Density-Dependent Rearrangement of Desmoplakin in Epithelial Junctions: Insights from Expansion Microscopy

Desmosomes mechanically couple neighboring epithelial cells to the intermediate filament cytoskeleton. However, how the nanoscale organization changes with epithelial cell density remains incompletely understood. Here, we used expansion microscopy (ExM) to examine Madin-Darby canine kidney epithelial monolayers. ExM revealed density-associated differences in the relative apicobasal organization of epithelial junctions, with increased separation between occludin-positive tight junctions and desmoglein 2-positive desmosomes in confluent monolayers. A comparison of subconfluent and confluent cultures revealed a density-associated reorganization of desmoplakin (DSP) within opposing desmosomal plaques, with a more pronounced change in C-terminal spacing than in N-terminal spacing. Independent STED analysis of non-expanded samples confirmed the density-associated increase in DSP C-terminal spacing. In keratin filament-deficient cells, this spacing was elevated in subconfluent cultures and did not increase with cell density. Pharmacological inhibition of myosin II or ROCK reduced this elevated spacing, indicating that keratin loss-associated reorganization of DSP is sensitive to actomyosin contractility. These findings show that epithelial cell density is associated with the remodeling of epithelial junction organization and DSP plaque architecture, demonstrating the utility of ExM for quantitative analysis of junctional architecture.