Rab-mediated membrane trafficking systems in terminal tracheal cells of Drosophila melanogaster

Abstract

The terminal cells of the tracheal system in Drosophila melanogaster are one of the few known cell types that form stable, long branches. Like all epithelial cells, terminal cells have an apical and a basal membrane domain. Throughout larval life, each terminal cell's basal membrane repeatedly bifurcates and elongates to establish an extensive branched network, and the apical membrane is invaginated to form a subcellular tube that expands a lumen in each branch. The lumen is contiguous with the tracheal system's lumen and carries air to deliver oxygen to internal organs. To achieve this elaborate structure, terminal cells expand their volume and surface area at least hundredfold over the course of a few days. The synthesis and distribution of membrane and proteins must thus pose a particular challenge. In addition to the necessary bulk of production, all material needs to be delivered to the appropriate membrane domain. In particular, the specialised apical extracellular matrix within the tracheal lumen must be supplied with the structural molecules that confer the tracheal system its physical stability and the ability to contain and exchange gas. My hypothesis was thus that terminal cells possess a customised membrane trafficking system that allows them to transport large amounts of newly synthesised proteins and lipids through secretory pathways to the apical and basal domains as appropriate. This directed the focus of my research to the Rab family of small GTPases, a group of proteins known as master regulators of membrane trafficking. Rabs bind to the plasma membrane or to intracellular membrane compartments and subsequently function as platforms to recruit effector proteins. These effectors execute virtually all mechanisms that direct membrane-bound and secreted molecules from one membrane compartment to the next, as well as to and from the cell's exterior environment. To explore membrane trafficking systems in terminal cells, I first conducted a systematic quantitative analysis of normal and abnormal terminal cell phenotypes to better understand how morphological defects should be interpreted. This was necessary because of the variability with which mutant phenotypes manifest in terminal cells. My results provide a reference for the range of natural variability in TC phenotypes, and identify phenomic analysis, currently only applied to plants, as a powerful method for extracting meaning from partially penetrant loss-of-function phenotypes. The main part of my work was a tracheal knockdown screen targeting all Drosophila Rabs. Rather than using specific RNAi constructs against each gene, I utilised a collection of endogenously YFP-tagged Rabs and knocked down the YFP tag in animals where all copies of a Rab are tagged. This indirect approach confers more confidence in its results because unlike conventional RNAi, its risk of false positives is minimal. Through this and supplementary experiments, I pinpointed a secretion route from endoplasmatic reticulum through Golgi and endosomes to the plasma membrane that ensures proper organisation of the extracellular matrix and allows the cell to grow. I identified only one Rab whose phenotype suggested a role in establishing the shape and size of the subcellular tube: Rab8. In my final experiments, I therefore investigated how Rab8 is involved in apical morphogenesis in terminal cells. While not definitive, my results suggest that Rab8 acts on apically-directed secretory endosomal traffic to retain basally-destined cargoes, thereby preventing their erroneous apical secretion.