Content release of extracellular vesicles in a cell‐free extract

Abstract

Extracellular vesicles (EVs) transfer molecules from donor to acceptor cells. The EV‐content delivery process within the acceptor cell is poorly characterized. We developed a new cell‐free assay to assess EV‐content release in vitro. We found that EV‐cytosolic cargoes are released from EVs when isolated vesicles are incubated with purified plasma membrane sheets at acidic pH, a characteristic of the endolysosomal environment. This process is protein dependent. Our results suggest that EV‐content delivery occurs within the endo/lysosomes of acceptor cells and is triggered by acidification. This process resembles virus content delivery and may require membrane fusion. The assay presented here will facilitate investigations into the core machinery and mechanisms underlying EV content delivery.

Characterization of brain‐derived extracellular vesicles reveals changes in cellular origin after stroke and enrichment of the prion protein with a potential role in cellular uptake

“…It has been reported that fusion of EVs with purified membrane sheets in a cell‐free system requires the presence of proteins at the surface of both entities and an acidic pH [106]. Our data suggest that PrP and PrP‐C1 may be relevant EVs residents in this initial EV‐to‐target cell interaction.…”

Uptake and Fate of Extracellular Membrane Vesicles: Nucleoplasmic Reticulum-Associated Late Endosomes as a New Gate to Intercellular Communication

“…An acidic milieu as observed in cancerous tissue microenvironment might favor EV-cell fusion [ 103 ]. The acidity-mediated direct fusion of EVs with tumor cells may be similar to the fusion of endocytosed EVs with limiting late endosomal membranes observed in the low pH endocytic compartment and/or the entry of certain enveloped viruses [ 104 , 105 , 106 ]. Under these extreme conditions, alterations in membrane fluidity and/or unmasking of fusogenic proteins may account for the fusion ability [ 103 ].…”

Environmental pH stress influences cellular secretion and uptake of extracellular vesicles

“…The findings regarding the cathepsin B resemble our results; however, their detailed molecular mechanisms are still unknown. In addition, there were established EV techniques including the anti‐GFP fluobody expressing system to assess membrane fusion of EVs and endosomes [36], EV cargo release technique using proteases [37], and intracellular visualization technique using Renilla luciferase [38], and the experimental systems will contribute for not only elucidation of CD63‐GFP cytosolic diffusion in the low pH condition but also cellular uptake, membrane fusion, and cytosolic release of EV contents.…”