Recombinant extracellular domain of CADM1 mimics the trans-homophilic interaction of CADM1 and induces cell spreading.

Parental MDCK cells or MDCK cells stably expressing CADM1-GFP (MDCK+CADM1-GFP) were incubated on coverslips coated with control IgG or recombinant proteins consisting of the extracellular fragment of CADM1 fused to Fc fragments of mouse IgG (CADM1-EC-Fc). After 60 min, the cells were visualized by staining the actin cytoskeleton with Alexa Fluor 568-labeled phalloidin. (A) Representative images of F-actin in cells incubated on control IgG- or CADM1-EC-Fc-coated glasses as indicated. Bars: 20 μm. (B) MDCK+CADM1-GFP cells were incubated on IgG or CADM1-EC-Fc in the presence or absence of control human IgG or anti-CADM1 antibody, 9D2 (10 μg/ml). Cell spreading was quantified by measuring the average surface area of cells. Relative value to cells on the IgG-coated glass without antibodies was shown. More than 100 cells were counted in the assay. *, p<0.05; NS, no significant difference (vs. cells on IgG without antibodies). #, p<0.05. (C) Cell spreading assay using MDCK+CADM1-GFP cells incubated on IgG or CADM1-EC-Fc with DMSO or 1 μM of Cytochalasin D (Cyto D). The area was normalized to that of cells on IgG with DMSO, and the relative value to cells on CADM1-EC-Fc with DMSO was shown. More than 180 cells were counted in the assay. *, p<0.05. (D) A schematic representation of CADM1 protein structure. The YFP-fusion proteins of full-length CADM1 (CADM1-FL) and its deletion mutant lacking the cytoplasmic fragment (CADM1-ΔCT) were shown. (E) Cell spreading assay of MDCK cells stably expressing CADM1-YFP-FL or CADM1-YFP-ΔCT that were incubated on IgG or CADM1-EC-Fc. Relative value of cell surface area to that of CADM1-YFP-FL cells on IgG-coated glass was shown. More than 230 cells were counted in the assay. **; p<0.01. (B, C, and E) The results presented are mean ± SD of three independent experiments.