Abstract
The fruit fly Drosophila melanogaster is widely used to study conserved developmental and cellular signaling pathways in vivo. Many signaling pathways, including Wnt signaling, depend on tightly regulated secretion, endocytosis, and intracellular trafficking events. However, visualizing the dynamics of membrane-associated proteins within intact epithelial tissues such as wing imaginal discs remains challenging because of the small size and densely packed columnar organization of these cells. Here, we present a protocol combining three-dimensional stimulated emission depletion (3D STED) nanoscopy with Uptake assays and pharmacological perturbations in Drosophila wing imaginal discs. This workflow enables super-resolution imaging of membrane-associated protein dynamics within intact tissue architecture at near-isotropic resolution. Using Wingless-secreting cells as an example, we visualize the localization and trafficking of Wingless (Wg) at the apical membrane. To analyze dynamic trafficking processes, the imaging workflow is combined with an uptake assay that selectively labels proteins undergoing secretion and reinternalization within a defined time window. In combination with chemical inhibitors and visualization using 3D STED nanoscopy, this approach enables functional analysis of membrane trafficking dynamics. Together, this workflow provides a versatile framework for studying membrane organization and protein trafficking at subcellular resolution in whole tissue samples of Drosophila.