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J Cell Biol. 2018 Mar 5;217(3):1129-1142. doi: 10.1083/jcb.201703206. Epub 2018 Jan 16.

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

Author information

1
Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, Netherlands frederik.verweij@curie.fr.
2
Institut Curie, Paris Sciences et Lettres Research University, Centre National de la Recherché Scientifique, UMR 144, Paris, France.
3
Cell and Tissue Imaging Core Facility PICT-IBiSA, Institut Curie, Paris, France.
4
Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, Netherlands.
5
Division of Medicinal Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, Amsterdam, Netherlands.
6
Department of Medical Oncology, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, Netherlands.
7
Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, Netherlands.
8
Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, Netherlands.
9
Department of Chemical Immunology, Leiden University Medical Center, Leiden, Netherlands.
10
Department of Clinical Genetics, VU University Medical Center, Amsterdam, Netherlands.
11
Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, VU University Amsterdam, Amsterdam, Netherlands.
12
Cancer Genomics Netherlands-Hubrecht Institute-Koninklijke Nederlandse Akademie van Wetenschappen, Utrecht, Netherlands.
13
University Medical Centre Utrecht, Utrecht, Netherlands.
14
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada.
15
Centre National de la Recherché Scientifique, UMR 144, Paris, France.
16
Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, Netherlands d.pegtel@vumc.nl.

Abstract

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.

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