splice variant expression plasmids indicated above. 48 hours after transfection extracts of COS-7 cells were prepared and separated by SDS-PAGE; each lane contains 20 µg of total protein. (B) Western blot analysis of splice variant FL-3/7 as EGFP-fusion protein. i28, MCF7, RT112, EJ28, HeLa and COS-7 cells (indicated above) were transfected with pEGFP-splice variant FL-3/7 expression plasmid. 48 hours after transfection cell extracts were prepared and separated by SDS-PAGE; each lane contains 20 µg of total protein. The western blots were probed with GFP-antibodies followed by incubation with alkaline phosphatase conjugated secondary antibody, and visualised by NBT/BCIP. (FL, full length).

View larger version (66K): [in a new window]   Fig. 4. Cellular localisation of mARVCF splice variants. Mouse myoblasts i28 (A), MCF7 cells (B), RT112 (C), EJ28 (D), HeLa cells (E) and COS-7 cells (F) were transfected with the eight potential isoforms as EGFP-fusion constructs and expression visualised by fluorescence microscopy. The localisation of the eight potential isoforms was seen at the cell membrane in all cell lines tested with the exception of EJ28 cells. Here, as a representative result for the different isoforms, the localisation of EGFP-FL-3/7 is shown. As controls, each cell line was transfected with the empty EGFP-plasmid (A-co to F-co). In i28 cells, the endogenous M-cadherin was detected with M-cadherin antibodies (Ai), in MCF7 (Bi) and RT112 (Ci) cells endogenous E-cadherin was detected with the E-cadherin antibody and N-cadherin was detected with the anti A-CAM antibody in EJ28 (Di) and HeLa cells (Ei). In COS-7 cells the anti-pan-cadherin antibody was used to detect endogenous cadherin(s) (Fi). (Am-Fm) merged images of (A-F) and (Ai-Fi). Bars, 10 µm.

View larger version (21K): [in a new window]   Fig. 5. GST binding assay. In vitro translated 35S-methionine-labeled mARVCF splice variants FL-C11 (lane 1) and FL-3/7 (lane 2) were incubated with the cytoplasmic domains of M- (lanes 4,8), E- (lanes 5,9) or N-cadherin (lanes 6,10) as GST-fusion proteins. As controls, in vitro translated 35S-methionine-labeled mARVCF splice variants FL-C11 (lane 3) and FL-3/7 (lane 7) were incubated with GST alone. (GST, glutathione-S-transferase).

View larger version (59K): [in a new window]   Fig. 6. MOM recruitment assay. Mouse myoblast i28 cells (A, Ai), MCF7 (B, Bi), RT112 (C, Ci), EJ28 (D, Di), HeLa (E, Ei) and COS-7 cells (F, Fi) were co-transfected with MOM-M-cadherin or MOM-E-cadherin plasmids and each mARVCF isoform as an EGFP-fusion expression plasmid. The MOM-construct, detected with a BP-antibody recognising the C-terminal birch profilin (BP) tag, is directed to the mitochondrial outer membrane due to the MOM-anchor and gives rise to a mitochondrial staining pattern of the fusion protein (A-F). The co-transfected mARVCF constructs showed the same mitochondrial staining pattern (Ai-Fi) indicating an interaction with the cytoplasmic domain of the respective cadherin. In MCF7 cells the membrane staining observed when mARVCF is transfected alone does not disappear as in the other cell lines (compare Fig. 4). The example shown here represents the MOM recruitment assay with MOM-M-cadherin and EGFP-FL-C11. Control, the empty EGFP-vector was co-transfected with MOM-M-cadherin (co and coi). Bars, 10 µm.

View larger version (60K): [in a new window]   Fig. 7. MOM recruitment assay with MOM-N-cadherin. Cells were co-transfected with MOM-N-cadherin and each mARVCF isoform as an EGFP-fusion plasmid. As an example, EGFP-FL-3/7 is shown in MCF7 cells, demonstrating the inhomogeneous interaction pattern between all of the mARVCF isoforms and the cytoplasmic domain of N-cadherin obtained in every cell line. (A) Indirect immunofluorescence with monoclonal birch profilin (BP) antibodies visualising the MOM-N-cadherin construct. (B) Localisation of EGFP-FL-3/7. (C) Merged image of (A) and (B). (D) 1.5x103 MCF7 cells expressing MOM-N-cadherin and EGFP-FL-3/7 were counted. In 36.7% of these cells a mitochondrial located interaction of both constructs was detected. In 63.3% of cells expressing MOM-N-cadherin and EGFP-FL-3/7 such an interaction did not take place. Error bars indicate standard deviation. Bar, 10 µm.

View larger version (92K): [in a new window]   Fig. 8. MOM recruitment assay with FL-3/7, p120(ctn) and MOM-N-cadherin. MCF7 cells were co-transfected with pMOM-N-cadherin, mARVCF splice variant FL-3/7 fused to an Xpress-tag and p120(ctn) as a EGFP-fusion protein. Cells were stained with a Xpress antibody to detect FL-3/7. As shown in B, FL-3/7-Xpress (a) and EGFP-p120(ctn) (b) can bind to MOM-N-cadherin (not stained) in the same cell. In c, the merged image of a and b is shown. (A) Cells were treated as in B and pictures were taken where both FL-3/7-Xpress (a) and EGFP-p120(ctn) (b) were detected at the plasma membrane. (c) The merged image of (a) and (b). To confirm that these cells were also positive for MOM-N-cadherin, the same coverslips were washed again and stained for MOM-N-cadherin with the birch profilin antibody (BP); the positive BP staining is given in d. (e) The merged image of a, b and d where the MOM-N-cadherin staining is coloured artificially blue. In f, 500 cells expressing FL-3/7-Xpress, EGFP-p120(ctn) and MOM-N-cadherin were counted. In 32.9% (mARVCF) or 35.2% (p120(ctn)) of these cells a mitochondrial located interaction of both constructs was detected. In 67.1% (mARVCF) or 64.8% (p120(ctn)) of cells expressing MOM-N-cadherin, EGFP-FL-3/7 and EGFP-p120(ctn) such an interaction did not take place. Error bars indicate s.d. Bar, 10 µm.