In the developing Drosophila trachea, the maturation of tracheal terminal cells involves the generation of gas-filled tubular branches. However, the mechanism underlying subcellular lumen formation in these cells remains unknown. By adapting high pressure freezing and freeze substitution techniques to Drosophila larvae and performing transmission electron microscopy, Mark Metzstein and Linda Nikolova (p. ) show that, contrary to previous belief, lumen formation is not achieved by the direct fusion of cytoplasmic vesicles. Instead, the authors find that it requires a previously undescribed intermediary membrane-lined multivesicular compartment. In this compartment, vesicles assemble and then fuse into a nascent lumen. By further adapting their ultrastructural imaging technique to preserve the fluorescence of protein reporters and performing correlative light and electron microscopy, the authors show that the resolution of the multivesicular intermediate into a mature lumen requires Rabconnectin-3-mediated acidification of the compartment by the V-ATPase proton pump. The tools developed in this study to analyse tubulogenesis in the trachea and the insights provided on the mechanisms underlying this process are likely to contribute to the understanding of lumen formation in other organs.
