The uptake of small extracellular vesicles by recipient cells is facilitated by paracrine adhesion signaling

Small extracellular vesicles (sEVs) play crucial roles in intercellular communication. However, the internalization of individual sEVs by recipient cells has not yet been directly observed. Here, we examined these mechanisms using state-of-the-art imaging techniques. Single-molecule imaging showed that tumor-derived sEVs can be classified into several subtypes. Simultaneous single-sEV particle tracking and observation of super-resolution movies of membrane invaginations in living cells revealed that all sEV subtypes were internalized via clathrin-independent endocytosis mediated by galectin-3 and LAMP-2C, while some subtypes that recruited raft markers were internalized through caveolae. Integrin {beta}1 and talin-1 accumulated in recipient cell plasma membranes beneath all sEV subtypes. Paracrine, but not autocrine, sEV binding triggered Ca2+ mobilization induced by the activation of Src family kinases and PLC{gamma}. Subsequent Ca2+-induced activation of calcineurin-dynamin promoted sEV internalization, leading to the recycling pathway. Thus, we have clarified the detailed mechanisms of sEV internalization driven by paracrine adhesion signaling.