Fig. 1

Ectopic TMIGD1 is localized in the cytoplasm of MDCKII cells but is efficiently transported to the cell surface after deletion of its D1 domain. a Schematic presentation of Flag-tagged TMIGD1 constructs expressed in MDCKII-TetOFF cells. The two ΔD1 mutants lack the membrane-distal Ig-like domain (D1 domain). The PDZ domain-binding motif (shown in red) is absent in the “Δ5” mutants. b MDCKII cell lines stably transfected with the TMIGD1 constructs depicted in (a) (TMIGD1/WT (WT), TMIGD1/Δ5 (Δ5), ΔD1-TMIGD1 (ΔD1), ΔD1-TMIGD1/Δ5 (ΔD1−/Δ5) were induced by doxycycline removal to express the transgenes and analyzed by Western blotting with antibodies against the Flag tag. c Flag-TMIGD1-expressing MDCKII-TetOFF cells were analyzed for cell surface localization of TMIGD1 constructs by flow cytometry using antibodies against the Flag tag. Cells were left untreated (Not permeabilized) to analyze cell surface proteins only, or were permeabilized by saponin treatment (Permeabilized) to analyze total protein levels. Note that the constructs with a complete extracellular domain (TMIGD1/WT, TMIGD1/Δ5) are predominantly localized in the cytoplasm, whereas constructs lacking the membrane-distal Ig-like domain (ΔD1-TMIGD1, ΔD1-TMIGD1/Δ5) are localized at the cell surface. Filled grey lines indicate unstained cells, dotted black lines indicate cells stained with secondary antibodies alone. d Immunofluorescence analysis of TMIGD1-expressing MDCKII cell lines depicted in panel (a). Cells were stained with antibodies against the Flag tag and against β-catenin. Nuclei were stained with DAPI (pseudocoloured in white). Note that the constructs lacking the D1 Ig-like domain are localized at cell-cell contacts. Scale bars: 5 μm. e Quantification of cell-cell contact localization of TMIGD1 constructs. Cells were visually inspected for Flag signals at cell-cell contacts. TMIGD1 signals were rated as cell-cell contact-localized when a clear overlap with β-catenin signals at linear cell-cell contact sites was observed. Statistical analysis was performed using unpaired Student’s t-test. Data were obtained from three independent experiments and are presented as arithmetic means ± SEM; ****P < 0.0001. The differences in cell-cell contact localization between the two ΔD1-TMIGD1 constructs (ΔD1-TMIGD1, ΔD1-TMIGD1/Δ5) and each of the two constructs with entire extracellular domains (TMIGD1, TMIGD1/Δ5) are highly significant (P < 0.0001; not indicated by asterisks in the graph). f Cell-cell contact localization of TMIGD1 - JAM-A domain-swapping mutants. Left panel: Schematic presentation of domain swapping mutants. Middle panel: TMIGD1 - JAM-A swapping mutants shown in the left panel were transiently expressed in MDCKII cells. The subcellular localization was analyzed by fluorescence microscopy based on the EGFP signals. Right panel: Quantification of cell-cell contact localization of the TMIGD1 - JAM-A domain-swapping mutants. Cells were visually inspected for the localization of EGFP fluorescence signals at linear cell-cell contacts defined by β-catenin-positive signals (not shown). Statistical analyses were performed using unpaired Student’s t-test and display comparisons of the TMIGD1 swapping mutants with the TMIGD1 full length protein (TMIGD1-EGFP). Data were obtained from three independent experiments and are presented as arithmetic means ± SEM; ***P < 0.001