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

Abstract

SUMMARY: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 10-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 10-nm gold, suggesting that new Dsg3 molecules were being delivered to the cell surface. Desmosomes were labeled with both 5-nm gold and 10-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. (Lab Invest 2000, 80:1583-1592.

150thAnniversary Series: Desmosomes and Autoimmune Disease, Perspective of Dynamic Desmosome Remodeling and Its Impairments in Pemphigus

“…This dynamic nature of desmosomes can be well illustrated in experiments using low -high Ca 2 ϩ -switching in culture medium (Hennings et al, 1980;Hennings & Holbrook, 1983;Sato et al, 2000). In low (below 0.1 mM) Ca 2 ϩ -cultured keratinocytes, desmosomes are not formed.…”

“…In low Ca 2 ϩ -grown cells, desmogleins form simple lateral aggregations without intracellular attachment plaques on the plasma membrane, followed by association with attachment plaques and keratin fi laments, forming " half-desmosomes " , but not complete desmosomes, as observed under electron microscopy. These halfdesmosomes appear to couple to form complete desmosomes following low -high Ca 2 ϩ -switching (Sato et al, 2000). These " half-desmosomes " are endocytosed in low Ca 2 ϩ medium, a process referred to as a " coordinated Sisyphus cycle " (Demlehner et al, 1995).…”

“…Using cultured keratinocytes, PVIgG and AK23 treatments induced internalization and depletion of Dsg 3 from cell membrane (Triton-X-100 soluble) fractions within 30 -60 min and depletion of Dsg3 from cytoskeleton (Triton-X-100 insoluble) fractions including desmosomes within later 24 -48 h Aoyama et al, 2010;Calkins et al, 2006: Sato et al, 2000Williamson et al, 2006;Yamamoto et al, 2007). These results suggest that the fi rst depletion of Dsg3 from non-desmosomal pool is linked to the secondary depletion of Dsg3 from desmosomes, causing a decrease in cell adhesion strength.…”

“…The second step in the process to generate acantholysis is the endocytosis of Dsg-autoantibody immune complex generated at the cell surface of dynamic weakadhesive cells, resulting in the depletion of Dsgs from desmosomes as shown in Figure 4 (Sato et al, 2000;Calkins et al, 2006;Delva et al, 2008;Mao et al, 2009). This PV IgG-induced internalization occurs via a membrane raft-mediated pathway (Delva et al, 2008), and is probably mediated by activation of EGFR (Bektas et al, 2013) and p38MAPK (Jolly et al, 2010;Mao et al, 2014).…”

New Functions of Polysialic Acid and Its Relationship to Schizophrenia

“…4A), as described above. In addition, we have demonstrated that a microglia cell line contains α2,8-linked polyNeu5Ac and that polySia disappears rapidly in these cells after inflammatory stimulation with lipopolysaccharide (LPS) (30,31). The rapid clearance of polySia on the microglia cell surface indicates that these cells can supply trophic factors which bind to polySia, immediately after LPS stimulation.…”