biostudies-literatureMorandi MIEuropean Research Councilpgac156https://www.ebi.ac.uk/biostudies/studies/S-EPMC9802263biostudies-literatureUnknown1(4)Extracellular vesicles (EVs) transfer bioactive molecules between cells in a process reminiscent of enveloped viruses. EV cargo delivery is thought to occur by protein-mediated and pH-dependent membrane fusion of the EV and the cellular membrane. However, there is a lack of methods to identify the fusion proteins and resolve their mechanism. We developed and benchmarked an <i>in vitro</i> biophysical assay to investigate EV membrane fusion. The assay was standardized by directly comparing EV and viral fusion with liposomes. We show that EVs and retroviruses fuse with liposomes mimicking the membrane composition of the late endosome in a pH- and protein-dependent manner. Moreover, we directly visualize the stages of membrane fusion using cryo-electron tomography. We find that, unlike most rPNAS nexusExtracellular vesicle fusion visualized by cryo-electron microscopy.PMC9802263757743851080Zarfati GRegev-Rudzki NNapso Shogan TMorandi MIAbou Karam PGinini LAvinoam OOzer-Partuk EBusko PElbaz-Alon YGil ZKhan SfalseExtracellular vesicle fusion visualized by cryo-electron microscopy.Extracellular vesicles (EVs) transfer bioactive molecules between cells in a process reminiscent of enveloped viruses. EV cargo delivery is thought to occur by protein-mediated and pH-dependent membrane fusion of the EV and the cellular membrane. However, there is a lack of methods to identify the fusion proteins and resolve their mechanism. We developed and benchmarked an <i>in vitro</i> biophysical assay to investigate EV membrane fusion. The assay was standardized by directly comparing EV and viral fusion with liposomes. We show that EVs and retroviruses fuse with liposomes mimicking the membrane composition of the late endosome in a pH- and protein-dependent manner. Moreover, we directly visualize the stages of membrane fusion using cryo-electron tomography. We find that, unlike most r2022-01-01T00:00:00Z2022 Sep2025-04-27T00:48:55.54Z2025-04-06T18:04:03.157ZS-EPMC98022633671484810.1093/pnasnexus/pgac156