Abstract Rationale Desmosomes are a type of cell-cell junction present in various tissues and epithelia. Defective desmosomes result in a variety of inherited and autoimmune diseases. Recent genome wide-association studies have increased interest in desmosome genes in the lung, particularly desmoplakin (DP), due to an association with pulmonary fibrosis. We sought to define desmosome organization in airway epithelium to better understand their function and pathophysiology in lung disease. To achieve this, we used immunofluorescence and high-resolution volume electron microscopy to visualize desmosome structure in two-dimensional and three-dimensional (3D) cell culture models. Methods 16HBE4o- cells, a bronchial epithelial cell line, was cultured in monolayer and in air-liquid interface (ALI) for immunofluorescence localization of DP. To define desmosome ultrastructure, we performed segmentations and 3-D quantitative analyses of desmosomes in a publicly available focused ion beam-scanning electron microscopy (FIB-SEM) dataset of airway basal stem cells grown in organotypic ALI culture. Results In monolayer culture, we observed DP staining along cell-cell interfaces with a characteristic “tram track” pattern as observed in other epithelia. In ALI culture imaged 28 days post-differentiation, DP staining was uniformly seen along cell-cell borders from the apical to basolateral surfaces. The FIB-SEM dataset consisted of airway epithelium composed of multiciliated cells (MCC) and mucin-producing goblet cells (GC). 3D segmentation identified 136 individual desmosomes. Analysis of the 3D map similarly demonstrated uniform distribution of desmosomes along the apical and basolateral cell surfaces. However, desmosomes near the apical region appeared longer than those in basolateral regions. We next compared desmosome characteristics at homotypic (MCC-MCC) and heterotypic (MCC-GC) cell-cell borders. They displayed similar plaque to plaquedistances (59nm vs 61nm) and similar maximum Feret diameter (MFD): median MFD 120.82nm (48-460nm) and median F = 0.86 (0.61-0.98) vs median MFD 127.64nm (64-420nm) and median F = 0.86 (0.68-0.96). Conclusions We visualized desmosomes in airway epithelia grown in monolayer and 3D culture models. In our models, desmosomes were uniformly spaced along the basolateral cell contact. Additionally, using a publicly available dataset, our 3D segmentation revealed that desmosomes in the apical region of lateral cell-cell borders appeared longer than desmosomes located more basally. Lastly, we report for the first time, a comparative analysis of desmosome ultrastructure at homotypic and heterotypic cell contacts, observing that they are similar in size. These results define desmosome morphology and distribution in the airway and provide a foundation for understanding how mutations in desmosome genes might contribute to lung pathology. This abstract is funded by: NIH
Chen et al. (Fri,) studied this question.