Development of a live-cell imaging assay to elucidate spatiotemporal dynamics of extracellular vesicle fusion with target cells

Abstract Cells communicate via extracellular vesicles (EVs) containing functional RNAs, proteins and lipids. Knowledge on the fate of internalized EVs, especially their capacity to fuse with target cell membranes and deliver luminal cargo, is limited. Currently available EV-cargo delivery assays are indirect and thus unlikely to uncover molecular players and conditions that specifically control the EV-fusion step. Here, we present a novel live-cell imaging assay for detection of EV-binding, -uptake, and -fusion in time and space. We employed the SunTag system for exceptional signal amplification. EV-donor cells were engineered to tag the luminal EV-membrane with a fluorescent label coupled to SunTag peptides. Recipient cells express fluorescent single-chain anti-SunTag antibody (STAb), which binds EV-enclosed SunTag upon its cytosolic exposure. Using SunTagged EVs carrying fusogen VSV-G, we visualize the EV-fusion process, quantify fusion kinetics and efficiency, and determine subcellular localization of fusion events. We term this methodology EV-FUSIM (Extracellular Vesicle Fusion Spatiotemporal Imaging Method). In the future, this technology can support identification of fusogenic EV-subsets, as well as molecular players and drugs that modulate EV-fusion, without confounding effects of post-fusion processes. This will extend knowledge on EV-biology and can aid in the engineering of EVs that efficiently deliver intraluminal therapeutic payloads. Competing Interest Statement The authors have declared no competing interest. Footnotes Data availability statement: the authors confirm that the data supporting the findings are available within the article or its supplementary material. Raw data that support the findings presented here can be made available upon reasonable request. We have submitted all relevant data regarding methodological reporting of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV250079) (EV-TRACK Consortium et al., 2017). Relevant data regarding methodological reporting of flow cytometry experiments has been attached as Supplementary Tables 2 and 3 in the form of completed MIFlowCyt and MIFlowCyt-EV checklists, respectively (Lee et al., 2008; Welsh et al., 2020). Funding statement: this work was supported by The Netherlands Organisation for Scientific Research under Grants NWO-VICI VI.C.212.072 and NWO-XS OCENW.XS23.1.151 awarded to E.N.M.N-‘tH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest disclosure: the authors have declared that no competing interests exist.