Understanding Secretory MVB Trafficking for the Release of Small Extracellular Vesicles

Abstract

The release of small extracellular vesicles (sEVs) from multivesicular bodies (MVBs) represents an integral part of intercellular communication during development, homeostasis and disease. While secretory MVBs are trafficked to the cell membrane to release intraluminal vesicles into the extracellular space, other subsets undergo lysosomal fusion for content degradation. This endosomal fate decision is highly relevant for intercellular communication, yet the molecular motors involved remain unknown. In this study, an in vivo RNAi screen identified the kinesin-3 family member Klp98A as the master regulator of endosomal transport in Drosophila wing imaginal discs. In this model system, Wg secretion for long-range signal transduction occurs after apical Wg entry into the endosomal system, followed by secretory transport and sEV-based Wg release. In the absence of Klp98A, apicobasal transport of Wg and the sEV marker Tsp96F was abrogated and MVBs accumulated in the apical cytosol. Despite the fact that basal Wg secretion was previously proposed as the main source of the Wg signal, Wg signal transduction and wing development occurred normally. Our data therefore challenge the previous model of Wg signal transfer and imply the existence of alternative routes for transcytosis-independent Wg secretion. We consequently demonstrate that Wg is indeed recycled from Rab4- and Rab11-endosomes at the apical cell side independently of apicobasal transport. To transfer our findings from Drosophila into a human model system, we analyzed the implication of kinesin-3 family members with endosomal trafficking and sEV release in HCT116 cells. To this end, motorless kinesin constructs were generated, whose inducible linkage to a minus end-directed motor allowed for the systematic generation of kinesin-endosome interaction profiles. Indeed, individual kinesin-3 members were distinctly recruited to specific endosomal compartments: We show that while KIF13A and KIF13B preferentially interact with early Rab11-endosomes, KIF1A and KIF1B predominantly interact with CD63-bearing late endosomes. As both of these compartments participate in the release of sEVs, we consequently probed for the involvement of KIF1 and KIF13 in secretory MVB trafficking. We confirm that especially KIF1A and KIF1B engage in transport of sEV marker-bearing late MVBs and show that correspondingly, their knockdown is associated with partially reduced sEV secretion levels. We hence propose both kinesins as promising candidates for sEV secretion. Understanding which kinesins mediate secretory MVB transport is a key step towards comprehending the balance between secretory versus degradative MVB trafficking and thus the underlying regulation of sEV biogenesis. By combining in vivo and in vitro model systems, we identified Drosophila Klp98A and the kinesin-3 family as evolutionary conserved trafficking factors critically involved in anterograde MVB and sEV-marker transport. Altogether, this thesis lays the groundwork to unravel the central role sEVs play during health and disease and contributes to a deeper understanding of the molecular machinery that governs intercellular communication.