Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

Frederik Johannes Verweij*, Maarten P. Bebelman, Connie R. Jimenez, Juan J. Garcia-Vallejo, Hans Janssen, Jacques Neefjes, Jaco C. Knol, Richard de Goeij-de Haas, Sander R. Piersma, S. Rubina Baglio, Matthijs Verhage, Jaap M. Middeldorp, Anoek Zomer, Jacco van Rheenen, Marc G. Coppolino, Ilse Hurbain, Graça Raposo, Martine J. Smit, Ruud F.G. Toonen, Guillaume van NielD. Michiel Pegtel

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


Exosomes are small endosome-derived extracellular vesicles implicated in cell-cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB-PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB-PM fusion using live total internal reflection fluorescence and dynamic correlative light-electron microscopy. Quantitative analysis demonstrates that MVB-PM fusion frequency is reduced by depleting the target membrane SNA REs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB-PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.

Original languageEnglish
Pages (from-to)1129-1142
Number of pages14
JournalJournal of Cell Biology
Issue number3
Early online date16 Jan 2018
Publication statusPublished - 5 Mar 2018


  • Journal Article


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