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 been directly observed. Here, we examined these mechanisms using state-of-the-art imaging techniques. Single-molecule imaging shows 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 reveal that all sEV subtypes are internalized via clathrin-independent endocytosis mediated by galectin-3 and lysosome-associated membrane protein-2C, while some subtypes that recruited raft markers are internalized through caveolae. Integrin β1 and talin-1 accumulate in recipient cell plasma membranes beneath all sEV subtypes. Paracrine, but not autocrine, sEV binding triggers Ca²⁺ mobilization induced by the activation of Src family kinases and phospholipase Cγ. Subsequent Ca²⁺-induced activation of calcineurin-dynamin promotes sEV internalization, leading to the recycling pathway. Thus, we clarified the detailed mechanisms of sEV internalization driven by paracrine adhesion signaling.