Acute manipulation and real-time visualization of membrane trafficking and exocytosis inDrosophila

Abstract

Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but essential tools for investigating the dynamics of trafficking have been limited to cultured cells. Here we present a system that enables acute manipulation and real-time visualization of membrane trafficking through reversible retention of proteins in the endoplasmic reticulum (ER) in living multicellular organisms. By adapting the retention using selective hooks (RUSH) approach to Drosophila, we show that trafficking of GPI-linked, secreted, and transmembrane proteins can be controlled with high temporal precision using streptavidin hooks and biotin-induced release in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and of apical secretion in tracheal tubes and the spatiotemporal dynamics of tricellular junction assembly in epithelia. Furthermore, hook-induced ER retention enables tissue-specific knockdown of secretory protein function. The system is compatible with available protein-traps and is widely applicable to investigate membrane trafficking in diverse cell types in vivo.