LAUSR

Abstract CD47 regulates the trafficking of specific coding and noncoding RNAs into extracellular vesicles (EVs), and the RNA contents of CD47+ EVs differ from that of CD63+ EVs released by the same cells. Single particle interferometric reflectance imaging sensing combined with immunofluorescent imaging was used to analyse the colocalization of tetraspanins, integrins, and CD47 on EVs produced by wild type and CD47‐deficient Jurkat T lymphoblast and PC3 prostate carcinoma cell lines. On Jurkat cell‐derived EVs, β1 and α4 integrin subunits colocalized predominantly with CD47 and CD81 but not with CD63 and CD9, conserving the known lateral interactions between these proteins in the plasma membrane. Although PC3 cell‐derived EVs lacked detectable α4 integrin, specific association of CD81 with β1 and CD47 was preserved. Loss of CD47 expression in Jurkat cells significantly reduced β1 and α4 levels on EVs produced by these cells while elevating CD9+, CD63+, and CD81+ EVs. In contrast, loss of CD47 in PC3 cells decreased the abundance of CD63+ and CD81+ EVs. These data establish that CD47+ EVs are mostly distinct from EVs bearing the tetraspanins CD63 and CD9, but CD47 also indirectly regulates the abundance of EVs bearing these non‐interacting tetraspanins via mechanisms that remain to be determined.