Genetic screening identifies WNT5A/RYK as a determinant of extracellular vesicle fate

Extracellular vesicles (EVs) are natural mediators of intercellular communication through the transfer of cytoplasmic material from donor to recipient cells. The molecular mechanisms governing EV uptake and cargo delivery in recipient cells remain largely elusive. To address this gap, we performed a genetic screening interrogating 300 genes of the dynamic surfaceome for their involvement in EV uptake. Activators found in the screening establish a previously unknown connection between EV uptake and Wnt pathways. We then observed that, unlike WNT-naïve EVs that are primarily trafficked to lysosomes in recipient cells, WNT5A-bearing EVs linger in early endocytic structures. This rerouting is dependent on the expression of RYK, an established co-receptor for WNT5A. Our results depict a mechanism underlying EV functional heterogeneity as well as reveal a potential regulation system for noncanonical WNT signaling.