FIGURE 1

Immunofluorescence micrographs showing pemphigus vulgaris immunoglobulin G (PV-IgG)-induced lateral aggregation (Panels a and b) and internalization (Panels c and d) of desmoglein 3 (Dsg3) on the cell surface of cultured DJM-1 cells. Cells were stimulated with PV-IgG-containing medium for a short pulse time of 3 minutes, followed by washing with PBS, and stained with antihuman IgG-FITC in culture medium for 5 minutes (Panel a). FITC is distributed as a linear line on the entire cell surface of cell-cell contact areas and diffusely, although very faintly, on the non-cell-cell contact cell surfaces (Panel a). However, when cells were further incubated in antibody-free medium for an additional 60 minutes after a pulse treatment with PV-IgG (3 min) and staining with antihuman IgG-FITC in culture medium (5 min), a distinct punctuated-label pattern was evident on the entire cell surface, including non-cell-cell contact (free cell surface) areas (Panel b). To determine whether these spots on the free cell surface of non-cell-cell contacts were actually located on the cell surface or internalized within the cells as endosomes, cells were briefly reincubated with PV-IgG (5 min) after the 60-minute chase in antibody-free culture medium and stained with a fresh medium containing antihuman IgG-Rod antibodies for 5 minutes. This time-lapsed double-staining showed that FITC-positive spots visible along the cell-cell contacts, which correspond to desmosomes, also were stained with red Rod fluorescence. Conversely, many of the FITC-positive sites in the central domains of cells were not co-stained with red Rod (Panels c and d), suggesting that FITC-positive, Rod-negative dots were internalized as endosomes, to which the second labeling of PV-IgG could not penetrate.