Genetic and metabolic labelling of Extracellular Vescicles: a new tool for biogenesis studies

It has been described that the Human Immunodeficiency Virus (HIV)-1 Nef protein associates with exosomes through anchoring its N-terminal myristoylation to lipid raft microdomains at the endosome membranes. To study Nef association with exosomes, we used a Nef protein mutant defective for all anti-cellular Nef functions that presents a N-terminal palmitoylation, fused at C-terminal with Green Fluorescent Protein (GFP). To demonstrate that Nefmut-GFP goes to compartments like late endosomes or other organelles involved in exosomes biogenesis, we used a novel methodology developed in our laboratory to metabolically label exosomes by incubating cells with a red fluorescent fatty acid BODIPY 558/568 C12 (C12). The lipid is readily taken up by cells and transformed into phospholipids that will ultimately form the exosome lipid bilayer. By transfecting HEK293 cells with the Nefmut-GFP vector and pulsing them with C12 we could purify exosomes containing Nefmut-GFP (Nef-GFP exo) and/or C12 (C12 exo). Fluorescent exosomes were characterized for typical exosomes markers and were analyzed by density gradients showing that Nef-GFP exo could be separated in two distinct peaks whereas C12 exo displayed only one fluorescent peak. Furthermore, they could be sorted by Fluorescence-activated Cell Sorting (FACS) that can be further characterized. In conclusion Nefmut-GFP efficiently incorporates into exosomes that display typical exosome markers (CD63, CD81, CD9, Alix, TSG101). NanoSight analysis showed that vesicles have a mean diameter of 124 nm, compatible with the dimensions of the exosomes reported in literature. Analysis pre-sorting by FACS of double labelled Nefmut-GFP/C12 exosomes showed that the majority of exosomes had incorporated the Nefmut-GFP protein, (92,4% ), of which 40% of the total labeled exosomes is double labeled with Nefmut-GFP and C12 fluorescent lipid. Density gradient separation of double labelled Nefmut-GFP/C12 exosomes showed two distinct peaks for Nefmut-GFP exo whereas C12 exo displayed only one fluorescent peak. Distribution analysis of exo markers by western blot suggested we may have unraveled two different populations of exosomes that can be further characterized.