Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function

Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function.