Extracellular vesicle fusion visualized by cryo-electron microscopy

Abstract

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 in vitro 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 retroviruses, EVs remain fusogenic after acidification and re-neutralization. These results provide novel insights into the EV cargo delivery mechanism and an experimental approach to identify the EV fusion machinery.

Plasma versus serum for extracellular vesicle (EV) isolation: A duel for reproducibility and accuracy for CNS‐originating EVs biomarker analysis

“…Regardless, in the blood, ptEVs also serve as the most abundant source of EVs (Tao et al, 2017). It is important to also note that EVs are thought to have a "corona," which consists of biomolecules carried on their outer lipid bilayer surface (Tóth et al, 2021), as well as engage in fusion events (Morandi et al, 2022).…”

Tuning the Extracellular Vesicles Membrane through Fusion for Biomedical Applications

“…These biochemical mediating fusions are structurally diverse, follow the merging of two bilayers, and appear as a common pathway involving a sequence of structurally distinct intermediates [ 90 , 91 , 92 ]. The process begins with loose protein-mediated bilayer membrane contact and progresses to the tight adhesion of the membranes while preserving the integrity of the bilayer [ 93 , 94 ]. In this section, we discuss the artificial fusion of EVs with both synthetic vesicles, such as liposomes, and natural membrane vesicles, such as EVs or cell-derived membranes.…”

Cell entry and release of quasi-enveloped human hepatitis viruses

“…An alternative model for eHAV entry involving fusion rather than degradation of the eHAV membrane was proposed recently 87 . Biochemical and cryo-EM studies provide evidence that exosomes originating from MVEs can fuse with low efficiency to endosomal membranes following endocytosis 88 , 89 . However, there is no direct experimental evidence supporting fusion of the quasi-envelope and endosomal membranes.…”

“…Moreover, a fusion model fails to explain why the membrane-degrading activities of LAL and NPC1 are required for quasi-enveloped viral entry 60 , 68 . Fusion would also deliver viral capsids or RNA directly to the cytoplasm 88 , 89 , making it difficult to understand why endosomal gangliosides are essential for eHAV infection 67 .…”