Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

Mathilde Mathieu; Nathalie Névo; Mabel Jouve; José Ignacio Valenzuela; Mathieu Maurin; Frederik J Verweij; Roberta Palmulli; Danielle Lankar; Florent Dingli; Damarys Loew; Eric Rubinstein; Gaëlle Boncompain; Franck Perez; Clotilde Théry

doi:10.1038/s41467-021-24384-2

Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik J Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew, Eric Rubinstein, Gaëlle Boncompain, Franck Perez, Clotilde Théry

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

Original language	English
Article number	4389
Number of pages	18
Journal	Nature Communications
Volume	12
Issue number	1
DOIs	https://doi.org/10.1038/s41467-021-24384-2
Publication status	Published - Dec 2021
Externally published	Yes

Bibliographical note

Funding Information:
We thank for fruitful discussions several team members, especially Drs Mercedes Tkach, Eleonora Grisard, Lorena Martin-Jaular, Jason Ecard, and for helpful discussions and tools, Dr Aurélien Dauphin, Institut Curie, Paris, Dr P. Zimmermann, KU Leuven, Belgium and CRCM Marseille, France, Dr G. van Niel, IPN Paris, France, Dr P Benaroch, Institut Curie, Paris, France, Dr Suresh Mathivanan, LaTrobe University, Melbourne, Australia. The H4A3 monoclonal antibody (anti-human LAMP1), developed by August, J.T. and Hildreth, J.E.K. from Johns Hopkins University School of Medicine, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH, and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was funded by INSERM, CNRS, Institut Curie, French IdEx and LabEx (ANR-10-INSB-04, ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-0038, ANR-11-LABX-0043, ANR-18-IDEX-0001 Université de Paris), grants from french ANR (ANR-18-CE13-0017-03; ANR-18-CE15-0008-01; ANR-18-CE16-0022-02), INCa (INCA-11548), Fon-dation ARC (PGA1 RF20180206962), FRM (FDT201904007945, EQU201903007925 and DGE20121125630), Cancéropôle Île-de-France (2013-2-EML-02-ICR-1), USA NIDA (DA040385), and a Long-Term EMBO Fellowship (ALTF 607-2015) co-funded by the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409) to J.I.V. We also acknowledge the following platforms of Institut Curie: PICT-IBiSA, member of the France-BioImaging national research infrastructure (ANR-10-INBS-04) for fluorescence and electron microscopy, flow cytometry for assistance in data acquisition, genomic for authentication of HeLa cells by STR analysis.

Publisher Copyright:
© 2021, The Author(s).

Keywords

Cell Communication
Cell Membrane/metabolism
Endosomes/metabolism
Exosomes/metabolism
Extracellular Vesicles/metabolism
Fusion Regulatory Protein 1, Heavy Chain
Gene Knockout Techniques
HeLa Cells
Humans
Membrane Proteins/metabolism
Protein Transport
Proteomics
Tetraspanin 29/metabolism
Tetraspanin 30/metabolism

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Mathieu, M., Névo, N., Jouve, M., Valenzuela, J. I., Maurin, M., Verweij, F. J., Palmulli, R., Lankar, D., Dingli, F., Loew, D., Rubinstein, E., Boncompain, G., Perez, F., & Théry, C. (2021). Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9. Nature Communications, 12(1), Article 4389. https://doi.org/10.1038/s41467-021-24384-2

@article{408a695f56004a59ae33cd8c6964605e,

title = "Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9",

abstract = "Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.",

keywords = "Cell Communication, Cell Membrane/metabolism, Endosomes/metabolism, Exosomes/metabolism, Extracellular Vesicles/metabolism, Fusion Regulatory Protein 1, Heavy Chain, Gene Knockout Techniques, HeLa Cells, Humans, Membrane Proteins/metabolism, Protein Transport, Proteomics, Tetraspanin 29/metabolism, Tetraspanin 30/metabolism",

author = "Mathilde Mathieu and Nathalie N{\'e}vo and Mabel Jouve and Valenzuela, {Jos{\'e} Ignacio} and Mathieu Maurin and Verweij, {Frederik J} and Roberta Palmulli and Danielle Lankar and Florent Dingli and Damarys Loew and Eric Rubinstein and Ga{\"e}lle Boncompain and Franck Perez and Clotilde Th{\'e}ry",

