Lack of involvement of CD63 and CD9 tetraspanins in the extracellular vesicle content delivery process.

Extracellular vesicles (EVs) are thought to mediate intercellular communication by transferring cargoes from donor to acceptor cells. The EV content-delivery process within acceptor cells is still poorly characterized and debated. CD63 and CD9, members of the tetraspanin family, are highly enriched within EV membranes and are respectively enriched within multivesicular bodies/endosomes and at the plasma membrane of the cells. CD63 and CD9 have been suspected to regulate the EV uptake and delivery process. Here we used two independent assays and different cell models (HeLa, MDA-MB-231 and HEK293T cells) to assess the putative role of CD63 and CD9 in the EV delivery process that includes uptake and cargo delivery. Our results suggest that neither CD63, nor CD9 are required for this function.

Quantitative Measurement of Extracellular Vesicle Content Delivery Within Acceptor Cells.

Extracellular vesicles (EVs), including exosomes and microvesicles, are thought to transport bioactive molecules from donor to acceptor cells. Although EV uptake has been qualitatively assessed through subcellular imaging, EV content delivery has been rarely addressed due to a lack of adequate methods. Here we present a sensitive bulk assay to quantitatively measure EV uptake and content delivery in mammalian cell. In this assay, EVs containing a NanoLuc luciferase-tagged cargo are mixed with unlabeled acceptor cells. Cell fractionation separates membrane and cytosolic fractions, and luciferase activity is measured within each fraction to determine the percentage of cytosolic release. This assay can be used to further decipher cellular and molecular mechanisms that regulate the EV delivery process or to quantitatively test specific pairs of donor-acceptor cells.

Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells.

Extracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.

Content release of extracellular vesicles in a cell-free extract.

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.