Cargando…

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

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, pr...

Descripción completa

Detalles Bibliográficos
Autores principales: Verweij, Frederik Johannes, Bebelman, Maarten P., Jimenez, Connie R., Garcia-Vallejo, Juan J., Janssen, Hans, Neefjes, Jacques, Knol, Jaco C., de Goeij-de Haas, Richard, Piersma, Sander R., Baglio, S. Rubina, Verhage, Matthijs, Middeldorp, Jaap M., Zomer, Anoek, van Rheenen, Jacco, Coppolino, Marc G., Hurbain, Ilse, Raposo, Graça, Smit, Martine J., Toonen, Ruud F.G., van Niel, Guillaume, Pegtel, D. Michiel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839777/
https://www.ncbi.nlm.nih.gov/pubmed/29339438
http://dx.doi.org/10.1083/jcb.201703206
_version_ 1783304460427067392
author Verweij, Frederik Johannes
Bebelman, Maarten P.
Jimenez, Connie R.
Garcia-Vallejo, Juan J.
Janssen, Hans
Neefjes, Jacques
Knol, Jaco C.
de Goeij-de Haas, Richard
Piersma, Sander R.
Baglio, S. Rubina
Verhage, Matthijs
Middeldorp, Jaap M.
Zomer, Anoek
van Rheenen, Jacco
Coppolino, Marc G.
Hurbain, Ilse
Raposo, Graça
Smit, Martine J.
Toonen, Ruud F.G.
van Niel, Guillaume
Pegtel, D. Michiel
author_facet Verweij, Frederik Johannes
Bebelman, Maarten P.
Jimenez, Connie R.
Garcia-Vallejo, Juan J.
Janssen, Hans
Neefjes, Jacques
Knol, Jaco C.
de Goeij-de Haas, Richard
Piersma, Sander R.
Baglio, S. Rubina
Verhage, Matthijs
Middeldorp, Jaap M.
Zomer, Anoek
van Rheenen, Jacco
Coppolino, Marc G.
Hurbain, Ilse
Raposo, Graça
Smit, Martine J.
Toonen, Ruud F.G.
van Niel, Guillaume
Pegtel, D. Michiel
author_sort Verweij, Frederik Johannes
collection PubMed
description 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.
format Online
Article
Text
id pubmed-5839777
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher The Rockefeller University Press
record_format MEDLINE/PubMed
spelling pubmed-58397772018-03-08 Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling Verweij, Frederik Johannes Bebelman, Maarten P. Jimenez, Connie R. Garcia-Vallejo, Juan J. Janssen, Hans Neefjes, Jacques Knol, Jaco C. de Goeij-de Haas, Richard Piersma, Sander R. Baglio, S. Rubina Verhage, Matthijs Middeldorp, Jaap M. Zomer, Anoek van Rheenen, Jacco Coppolino, Marc G. Hurbain, Ilse Raposo, Graça Smit, Martine J. Toonen, Ruud F.G. van Niel, Guillaume Pegtel, D. Michiel J Cell Biol Research Articles 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. The Rockefeller University Press 2018-03-05 /pmc/articles/PMC5839777/ /pubmed/29339438 http://dx.doi.org/10.1083/jcb.201703206 Text en © 2018 Verweij et al. https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Articles
Verweij, Frederik Johannes
Bebelman, Maarten P.
Jimenez, Connie R.
Garcia-Vallejo, Juan J.
Janssen, Hans
Neefjes, Jacques
Knol, Jaco C.
de Goeij-de Haas, Richard
Piersma, Sander R.
Baglio, S. Rubina
Verhage, Matthijs
Middeldorp, Jaap M.
Zomer, Anoek
van Rheenen, Jacco
Coppolino, Marc G.
Hurbain, Ilse
Raposo, Graça
Smit, Martine J.
Toonen, Ruud F.G.
van Niel, Guillaume
Pegtel, D. Michiel
Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling
title Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling
title_full Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling
title_fullStr Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling
title_full_unstemmed Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling
title_short Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling
title_sort quantifying exosome secretion from single cells reveals a modulatory role for gpcr signaling
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839777/
https://www.ncbi.nlm.nih.gov/pubmed/29339438
http://dx.doi.org/10.1083/jcb.201703206
work_keys_str_mv AT verweijfrederikjohannes quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT bebelmanmaartenp quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT jimenezconnier quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT garciavallejojuanj quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT janssenhans quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT neefjesjacques quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT knoljacoc quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT degoeijdehaasrichard quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT piersmasanderr quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT bagliosrubina quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT verhagematthijs quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT middeldorpjaapm quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT zomeranoek quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT vanrheenenjacco quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT coppolinomarcg quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT hurbainilse quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT raposograca quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT smitmartinej quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT toonenruudfg quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT vannielguillaume quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling
AT pegteldmichiel quantifyingexosomesecretionfromsinglecellsrevealsamodulatoryroleforgpcrsignaling