note = "Funding Information: We thank for fruitful discussions several team members, especially Drs Mercedes Tkach, Eleonora Grisard, Lorena Martin-Jaular, Jason Ecard, and for helpful discussions and tools, Dr Aur{\'e}lien Dauphin, Institut Curie, Paris, Dr P. Zimmermann, KU Leuven, Belgium and CRCM Marseille, France, Dr G. van Niel, IPN Paris, France, Dr P Benaroch, Institut Curie, Paris, France, Dr Suresh Mathivanan, LaTrobe University, Melbourne, Australia. The H4A3 monoclonal antibody (anti-human LAMP1), developed by August, J.T. and Hildreth, J.E.K. from Johns Hopkins University School of Medicine, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH, and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was funded by INSERM, CNRS, Institut Curie, French IdEx and LabEx (ANR-10-INSB-04, ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-0038, ANR-11-LABX-0043, ANR-18-IDEX-0001 Universit{\'e} de Paris), grants from french ANR (ANR-18-CE13-0017-03; ANR-18-CE15-0008-01; ANR-18-CE16-0022-02), INCa (INCA-11548), Fon-dation ARC (PGA1 RF20180206962), FRM (FDT201904007945, EQU201903007925 and DGE20121125630), Canc{\'e}rop{\^o}le {\^I}le-de-France (2013-2-EML-02-ICR-1), USA NIDA (DA040385), and a Long-Term EMBO Fellowship (ALTF 607-2015) co-funded by the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409) to J.I.V. We also acknowledge the following platforms of Institut Curie: PICT-IBiSA, member of the France-BioImaging national research infrastructure (ANR-10-INBS-04) for fluorescence and electron microscopy, flow cytometry for assistance in data acquisition, genomic for authentication of HeLa cells by STR analysis. Publisher Copyright: {\textcopyright} 2021, The Author(s).",

year = "2021",

month = dec,

doi = "10.1038/s41467-021-24384-2",

language = "English",

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journal = "Nature Communications",

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Mathieu, M, Névo, N, Jouve, M, Valenzuela, JI, Maurin, M, Verweij, FJ, Palmulli, R, Lankar, D, Dingli, F, Loew, D, Rubinstein, E, Boncompain, G, Perez, F & Théry, C 2021, 'Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9', Nature Communications, vol. 12, no. 1, 4389. https://doi.org/10.1038/s41467-021-24384-2

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T1 - Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

AU - Mathieu, Mathilde

AU - Névo, Nathalie

AU - Jouve, Mabel

AU - Valenzuela, José Ignacio

AU - Maurin, Mathieu

AU - Verweij, Frederik J

AU - Palmulli, Roberta

AU - Lankar, Danielle

AU - Dingli, Florent

AU - Loew, Damarys

AU - Rubinstein, Eric

AU - Boncompain, Gaëlle

AU - Perez, Franck

AU - Théry, Clotilde

N1 - Funding Information: We thank for fruitful discussions several team members, especially Drs Mercedes Tkach, Eleonora Grisard, Lorena Martin-Jaular, Jason Ecard, and for helpful discussions and tools, Dr Aurélien Dauphin, Institut Curie, Paris, Dr P. Zimmermann, KU Leuven, Belgium and CRCM Marseille, France, Dr G. van Niel, IPN Paris, France, Dr P Benaroch, Institut Curie, Paris, France, Dr Suresh Mathivanan, LaTrobe University, Melbourne, Australia. The H4A3 monoclonal antibody (anti-human LAMP1), developed by August, J.T. and Hildreth, J.E.K. from Johns Hopkins University School of Medicine, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH, and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was funded by INSERM, CNRS, Institut Curie, French IdEx and LabEx (ANR-10-INSB-04, ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-0038, ANR-11-LABX-0043, ANR-18-IDEX-0001 Université de Paris), grants from french ANR (ANR-18-CE13-0017-03; ANR-18-CE15-0008-01; ANR-18-CE16-0022-02), INCa (INCA-11548), Fon-dation ARC (PGA1 RF20180206962), FRM (FDT201904007945, EQU201903007925 and DGE20121125630), Cancéropôle Île-de-France (2013-2-EML-02-ICR-1), USA NIDA (DA040385), and a Long-Term EMBO Fellowship (ALTF 607-2015) co-funded by the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409) to J.I.V. We also acknowledge the following platforms of Institut Curie: PICT-IBiSA, member of the France-BioImaging national research infrastructure (ANR-10-INBS-04) for fluorescence and electron microscopy, flow cytometry for assistance in data acquisition, genomic for authentication of HeLa cells by STR analysis. Publisher Copyright: © 2021, The Author(s).

PY - 2021/12

Y1 - 2021/12

N2 - Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

AB - Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

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KW - Cell Membrane/metabolism

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KW - Exosomes/metabolism

KW - Extracellular Vesicles/metabolism

KW - Fusion Regulatory Protein 1, Heavy Chain

KW - Gene Knockout Techniques

KW - HeLa Cells

KW - Humans

KW - Membrane Proteins/metabolism

KW - Protein Transport

KW - Proteomics

KW - Tetraspanin 29/metabolism

KW - Tetraspanin 30/metabolism

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DO - 10.1038/s41467-021-24384-2

M3 - Article

C2 - 34282141

SN - 2041-1723

VL - 12

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 4389

ER -

Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

Abstract

Bibliographical note

Keywords

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