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.
Highlights
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, or at the plasma membrane (PM) remains unclear
Our results demonstrate that both CD63 and CD9 can be released in small ectosomes formed at the plasma membrane, and that in HeLa cells, exosomes represent a minor subpopulation of small EVs that bear CD63 together with other late endosomal molecules such as LAMP1/2
CD63 and CD9 are secreted abundantly in small EVs, where an EV subpopulation bearing CD63 with CD9 and/or CD81 seemed to correspond to endosome-derived exosomes, but both are detected in larger EVs7
Summary
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. We have adapted CD63 and CD9 to the Retention Using Selective Hook (RUSH) system, which has been used to follow and control trafficking of numerous transmembrane and secreted proteins, and identify atypical pathways of secretion[8] This allows us to synchronize the release of CD9- or CD63-containing EVs, to determine the composition of a more homogenous mixture of newly synthesized EVs. Our results demonstrate that both CD63 and CD9 can be released in small ectosomes formed at the plasma membrane, and that in HeLa cells, exosomes represent a minor subpopulation of small EVs (sEVs) that bear CD63 together with other late endosomal molecules such as LAMP1/2. The markers and molecular mechanisms specific of exosomes versus ectosomes identified here will pave the way for further studies to decipher their respective functions
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