Two distinct inhibitors of PI3K suppress cell spreading mediated by <i>trans</i>-homophilic interaction of CADM1.

<p>(A) Cell spreading assay of MDCK+CADM1-GFP cells incubated on IgG or CADM1-EC-Fc in the presence of an inhibitor of PI3K, LY294002, from concentrations of 0.01 μM to 10 μM as indicated. *, p<0.05; NS, no significant difference (vs. cells on CADM1-EC-Fc with DMSO). (B) Cell spreading assay was performed using MDCK+CADM1-GFP cells cultured on CADM1-EC-Fc with 1 μM of LY294002 for 45 min and then washed and further incubated for 45 min in the presence of the same concentration of LY294002 (LY294002) or DMSO (LY294002+washout). Representative images of CADM1-GFP are shown at the top of each bar graph. The area was normalized to that of cells on IgG with DMSO, and the relative value of the cell surface area to that of cells on CADM1-EC-Fc with DMSO was shown. *, p<0.05. (A and B) The results presented are mean ± SD of three independent experiments. More than 200 and 280 cells were counted in A and B, respectively. (C) Aggregation assay of MDCK+CADM1-GFP cells in Ca²⁺- and Mg²⁺-free condition in the presence of LY294002 at the concentrations indicated. The cell aggregation was represented by the ratio of the total particle number at time <i>t</i> of incubation (Nt) to the initial particle number (N0). The data shown here indicate the average Nt/N0 in triplicate experiments.</p

<i>Trans</i>-Homophilic Interaction of CADM1 Activates PI3K by Forming a Complex with MAGuK-Family Proteins MPP3 and Dlg

<div><p>CADM1 (Cell adhesion molecule 1), a cell adhesion molecule belonging to the immunoglobulin superfamily, is involved in cell-cell interaction and the formation and maintenance of epithelial structure. Expression of CADM1 is frequently down-regulated in various tumors derived from epithelial cells. However, the intracellular signaling pathways activated by CADM1-mediated cell adhesion remain unknown. Here, we established a cell-based spreading assay to analyze the signaling pathway specifically activated by the <i>trans</i>-homophilic interaction of CADM1. In the assay, MDCK cells expressing exogenous CADM1 were incubated on the glass coated with a recombinant extracellular fragment of CADM1, and the degree of cell spreading was quantified by measuring their surface area. Assay screening of 104 chemical inhibitors with known functions revealed that LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), efficiently suppressed cell spreading in a dose-dependent manner. Inhibitors of Akt and Rac1, downstream effectors of PI3K, also partially suppressed cell spreading, while the addition of both inhibitors blocked cell spreading to the same extent as did LY294002. Furthermore, MPP3 and Dlg, membrane-associated guanylate kinase homologs (MAGuK) proteins, connect CADM1 with p85 of PI3K by forming a multi-protein complex at the periphery of cells. These results suggest that <i>trans</i>-homophilic interaction mediated by CADM1 activates the PI3K pathway to reorganize the actin cytoskeleton and form epithelial cell structure.</p></div

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

<p>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.</p

Activation of PI3K signaling is necessary for CADM1-mediated cell spreading.

<p>(A) MDCK cells stably expressing CADM1 were transiently transfected with GFP-Akt-PH and incubated on control IgG or CADM1-EC-Fc. Then, cells were visualized by staining with Alexa Fluor 568-labeled phalloidin. GFP-Akt-PH was observed at the periphery of the spreading cell where actin-rich lamellipodia were generated. High-magnification images of the region indicated by arrowhead were shown in the right panels. (B) Representative results of Western blotting analysis of phosphorylated-Akt, total Akt, and CADM1 using the lysates of MDCK+CADM1-GFP cells incubated on IgG or on CADM1-EC-Fc with DMSO (−) or with 10 μM of LY294002 (+). Note that the difference of signal intensities of Akt and p-Akt was due to the different sensitivities of antibodies and exposure time. The membrane was stained by Amido Black to confirm the equal loading of proteins. The amount of phosphorylated-Akt was normalized to that of the total Akt in each lane, and the relative value to cells on control IgG without LY294002 was calculated. The average scores of the relative values in 3 independent experiments are indicated in the lower panel. (C) MDCK+CADM1-GFP cells were incubated on control IgG or CADM1-EC-Fc in the presence of DMSO or 1 μM of the inhibitors of PI3K, Rac1 and/or Akt as indicated. The surface 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. The results presented are mean ± SD of five independent experiments. More than 470 cells were counted in the assay. *; p<0.05, **; p<0.01 (vs. cells on CADM1-EC-Fc with DMSO). #; p<0.05, NS; no significant difference (vs. cells on CADM1-EC-Fc with LY294002).</p

Membrane-associated guanylate kinase homologs (MAGuKs), MPP3 and Dlg, link CADM1 with p85 by forming a multi-protein complex.

