DNA-PAINT resolves E-cadherin-independent cross-junctional F-actin organization in <i>Drosophila</i> embryonic tissue

Cell junction remodeling is central to epithelial morphogenesis and tissue rheology and depends on the interplay between adhesion molecules and the actomyosin cortex. E-cadherin constitutes the molecular basis for epithelial cell adhesion, whereas cortical actomyosin plays a major role in intracellular force generation. However, the precise nanoscale organization and relationship between F-actin and E-cadherin at the cell interface still remain insufficiently understood. Here, we applied super-resolution DNA/peptide-PAINT microscopy to reveal the nanoscopic clustering of E-cadherin and its junctional distribution in relation to cortical F-actin at adherens junctions in the Drosophila embryonic epidermis. We were able to resolve distinct pairs of E-cadherin clusters approximately 45 nm apart on opposite sides of the adherens junctions. Intriguingly, these paired clusters were interspersed with unpaired clusters, lacking corresponding counterparts across the junction. We observed that cluster size, spatial arrangement, and cross-junction matching change during development and depend on N-glycosylation, a posttranslational modification affecting E-cadherin. Moreover, the organization of F-actin cortices between neighboring cells was found to be strongly correlated at junctions. Contrary to expectations, this intercellular F-actin correlation was observed independently of E-cadherin. Our study provides new insights into the nanoscale organization of adherens junctions, opening a window into the molecular mechanism of adhesion and mechanics of epithelial cells during morphogenesis.