Figure 4: Visualization of EVs and packaged mRNAs.

(a) Dual-function EV-RNA reporter system enabling EV and EV-RNA labelling; two constructs encoding either PalmtdTomato protein with RNA reporter transcripts containing repeat MS2 RNA binding sequences (MS24X; PalmtdTomato-MS24X; top), or MS2-GFP coat protein (MS2CP) with a nuclear localization signal (NLS) (bottom). (b) Diagram of vesicle RNA labelling process: [1] RNA reporter (blue bar) allows detection of EV-RNA reporter transcripts through binding of MS2 coat protein fused with GFP (MS2CP-GFP-NLS, green dewdrop) to PalmtdTomato-MS24X sequence in mRNA; [2] PalmtdTomato-MS24X:MS2CP-GFP RNA reporter complexes are packaged into EVs; [3] EVs are released from cells by budding from the cell membrane and/or fusion of multivesicular bodies with the cell membrane; and [4] nonspecific EV packaging of MS2CP-GFP-NLS is minimized by translocating the unbound RNA MS2CP-GFP reporter into the nucleus via the NLS signal. CMV, cytomegalovirus enhancer/promoter; HA, haemagglutinin tag; NLS, nuclear localization signal; UTR, untranslated region. (c) EVs were isolated from human Gli36 glioma cells expressing MS2CP-GFP with either PalmtdTomato-MS24X or PalmtdTomato alone (negative control for MS24X mRNA binding sites). Confocal microscopy imaging showed that only EVs isolated from cells co-expressing MS2CP-GFP and PalmtdTomato-MS24X exhibited co-localization between EVs (tdTomato) and MS2CP-GFP:MS24X mRNA complexes (GFP; top row). The negative control for MS24X (PalmtdTomato) showed only membrane labelled EVs (tdTomato). Scale bar, 10 μm.