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

TLDR

Extracellular vesicle populations and their secretion mechanisms—exosomes forming as intraluminal vesicles of endocytic compartments versus ectosomes budding from the plasma membrane—are not fully characterized. The study aims to trace the intracellular trafficking of CD9 and CD63 from the ER to their respective residency compartments and to establish a framework for distinguishing exosomes from small ectosomes. Using live‑cell imaging of CD9 and CD63, comparative proteomics, and endosomal pH neutralization, the authors mapped marker localization and identified surface proteins specific to exosomes (LAMP1) or ectosomes (BSG, SLC3A2). They observed transient co‑localization of CD9 and CD63 before segregation, with CD9 and a PM‑stabilized CD63 mutant released more abundantly, demonstrating that ectosomes predominate over exosomes in HeLa cells.

Abstract

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.