Reporter gene assay for membrane fusion of extracellular vesicles.

Abstract

Extracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells. Here, we describe a reporter gene assay that can measure the membrane fusion efficiency of EVs during cargo delivery to recipient cells. When EVs containing tetracycline transactivator (tTA)-fused tetraspanins are internalized by recipient cells and fuse with cell membranes, the tTA domain is exposed to the cytoplasm and cleaved by tobacco etch virus protease to induce tetracycline responsive element (TRE)-mediated reporter gene expression in recipient cells. This assay (designated as EV-mediated tetraspanin-tTA delivery assay, ETTD assay), enabled us to assess the cytoplasmic cargo delivery efficiency of EVs in recipient cells. With the help of a vesicular stomatitis virus-derived membrane fusion protein, the ETTD assay could detect significant enhancement of cargo delivery efficiency of EVs. Furthermore, the ETTD assay could evaluate the effect of potential cargo delivery enhancers/inhibitors. Thus, the ETTD assay may contribute to a better understanding of the underlying mechanism of the cytoplasmic cargo delivery by EVs.

• Authors: Masaharu Somiya, Shun'ichi Kuroda
• Journal: Journal of extracellular vesicles (J Extracell Vesicles) Vol. 10 Issue 13 Pg. e12171 (11 2021) ISSN: 2001-3078 [Electronic] United States
• PMID: 34807503 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.

Chemical References

• Tetraspanins
• Trans-Activators
• enhanced green fluorescent protein
• Green Fluorescent Proteins
• Endopeptidases
• TEV protease
• Tetracycline

Topics

• Biological Transport (genetics)
• Cell Communication (genetics)
• Cell Membrane (metabolism)
• Cytoplasm (metabolism)
• Endopeptidases (genetics, metabolism)
• Extracellular Vesicles (metabolism)
• Gene Expression Profiling (methods)
• Genes, Reporter
• Green Fluorescent Proteins (genetics)
• HEK293 Cells
• Humans
• Membrane Fusion (genetics)
• Signal Transduction (genetics)
• Tetracycline (metabolism)
• Tetraspanins (metabolism)
• Trans-Activators (metabolism)
• Transfection