Abstract
Extracellular vesicles (EVs), including exosomes, are widely believed to mediate intercellular communication by delivering molecular cargo into the cytosol of recipient cells. However, the efficiency and mechanisms of such cargo delivery remain unclear. In this study, we investigated whether EVs bearing the canonical tetraspanin EVs marker CD63 are capable of fusing with recipient cell membranes to release their contents. Using the highly sensitive NanoBiT split luciferase system, we tracked potential fusion events between HiBiT-tagged CD63-EVs and HEK293 cells expressing cytosolic or endosome-localized LgBiT. While HiBiT and LgBiT reconstitution was readily detected when EVs carried the viral fusion protein VSV-G, no luminescence was observed with HiBiT tagged CD63-EVs in absence of VSV-G, despite their uptake or binding by recipient cells. Similarly, EVs carrying HiBiT-HSP70 failed to show evidence of cargo release. These findings demonstrate that unmodified HiBiT-CD63-EVs do not fuse with the plasma or endosomal membranes of recipient cells, suggesting that EV-mediated cargo delivery via membrane fusion is inefficient or absent under standard culture conditions. Our results challenge the prevailing view of EV function in intercellular communication and underscore the need for re-evaluation of EV cargo transfer mechanisms.
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The datasets generated during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank Rose-Laure Revel-Goyet, Oleksandr Glushonkov, Jean-Philippe Kleman (Institut de Biologie Structurale, Grenoble) for the support and access to the Cell imaging Platform, Delphine Guilligay (Institut de Biologie Structurale, Grenoble) for TEM of the EVs.We thank B. Blot for helping with some of the cDNA constructs, M.O. Fauvarque, J. Fauré, S. Fraboulet for helpful discussions throughout this work. We also thank K. Sadoul for critically reading the manuscript.
Funding
We acknowledge support from the ‘Fondation pour la Recherche Medicale (FRM, Equipe FRM 2023, project EQU202303016333) and access to the platforms of the Grenoble Instruct-ERIC center (IBS and ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), with support from FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (EcolesUniversitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). A.A.S. was supported by funding from GRAL. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA) and financial support from CEA, CNRS and UGA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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A.A.S. performed the EV purification and characterization (NTA), did most of NanoLuc assays, made DNA constructions, W.W. and R.S. funding acquisition, R.S. supervised the project and wrote the manuscript, C.C. performed all experiments with VSV-G-EVs, made statistical analysis and co-supervised the project. A.A.S. and C.C. writing original draft preparation.
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Askarian-Amiri, S., Weissenhorn, W., Sadoul, R. et al. Lack of evidence for cargo release of CD63-EVs into recipient cells. Sci Rep (2026). https://doi.org/10.1038/s41598-026-45021-2
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DOI: https://doi.org/10.1038/s41598-026-45021-2
