Assembly pathway of desmoglein 3 to desmosomes and its perturbation by pemphigus vulgaris-IgG in cultured keratinocytes, as revealed by time-lapsed labeling immunoelectron microscopy

To determine the assembly pathway of desmoglein 3 (Dsg3) into desmosomes and the subsequent effects of pemphigus vulgaris immunoglobulin G (PV-IgG) on such, we employed a time-lapsed labeling for FITC/Rhodamine (Rod) double-stained immunofluorescence and 5-nm/10-nm gold double-stained immunoelectron microscopy by using PV-IgG, which was confirmed to react specifically Dsg3. Cells from a human squamous cell carcinoma cell line (DJM-1) were first treated briefly with PV-IgG (3 min), then incubated in either anti-human IgG-FITC or 5-nm gold antibody-containing medium (5 min), followed by a 60-minute chase in normal medium without antibodies. The same cells were reincubated with PV-IgG medium for 3 minutes, followed by either anti-human IgG-Rod or IO-nm gold antibodies for 5 minutes. Using this method, FITC and 5-nm gold particles show the fate of Dsg3-PV-IgG complexes during the following 60-minute chase. IgG-Rod or 10-nm gold particles, which are bound during the last 5 minutes of the chase, show Dsg3 molecules newly expressed on the cell surface during the 60-minute-chase period. Initially, Dsg3 formed two types of small clusters on the nondesmosomal plasma membrane, ie, either half-desmosome-like clusters with keratin intermediate filament (KIF) attachment or simple clusters without KIF attachment. The PV-IgG binding to Dsg3 caused the internalization of the simple clusters into endosomes, but not the half-desmosome-like clusters. After the 60-minute-chase period, both types of cell surface Dsg3 clusters were labeled with only IO-nm gold, suggesting that new Dsg3 molecules were being delivered to the cell surface. Desmosomes were labeled with both 5-nm gold and IO-nm gold, whereas the half-desmosome-like clusters were labeled with only 10-nm gold, suggesting that the desmosomes themselves were not split. These results suggest that Dsg3 first forms simple clusters, followed by KIF-attachment, and then becomes integrated into desmosomes, and that PV-IgG-induced internalization of the nondesmosomal simple clusters of Dsg3 may represent the primary effects of PV-IgG on keratinocytes.