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

Small extracellular vesicles (sEVs) play critical roles in intercellular communication. However, the mechanisms by which sEVs are internalized by recipient cells remain unclear. Here, we investigated these mechanisms through state-of-the-art imaging techniques. Single-molecule imaging revealed that tumor-derived sEVs can be divided into several subtypes. By simultaneously performing single sEV-particle tracking and super-resolution movie observation of membrane invaginations in living cells, we discovered that all sEV subtypes were internalized via phagocytosis, while some subtypes that recruited raft markers were endocytosed via caveolae. Furthermore, we demonstrated that integrin β1 and talin-1 accumulated in recipient cell plasma membranes underneath all sEV subtypes. Paracrine, but not autocrine, sEV binding triggers Ca ²⁺ mobilization, which is induced by the activation of Src family kinases and PLCγ. Ca ²⁺ -induced activation of calcineurin-dynamin subsequently promoted sEV internalization, leading to the recycling pathway. Thus, we elucidated the detailed mechanisms of sEV internalization, which is facilitated by paracrine adhesion signaling.