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 f...

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Autores principales:	Masaharu Somiya, Shun'ichi Kuroda
Formato:	article
Lenguaje:	EN
Publicado:	Taylor & Francis Group 2021
Materias:	cargo transfer extracellular vesicles membrane fusion NanoLuc VSV‐G Cytology QH573-671
Acceso en línea:	https://doaj.org/article/b424bd44e3e14b729973ab6b5a5a0520
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spelling	oai:doaj.org-article:b424bd44e3e14b729973ab6b5a5a05202021-11-24T14:04:30ZReporter gene assay for membrane fusion of extracellular vesicles2001-307810.1002/jev2.12171https://doaj.org/article/b424bd44e3e14b729973ab6b5a5a05202021-11-01T00:00:00Zhttps://doi.org/10.1002/jev2.12171https://doaj.org/toc/2001-3078Abstract 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.Masaharu SomiyaShun'ichi KurodaTaylor & Francis Grouparticlecargo transferextracellular vesiclesmembrane fusionNanoLucVSV‐GCytologyQH573-671ENJournal of Extracellular Vesicles, Vol 10, Iss 13, Pp n/a-n/a (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	cargo transfer extracellular vesicles membrane fusion NanoLuc VSV‐G Cytology QH573-671
spellingShingle	cargo transfer extracellular vesicles membrane fusion NanoLuc VSV‐G Cytology QH573-671 Masaharu Somiya Shun'ichi Kuroda Reporter gene assay for membrane fusion of extracellular vesicles
description	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.
format	article
author	Masaharu Somiya Shun'ichi Kuroda
author_facet	Masaharu Somiya Shun'ichi Kuroda
author_sort	Masaharu Somiya
title	Reporter gene assay for membrane fusion of extracellular vesicles
title_short	Reporter gene assay for membrane fusion of extracellular vesicles
title_full	Reporter gene assay for membrane fusion of extracellular vesicles
title_fullStr	Reporter gene assay for membrane fusion of extracellular vesicles
title_full_unstemmed	Reporter gene assay for membrane fusion of extracellular vesicles
title_sort	reporter gene assay for membrane fusion of extracellular vesicles
publisher	Taylor & Francis Group
publishDate	2021
url	https://doaj.org/article/b424bd44e3e14b729973ab6b5a5a0520
work_keys_str_mv	AT masaharusomiya reportergeneassayformembranefusionofextracellularvesicles AT shunichikuroda reportergeneassayformembranefusionofextracellularvesicles
_version_	1718415069430153216

Reporter gene assay for membrane fusion of extracellular vesicles

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