Hollmann, M. | bioRxiv Science

bioRxiv2022

Acute manipulation and real-time visualization of membrane trafficking and exocytosis in Drosophila

Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but tools for investigating dynamics of membrane 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 in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and apical secretion and the spatiotemporal dynamics of tricellular junction assembly in epithelia of living embryos. Furthermore, we show that controllable ER-retention enables tissue-specific depletion of secretory protein function. The system is broadly applicable to visualize and manipulate membrane trafficking in diverse cell types in vivo.

cell biology↗

bioRxiv2020

The transmembrane proteins M6 and Anakonda cooperate to initiate tricellular junction assembly in epithelia of Drosophila

Cell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1, 2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3-5], how they are assembled, maintained and remodeled is poorly understood. In Drosophila the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembrane-domain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8, 9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10, 11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here we show that M6 is essential for the assembly of tricellular, but not bicellular occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a low-mobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices.

cell biology↗