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

Desmosomes mechanically couple neighboring epithelial cells to the intermediate filament cytoskeleton. However, the nanoscale organization of opposing cytoplasmic plaques and their dynamic remodeling during epithelial maturation remain poorly understood owing to the resolution limits of conventional optical microscopy. In this study, we applied expansion microscopy (ExM) to Madin-Darby canine kidney epithelial monolayers to resolve the spatial distribution of the core desmosomal proteins within opposing plaques for quantitative mapping. A comparative study of subconfluent and confluent cultures revealed a density-dependent transition of desmoplakin (DSP) from a compact "closed" state to an extended "open" conformation. Specifically, the spacing between opposing DSP C-terminal regions increased significantly with cell density. In contrast, desmoglein 2, plakoglobin and plakophilin 2 remained spatially stable, while the DSP N-terminus exhibited a comparatively subtle cytoplasmic shift. Notably, keratin filament-deficient cells adopt a constitutively open conformation regardless of cell density, demonstrating that keratin anchoring is indispensable for structural remodeling. Our findings reveal keratin-dependent conformational remodeling of DSP as a hallmark of junctional maturation and establish ExM as a robust, accessible approach for deciphering the functional nanoscale architecture of cell-cell junctions.