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

Frederik Verweij (1, 2, 3) , Maarten P. Bebelman (1) , Connie R. Jimenez (1) , Juan J. Garcia-Vallejo (1) , Hans Janssen (4) , Jacques Neefjes (5) , Jaco C. Knol (1) , Richard de Goeij-De Haas (1) , Sander R. Piersma (1) , S. Rubina Baglio (1) , Matthijs Verhage (1) , Jaap M. Middeldorp (1) , Anoek Zomer (6, 7) , Jacco Van Rheenen (7, 6) , Marc G. Coppolino (8) , Ilse Hurbain (2, 3) , Graça Raposo (9) , Martine J. Smit (9) , Ruud F.G. Toonen (7, 9) , Guillaume van Niel (2, 3) , D. Michiel Pegtel (9)

1 VU University Medical Center [Amsterdam]
2 Biologie Cellulaire et Cancer
3 PICT-IBiSA - BioImaging Cell and Tissue Core Facility
4 NKI - Netherlands Cancer Institute
5 LUMC - Leiden University Medical Center
6 Hubrecht Institute [Utrecht, Netherlands]
7 University Medical Center [Utrecht]
8 University of Guelph
9 VU - Vrije Universiteit Amsterdam [Amsterdam]

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 SNAREs 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.

Domaines: Sciences du Vivant [q-bio]; Biologie cellulaire; Interactions cellulaires [q-bio.CB]

Frederik Verweij, Maarten P. Bebelman, Connie R. Jimenez, Juan J. Garcia-Vallejo, Hans Janssen, et al.. Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling. Journal of Cell Biology, 2018, 217 (3), pp.1129-1142. ⟨10.1083/jcb.201703206⟩. ⟨hal-02359707⟩