NLuc-Hsp70 plasmid vector胞質標記NanoLuc熒光表達質粒載體

NLuc-Hsp70(胞質標記)
NanoLuc luciferase-tagged Hsp70.
We focused on Hsp70, an established generic EV marker22,23,24, thus a good candidate for cytosolic release assessment, in contrast with most of the EV markers that are membrane-associated. Importantly, we successfully validated and used this EV cargo in our recent cell-free reconstitution study14. We used NanoLuc luciferase, a recently engineered luciferase that has superior signal-to-noise ratio, to ensure high sensitivity detection of our assay25. In-gel luciferase activity from lysates of cells that stably expressed NLuc-Hsp70, revealed that the chimeric proteins migrated, as expected, as a unique band corresponding to a >75?kDa protein (Fig. 1a). The absence of detectable partial degradation of NLuc-Hsp70 validated the relevance to monitor NLuc-Hsp70 behavior/fate exclusively through its enzymatic/NLuc activity.
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.

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