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Fourth Featured Abstract at ISEV 2020 Virtual Annual Meeting Reports That, Following Endocytosis by Acceptor Cells, Extracellular Vesicles (EVs) Release Their Cargo from Endosomes; New Analytical Methodology Enables Key Finding

Today (Wednesday, June 22), in the last of four Featured Abstracts presented by graduate students during the ISEV 2020 Virtual Annual Meeting (July 20-22) (https://www.eventscribe.com/2020/ISEV/), Bhagyashree Joshi (photo), of the Department of Biomedical Engineering, University of Groningen, University Medical Center Groningen, The Netherlands, presented her group’s abstract (FA03) “Genetically Encoded Probes Provide Insight into Extracellular Vesicle Cargo Release in Cells.” Ms. Joshi is a PhD candidate in the laboratory of Inge Zuhorn, PhD, Associate Professor, Department of Biomedical Engineering, University of Groningen, University Medical Center Groningen University Medical Center Groningen. In her introduction, Ms. Joshi noted that extracellular vesicles (EVs) are known to modulate tissue development, regeneration, and disease through the transfer of proteins, nucleic acids, and lipids between cells. Currently, however, the mechanism of cytosolic delivery of EV cargo is largely unknown, she said. It has been speculated that EVs undergo back fusion at multi-vesicular bodies (MVBs) in recipient cells to release their functional cargo. However, Ms. Joshi said, evidence for this is lacking. She remarked that tracing the cellular uptake of EVs with high resolution, as well as acquiring direct evidence for the release of EV cargo, is challenging, chiefly because of technical limitations. To address this problem, Ms. Joshi and colleagues developed an analytical methodology that combined state-of-the-art molecular tools and correlative light and electron microscopy (CLEM) to identify the intracellular site for EV cargo release. Green fluorescent protein (GFP) was loaded inside EVs through the expression of GFP-CD63, a fusion of GFP to the cytosolic tail of CD63, in EV producer cells. In addition, Ms, Joshi and colleagues genetically engineered a cell line that expresses anti-GFP fluobody that specifically recognizes the EV cargo (GFP).

In experiments, the researchers showed that incubation of anti-GFP fluobody-expressing cells with GFP-CD63 EVs resulted in the formation of fluobody punctae, designating cytosolic exposure of GFP. Ultrastructural analysis of the underlying structures at GFP/fluobody double-positive punctae demonstrated that EV cargo release occurs from endosomes/lysosomes. Further, Ms. Joshi added that endosomal damage was not observed in the EV acceptor cells, as shown by the absence of galectin3 recruitment (which detects endosomal permeabilization) in the CD63-RFP EV treated recipient cells expressing fluorescently tagged galectin3.

In addition, the researchers showed that neutralization of endosomal pH and cholesterol accumulation in endosomes leads to blockage of EV cargo exposure, indicating that EV cargo release is dependent on endosomal pH and cholesterol level.

Based on their results, Ms. Joshi and colleagues concluded that a fraction of internalized EVs fuse with the limiting membrane of endosomes/lysosomes in an acidification-dependent manner, which results in EV cargo exposure to the cell cytosol.

They further concluded that genetically encoded cytosolic probes and CLEM offer an excellent approach to study both the mechanism and efficiency of EV cargo release in cells.

For additional details of this exciting work, please see the recent article “Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes,” by Ms. Joshi et al. in ACS Nano (https://pubs.acs.org/doi/pdf/10.1021/acsnano.9b10033).

In addition to having her work selected as one of ISEV 2020's four Featured Abstracts, Ms. Joshi is a proud ISEV 2020 Scholarship Awardee.

A 5-minute Q&A session on this Featured Abstract was moderated by Ken Witwer, PhD, Associate Professor, Molecular and Comparative Pathobiology, Johns Hopkins, and ISEV Executive Chair for Science & Meetings.