<p>(A) Schematic representation of the structures of CADM1, MPP3, and Dlg proteins. In amino acid sequences of the cytoplasmic domain of CADM1, consensus sequences of 4.1- and class II PDZ-binding motifs are highlighted by grey and black, respectively. The GST-fusion protein of CADM1 with an entire cytoplasmic fragment (GST-CADM1-C) and a mutant form lacking a class II PDZ-binding motif (GST-CADM1- CΔ4) are schematically represented below. N-terminal fragments of MPP3 and Dlg were purified as His-fusion proteins and used for an <i>in vitro</i> binding assay. (B) Interaction of GST-CADM1 with His-MPP3 and/or His-Dlg was examined by GST pull-down assay. Binding proteins were detected by Western blotting using anti-His tag antibodies, whereas GST-fusion proteins were detected by staining the membrane with Coomassie Brilliant Blue (CBB). (C) Localization of CADM1, MPP3, Dlg, and p85 in confluent Caco-2 cells. Confluent Caco-2 cells were fixed and stained with anti-CADM1 antibodies (green) and anti-MPP3 (upper), anti-Dlg (middle), or anti-p85 (lower) antibodies (red). Merged images are shown in the right panel. Bars: 20 μm. (D) Lysates of Caco-2 cells expressing MPP3-HA were immunoprecipitated (IP) with control rabbit IgG and anti-CADM1 (left) or anti-Dlg (right) antibodies and analyzed by Western blotting with antibodies against HA, Dlg, p85, and CADM1, as indicated. Black arrowheads indicate signals found in both the input and immunoprecipitates, whereas white arrowheads indicate signals only found in the input. Asterisks show non-specific bands.</p

Dynamic Regulation of a Cell Adhesion Protein Complex Including CADM1 by Combinatorial Analysis of FRAP with Exponential Curve-Fitting

<div><p>Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation.</p></div

A schematic representation of the dynamics of the CADM1 complex.

<p>In confluent MDCK cells, CADM1-Y forms <i>cis</i>-dimers on plasma membranes and <i>trans</i>-interacts with CADM1-Y expression in the adjacent cell. (<b>A</b>) In the single transfectant, CADM1-Y interacted with endogenous proteins 4.1s and MPPs and stably localized at the cell-cell contact sites with a time constant of approximately 16 min. In double transfectants of CADM1-Y and G-4.1B (<b>B</b>) or G-MPP3 (<b>C</b>), CADM1-Y was still stably localized; in contrast, G-4.1B or G-MPP3 was present as a free pool or as a complex with CADM1-Y with short- or long-time constants, respectively. The time constant of each protein is indicated.</p

Conventional FRAP analysis of CAMs expressed in confluent MDCK cells.

<p>MDCK cells expressing CADM1-Y, E-cadherin-G, or G-β-actin were analyzed using FRAP for 600 sec (short, <b>A</b>–<b>D</b>) after photobleaching. (<b>A</b>) Representative images before and at the time points indicated after photobleaching are shown. ROIs for photobleaching are indicated by red boxes. Bars, 5 μm. (<b>B</b>) Fluorescence recovery curve of FRAP analysis. (<b>C</b> and <b>D</b>) Halftime of recovery (t_1/2, <b>C</b>) and mobile fraction (Mf, <b>D</b>). Data are mean ± SEM. Statistical differences in t_1/2 and Mf in FRAP analysis for 10min were determined by Student’s t-test. *, p< 0.05; **, p< 0.01; NS, no significant difference. (<b>B</b>–<b>D</b>) n = 10, 5, and 8 for CADM1-Y, E-cadherin-G, and G-β-actin, respectively.</p

Dynamics of CADM1 and its binding proteins, 4.1B and MPP3, at cell-cell contact sites.

<p>MDCK cells expressing CADM1-Y and G-4.1B (<b>A</b> and <b>C</b>) or G-MPP3 (<b>B</b> and <b>D</b>) were analyzed using FRAP until 3,600 sec after photobleaching. (<b>A</b> and <b>B</b>) Representative images before and at the time points indicated after photobleaching are shown. ROIs for photobleaching are indicated by red boxes. Bars, 5 μm. (<b>C</b> and <b>D</b>) Single or double exponential curve fitting of fluorescence intensities of cells expressing CADM1-Y/G-4.1B (<b>C</b>, n = 7) and CADM1-Y/G-MPP3 (<b>D</b>, n = 8) as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116637#pone.0116637.t001" target="_blank">Table 1</a>.</p

Exchange of CAMs within cell-cell contact regions analyzed using FLIP assay.

<p>MDCK cells expressed with CADM1-Y and G-4.1B (<b>A</b> and <b>C</b>) or G-MPP3 (<b>B</b> and <b>D</b>) were continuously photobleached at the cell-cell contact site (CS, upper) or intracellular region (IC, lower) indicated by asterisk. (<b>A</b> and <b>B</b>) Representative images of CS- or IC-bleached cells before and at the time points indicated after photobleaching. Intensity of fluorescent protein was monitored at ROIs (regions of interest) 1–3 in CS-bleached cells and ROI 4 in IC-bleached cells for 600 sec. (<b>C</b> and <b>D</b>) Quantitative analysis of the fluorescence intensities in the cell-cell contact sites adjacent to the photobleached regions shown in <b>A</b> and <b>B</b> as ROI 1–4. Quantification was performed in at least three independent experiments, and a representative graph was shown.</p