Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies.

Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. While the formation and functions of EVs have been investigated extensively in cultured cells, studies of EVs in vivo have remained scarce. We report here that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. We define two distinct EV subpopulations, one of which contains the Munc13-4 (also known as UNC13D) homolog Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion (anastomosis) events. The formation of Stac-positive luminal EVs depends on the tracheal tip-cell-specific GTPase Arl3 (also known as Dnd in Drosophila), which is also required for the formation of Stac-positive multivesicular bodies (MVBs), suggesting that Stac-positive EVs derive from fusion of Stac-positive MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-positive MVB formation and tube fusion. We propose that Stac-positive MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35 and Stac. This article has an associated First Person interview with the first author of the paper.