Fig 1.
Conventional FRAP analysis of CAMs expressed in confluent MDCK cells.
MDCK cells expressing CADM1-Y, E-cadherin-G, or G-β-actin were analyzed using FRAP for 600 sec (short, A–D) after photobleaching. (A) 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) Fluorescence recovery curve of FRAP analysis. (C and D) Halftime of recovery (t1/2, C) and mobile fraction (Mf, D). Data are mean ± SEM. Statistical differences in t1/2 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–D) n = 10, 5, and 8 for CADM1-Y, E-cadherin-G, and G-β-actin, respectively.
Fig 2.
Dynamics of CAMs, CADM1, and E-cadherin in confluent MDCK cells.
MDCK cells expressing CADM1-Y or E-cadherin-G were analyzed using FRAP for 3,600 sec after photobleaching. (A) 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) Fluorescence intensity in the control ROI without photobleaching. (C) Fluorescence recovery curve of FRAP analysis. (D and E) Halftime of recovery (t1/2, D) and mobile fraction (Mf, E). Data are mean ± SEM. Statistical differences in t1/2 and Mf in FRAP analysis for 60min were determined by Student’s t-test. NS, no significant difference. (F–H) Single or double exponential curve fitting of fluorescence intensities of CADM1-Y (F, n = 5), E-cadherin-G (G, n = 9), and G-β-actin (H, n = 8). Exponential fitting for FRAP signals was performed as described previously [24]. To determine the time constant of fluorescence recovery at the cell-cell contact sites, the experimental data were plotted in a semi-logarithmic scale, and regression analysis was performed using the following equation:y = y0 + A1 ·exp(—x/τ1) + A2 exp(—x/τ2)
Table 1.
The time constant (τ) of E-cadherin, CADM1, 4.1B and MPP3 analyzed by long-time.
Fig 3.
Dynamics of CADM1 and its binding proteins, 4.1B and MPP3, at cell-cell contact sites.
MDCK cells expressing CADM1-Y and G-4.1B (A and C) or G-MPP3 (B and D) were analyzed using FRAP until 3,600 sec after photobleaching. (A and 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. (C and D) Single or double exponential curve fitting of fluorescence intensities of cells expressing CADM1-Y/G-4.1B (C, n = 7) and CADM1-Y/G-MPP3 (D, n = 8) as indicated in Table 1.
Fig 4.
A schematic representation of the dynamics of the CADM1 complex.
In confluent MDCK cells, CADM1-Y forms cis-dimers on plasma membranes and trans-interacts with CADM1-Y expression in the adjacent cell. (A) 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) or G-MPP3 (C), 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.
Fig 5.
Exchange of CAMs within cell-cell contact regions analyzed using FLIP assay.
MDCK cells expressed with CADM1-Y and G-4.1B (A and C) or G-MPP3 (B and D) were continuously photobleached at the cell-cell contact site (CS, upper) or intracellular region (IC, lower) indicated by asterisk. (A and 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. (C and D) Quantitative analysis of the fluorescence intensities in the cell-cell contact sites adjacent to the photobleached regions shown in A and B as ROI 1–4. Quantification was performed in at least three independent experiments, and a representative graph was shown.