Biology subjects

Somiya, M.

Publications and source records attributed to Somiya, M..

2 recordsLinked to original sources

bioRxiv2021

Reporter gene assay for membrane fusion of extracellular vesicles

AbstractExtracellular 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 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.

cell biology↗

bioRxiv2020

Real-time luminescence assay for cytoplasmic cargo delivery of extracellular vesicles

Extracellular vesicles (EVs) have been considered to deliver biological cargos between cells and mediate intercellular communication. However, the mechanisms that underlie the biological process of EV uptake and cytoplasmic cargo release in recipient cells are largely unknown. Quantitative and real-time assays for assessment of the cargo delivery efficiency inside recipient cells have not been feasible. In this study, we developed an EV cargo delivery (EVCD) assay using a split luciferase called the NanoBiT system. Recipient cells expressing LgBiT, a large subunit of luciferase, emit luminescence when the EV cargo proteins fused with a small luminescence tag (HiBiT tag) that can complement LgBiT are delivered to the cytoplasm of recipient cells. Using the EVCD assay, the cargo delivery efficiency of EVs could be quantitatively measured in real time. This assay was highly sensitive in detecting a single event of cargo delivery per cell. We found that modification of EVs with a virus-derived fusogenic protein significantly enhanced the cytoplasmic cargo delivery; however, in the absence of a fusogenic protein, the cargo delivery efficiency of EVs was below the threshold of the assay. The EVCD assay could assess the effect of entry inhibitors on EV cargo delivery. Furthermore, using a luminescence microscope, the cytoplasmic cargo delivery of EVs was directly visualized in living cells. This assay could reveal the biological mechanism of the cargo delivery processes of EVs.

cell biology↗